PHYLOGENETICS OF EFFECTOR MOLECULES INVOLVED IN CELL-MEDIATED
CYTOTOXICITY
by
KESAVANNAIR PRAVEEN
(Under the Direction of Liliana Jaso-Friedmann)
ABSTRACT
Cytotoxic lymphocytes induce target cell death using common effector pathways that involve
either the components of their cytotoxic granules or members of TNF superfamily ligands. We
present the first evidence for the existence of both granule exocytosis as well as TNF family
ligand mediated killing in cytotoxic cells from ectothermic vertebrates. Nonspecific cytotoxic
cells (NCC) are the first identified killer cell population in teleosts. This study presents the
molecular evidence for expression of multiple granzymes with different substrate specificities as
well as other components of cytotoxic granules in NCC, providing the sequence information for
such molecules for the first time in a non-mammalian species. The main substrate specificities
for granzymes found in these cells were chymase and tryptase. Teleost granzymes conserve the
genomic organization described for mammalian granzymes. Teleost granzymes with chymase
activity have novel S1 specificity triplet residues. This study also demonstrates the role of TNFalpha in NCC functions as an effector molecule of cytotoxicity and in regulation of NCC
functions. Teleost cytotoxic cells express both membrane-bound and secreted forms of TNFalpha, which can induce cell death in susceptible target cells. Membrane-bound TNF-alpha on
NCC can mediate activation-induced cell death in other NCCs in a paracrine manner, while the
soluble TNF-alpha can inhibit this and protect the killer cells from activation-induced cell death.
Soluble TNF-alpha upregulates the expression of other cytotoxic effector molecules like
granzymes in teleost killer cells, indicating a major role played by this molecule in NCC
functions. TNF-alpha-induced protection of NCC was shown to be mediated though different
regulators of apoptosis. One of the important mediators of such function was identified as
cellular apoptosis susceptibility protein, which is upregulated upon treatment of NCC with
recombinant TNF. These findings suggest a parallel evolution of cell-mediated cytotoxicity in
teleosts, leading to effector functions comparable to mammalian counterparts.
INDEX WORDS:
Cell-mediated Cytotoxicity, NK cells, Cytotoxic T Lymphocytes (CTL),
Granule Exocytosis, Granzymes, Perforin, Granulysin, Tumor Necrosis
Factor (TNF) alpha, Activation Induced Programmed Cell Death
(AIPCD).
PHYLOGENETICS OF EFFECTOR MOLECULES INVOLVED IN CELL-MEDIATED
CYTOTOXICITY
by
KESAVANNAIR PRAVEEN
M.S., University of Arkansas at Pine Bluff, 2001
M.F.Sc., Central Institute of Fisheries Education, India, 1998
B.F.Sc., Kerala Agricultural University, India, 1996
A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial
Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
ATHENS, GEORGIA
2005
© 2005
Kesavannair Praveen
All Rights Reserved
PHYLOGENETICS OF EFFECTOR MOLECULES INVOLVED IN CELL-MEDIATED
CYTOTOXICITY
by
KESAVANNAIR PRAVEEN
Electronic Version Approved:
Maureen Grasso
Dean of the Graduate School
The University of Georgia
May 2005
Major Professor:
Liliana Jaso-Friedmann
Committee:
Donald L. Evans
David S. Peterson
David J. Hurley
Zhen F. Fu
DEDICATION
For my beloved wife Manju and my family. I am thankful for your unconditional love and
support, without which this would not have been possible.
iv
ACKNOWLEDGEMENTS
Heartfelt thanks to Dr. Liliana Jaso-Friedmann, my major professor and mentor, for being
my constant source of guidance and encouragement all through this time. You have been above
and beyond what I would have ever wished for as a mentor. I owe you a lot for all my
achievements both at the professional as well as personal level. I am extremely thankful to my
committee members, Dr. Donald Evans, Dr. David Peterson, Dr. David Hurley, and Dr. Zhen Fu
for their continuous support, encouragement and invaluable suggestions.
Many thanks to Dr. Dorothy Hudig, Dr. Dieter Jenne, Dr. James Powers, and Dr. Jan
Potempa for their interest in my work, and for being there to offer helpful suggestions. Special
thanks to John Leary for helping me with the experiments. Many thanks to Dr. Okinaga for
sharing the reagents and protocols. Financial assistance from UGA's graduate school in the form
of Dissertation Completion Assistantship is highly appreciated.
I am quite fortunate to have wonderful friends like Jayakumar, Ritesh, Tina, Niraj,
Naomi, Praveen, Neelesh, and Chen. My heartfelt appreciation for all their help and friendship.
Words are not enough to express my gratitude and sincere appreciation for the Friedmann
family for everything they have done for me. I feel really fortunate to have known you. I am
obliged to Dr. Joseph Burleigh for his guidance and kindness. Special word of appreciation goes
to Goodwin family for all their help and friendship.
I would like to thank each and everyone in my family for their unconditional love and
support. Finally I would like to express my sincere appreciation for the guidance and blessings
from all my teachers. Your love and affection is my greatest strength.
v
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS.............................................................................................................v
LIST OF TABLES......................................................................................................................... ix
LIST OF FIGURES ........................................................................................................................ x
CHAPTER
1
INTRODUCTION .........................................................................................................1
2
LITERATURE REVIEW ..............................................................................................7
CELL-MEDIATED CYTOTOXICITY ....................................................................8
GRANULE-MEDIATED PATHWAYS OF CYTOTOXICITY............................11
CYTOTOXIC PATHWAYS MEDIATED BY TNF-SUPERFAMILY.................31
REGULATION OF CELL DEATH IN CYTOTOXIC CELLS .............................38
REFERENCES........................................................................................................41
3
EVIDENCE FOR THE EXISTENCE OF GRANZYME-LIKE SERINE
PROTEASES IN TELEOST CYTOTOXIC CELLS ..................................................71
ABSTRACT ............................................................................................................72
INTRODUCTION...................................................................................................73
MATERIALS AND METHODS ............................................................................76
RESULTS................................................................................................................79
DISCUSSION .........................................................................................................84
ACKNOWLEDGEMENTS ....................................................................................87
vi
REFERENCES........................................................................................................87
4
NONSPECIFIC CYTOTOXIC CELLS OF TELEOSTS ARE ARMED WITH
MULTIPLE GRANZYMES AND OTHER COMPONENTS OF THE GRANULE
EXOCYTOSIS PATHWAY......................................................................................106
ABSTRACT ..........................................................................................................107
INTRODUCTION.................................................................................................108
MATERIALS AND METHODS ..........................................................................110
RESULTS..............................................................................................................114
DISCUSSION .......................................................................................................119
ACKNOWLEDGEMENTS ..................................................................................124
REFERENCES......................................................................................................124
5
MOLECULAR CHARACTERIZATION AND EXPRESSION OF A GRANZYME
WITH CHYMASE ACTIVITY FROM CYTOTOXIC CELLS OF AN
ECTOTHERMIC VERTEBRATE ............................................................................141
ABSTRACT ..........................................................................................................142
INTRODUCTION.................................................................................................143
MATERIALS AND METHODS ..........................................................................145
RESULTS..............................................................................................................150
DISCUSSION .......................................................................................................156
REFERENCES......................................................................................................161
6
CONSTITUTIVE EXPRESSION OF TUMOR NECROSIS FACTOR-ALPHA IN
CYTOTOXIC CELLS OF TELEOSTS AND ITS ROLE IN REGULATION OF
CELL-MEDIATED CYTOTOXICITY.....................................................................190
vii
ABSTRACT ..........................................................................................................191
INTRODUCTION.................................................................................................192
MATERIALS AND METHODS ..........................................................................194
RESULTS..............................................................................................................200
DISCUSSION .......................................................................................................204
ACKNOWLEDGEMENTS ..................................................................................209
REFERENCES......................................................................................................210
7
MOLECULAR CLONING OF CELLULAR APOPTOSIS SUSCEPTIBILITY
(CAS) GENE IN OREOCHROMIS NILOTICUS AND ITS ROLE IN REGULATION
OF NONSPECIFIC CYTOTOXIC CELL (NCC) FUNCTIONS .............................234
ACKNOWLEDGEMENTS ..................................................................................238
REFERENCES......................................................................................................238
8
SUMMARY AND CONCLUSIONS ........................................................................255
viii
LIST OF TABLES
Page
Table 3.1: Oligonucleotide primers used in the cloning of catfish granzyme and for expression
studies by RT-PCR......................................................................................................104
Table 3.2: Amino acid identity and similarity of mature catfish granzyme to other known
granzymes and related proteins ...................................................................................105
Table 5.1: Organization of TLGR-1 gene....................................................................................188
Table 5.2: Pairwise comparison of TLGR-1 with related serine proteases .................................189
ix
LIST OF FIGURES
Page
Figure 3.1: Cloning strategy used to identify and characterize the cDNA for catfish granzyme ..91
Figure 3.2: Compiled full-length catfish granzyme cDNA sequence............................................93
Figure 3.3: Multiple sequence alignment of predicted catfish granzyme sequence with known
granzymes and related proteins .....................................................................................95
Figure 3.4: Phylogenetic analysis of catfish granzyme .................................................................98
Figure 3.5: Analysis of tissue expression of catfish granzyme....................................................100
Figure 3.6: Alignment of other fish granzyme sequences with catfish granzyme.......................102
Figure 4.1: Production of active and pro-CFGR-1 in Pichia pastoris.........................................127
Figure 4.2: Purification of recombinant proteins.........................................................................129
Figure 4.3: Enzymatic activity of recombinant CFGR-1.............................................................131
Figure 4.4: Correlation between cytotoxicity and exocytosis of granzymes with tryptase activity
in catfish NCC .............................................................................................................133
Figure 4.5: Expression of other proteases in the cytotoxic granules of catfish NCC ..................135
Figure 4.6: Identification of multiple granzymes from catfish NCC...........................................137
Figure 4.7: Expression of other granule components in catfish NCC .........................................139
Figure 5.1: Genomic organization of TLGR-1 gene and promoter region ..................................167
Figure 5.2: Predicted three dimensional structure of TLGR-1 ...................................................170
Figure 5.3: Comparison of TLGR-1 with other granzyme sequences ........................................172
Figure 5.4: Phylogenetic analysis of TLGR-1 .............................................................................174
x
Figure 5.5: Analysis of tissue expression of TLGR-1 .................................................................176
Figure 5.6: Transcriptional regulation of TLGR-1 in NCC.........................................................178
Figure 5.7: Production of recombinant TLGR-1 in Pichia pastoris............................................180
Figure 5.8: Purification of pro and mature TLGR-1 recombinant proteins .................................182
Figure 5.9: Enzymatic activity of recombinant TLGR-1.............................................................184
Figure 5.10: Substrate specificity of TLGR-1 .............................................................................186
Figure 6.1: Nucleotide and deduced amino acid sequence of tilapia TNF ..................................215
Figure 6.2: Multiple sequence alignment and molecular modeling for tilapia TNF ...................217
Figure 6.3: Phylogenetic analysis of tilapia TNF ........................................................................220
Figure 6.4: Expression of tilapia TNF gene.................................................................................222
Figure 6.5: Purification of recombinant tilapia TNF using Ni-affinity chromatography ............224
Figure 6.6: Expression of TNF on tilapia NCC membranes........................................................226
Figure 6.7: Cytotoxicity induced by tilapia TNF on susceptible WEHI cells .............................228
Figure 6.8: Recombinant TNF protects activation-induced cell death in NCC...........................230
Figure 6.9: Recombinant TNF upregulates the transcription of granzyme in tilapia NCC .........232
Figure 7.1: Compiled full-length cDNA sequence of tilapia CAS ..............................................242
Figure 7.2: Multiple sequence alignment of tilapia CAS ............................................................246
Figure 7.3: Phylogenetic analysis of tilapia CAS ........................................................................249
Figure 7.4: Expression of tilapia CAS in different tissues...........................................................251
Figure 7.5: Transcriptional regulation of tilapia CAS in NCC....................................................253
xi
CHAPTER 1
INTRODUCTION
1
The significance of cell-mediated immunity became apparent with the discovery that
professional killer cells were responsible for the clearance of virus infected and tumor cells and
that these killing processes were not mediated though antibodies or complement. During the past
few years, vast resources in time and effort were devoted to understanding the effector
mechanisms used by those cytotoxic cells. The major players in cell-mediated cytotoxicity
include cells of the adaptive immune system (cytotoxic T lymphocytes, CTLs) and natural killer
cells (NK cells) that belong to the innate immune system. The molecular mechanisms by which
these cells kill other cells have been investigated extensively and the importance of this process
in innate and adaptive immune responses has been well established.
Three stages have been identified in the cytolytic process mediated by professional killer
cells. In the recognition stage, the target cells have to be identified by the effector cells. The
second stage involves the lethal hit by which the effector cell kills the target cell. Lastly, the
target cell disintegrates. Although some of the molecular mechanisms associated with these three
crucial events have been identified, many more questions remain to be answered. Recognition of
target cells by CTL involves presentation of foreign peptides in the context of class I MHC
molecules and co-stimulators. Multiple receptors have been identified in target cell recognition
by NK cells, and more are yet to be discovered. Following recognition, the signals initiated by
the engagement of the receptors are a complex series of events that have not been fully
deciphered, but are known to lead to the lethal hit of the targets. At least two independent
pathways of killing have been identified in both CTL and NK cells: the granule exocytosis
pathway with release of lethal molecules, and the binding of death ligands of the TNF
superfamily to death receptors on target cells. It appears that the effector mechanism used by the
cytotoxic cell varies with the type of target it has to destroy. Both pathways play significant roles
2
in inducing target cell death and can act synergistically, but the granule exocytosis pathway
alone is sufficient to kill target cells even in the absence of death receptors. One of the aspects in
the lethal hit that is not fully understood is the mechanism by which the cytotoxic activity is
regulated in the activated killer cells.
Much effort has been dedicated to understand the molecules involved in the killing of
targets because, although CTL and NK cells employ different recognition mechanisms to
identify their targets, the pathways to induce death are shared. Still, many questions remain to be
answered about each of these pathways. Significant advances have been made in understanding
the signaling pathways that are initiated by the ligation of TNF superfamily ligands to their
corresponding death receptors. The granule exocytosis pathway is still poorly understood, due in
part to the lack of information about the molecular identity of many of the granular components,
as well as their physiological significance.
Serine proteases of the granzyme family and other associated components of cytotoxic
granules are the lethal components of the granule exocytosis pathway. Gene sequences for these
molecules have been identified in mammals, but there is no such information available for coldblooded vertebrates. This fact represents a significant immunologic and evolutionary void in our
knowledge, since granule exocytosis remains the preferred mechanism employed by T-cells and
NK cells in the killing of tumor cells, protozoan parasites or virus-infected cells.
One of the major obstacles immunologists and evolutionary biologists face in
understanding the granule exocytosis pathway of killing is in trying to explain the role of
multiple granzymes with overlapping substrate specificities. It is known that new highly
specialized primary specificities are gained by recently evolved proteases, while the predicted
evolutionary precursor already had very restricted substrate specificity. Thus, the evolution of
3
serine protease family members is perplexing and the events that have led to the despecialization
steps in generation of newer specificities are still not clear. Granzymes are considered as newly
evolved members of the serine protease family. They can be divided into four sub-families based
on their specificity: Tryptase, Asp-ase, Chymase and Met-ase. Members of these subfamilies
have co-evolved in separate genetic loci and their relative significance is not known. It has been
well established that granzymes A and B induce target cell death, while the functional
importance of other granzymes is unclear. These studies have been complicated by the
realization that silencing of one or two granzyme genes alone does not inhibit all the cytotoxic
activity of the effector cell. Clearly, simpler models of granule exocytosis killing mechanisms
would aid in answering some of the fundamental questions about this pathway of cytotoxicity.
Fish are the first group of animals in the evolutionary tree to develop a fully functional
immune system with innate and adaptive immunity comparable to mammals. As such, fish
represent an ideal model for the study of immunological processes in addition to understand
unsolved questions regarding the development and function of the immune system in higher
vertebrates. Moreover, the comparative study of different, evolutionarily distant fish species
provides an excellent opportunity to understand the phylogeny of effector molecules.
The first killer cell population of teleosts was identified and characterized in channel
catfish and was called nonspecific cytotoxic cells (NCC). Subsequently, the presence of teleost
cytotoxic cells other than NCC (as well as similar to NCC) has been reported from many species
of fish. Initial observations suggested that NCC were non-granular. However, their dependence
on calcium in inducing target cell death suggested the existence of the granule exocytosis
pathway. Although the effector molecules involved in cytotoxic function and their regulation
were not fully understood, killing of mammalian tumor target cells by NCC suggested that the
4
molecules involved in the lethal hit would be evolutionarily conserved. This theory was
supported by functional studies that demonstrated the presence of components of the death
ligand pathway in NCC from different species. While the role of Fas ligand in NCC cytotoxicity
has been demonstrated, the functions of other members of the TNF super family remain poorly
understood.
Significance: The present study was undertaken to delineate the pathways of cytotoxicity
employed by NCC in the delivery of the lethal hit. Identification of the components that mediate
NCC function is important for several reasons. The identification of genes coding for
components of cytotoxic granules from ectotherms would provide the first step in understanding
the phylogenetic origin and the mechanisms of action of the many members of the granzyme
family. This information could be used to understand the biological roles, substrates and
pathways of death employed by different granzymes in organisms, where acquired immune
responses appear to be less advanced. Aside from the basic knowledge in immunology gained by
these studies, a practical application of this work is that characterization of the molecules
involved in cell mediated cytotoxicity of teleosts will provide more insights into developing
strategies to improve fish health. This is exemplified by the finding that unlike mammalian NK
cells that can recycle and attack multiple targets, NCC lack the ability to recycle. It has been
demonstrated that NCC undergo activation-induced cell death (AICD) following conjugate
formation with the target cell. The role of death ligands as apoptosis regulatory factors has been
suggested as a mechanism to balance the signals for AICD and cell survival. This mechanism
could provide a way to enhance the NCC functions during episodes of acute stress.
Hypothesis: The central hypothesis for this study was that NCC use pathways involving
both granule exocytosis and TNF superfamily ligands to induce target cell death. This would
5
provide evidence for a parallel evolution of effector molecules in ectothermic vertebrates. The
objective of this study was to identify and characterize the molecular components involved in
NCC functions. To accomplish this, the following specific aims were pursued.
1
Identification of the components of cytotoxic granules in NCC from channel catfish
(Ictalurus punctatus) and tilapia (Oreochromis niloticus) to compare the pattern of
evolution in two phylogenetically distant species. This aim included:
•
Identification of granzyme gene sequences
•
Assessment of the expression pattern and substrate specificities of teleost
granzymes
•
Identification of genes coding for accessory molecules present in cytotoxic
granules
2
Identification of the role of death ligand TNF-alpha in functions of NCC in tilapia
(Oreochromis niloticus). This aim included:
•
Identification of the tilapia TNF gene sequence
•
Assessment of the expression pattern of TNF in tilapia NCC (membrane
and secreted forms)
•
3
Functional characterization of TNF in tilapia NCC
Identify the role of regulators of apoptosis in modulating NCC functions during
episodes of acute stress. This aim included:
•
Identification of apoptosis regulatory genes expressed under acute stress
•
Assessment of their expression patterns
6
CHAPTER 2
LITERATURE REVIEW
7
CELL-MEDIATED CYTOTOXICITY
The first report of the in vitro toxic effects of immune cells on target cells dates back to
the 1960’s (1). In these early studies, the aims were to understand the physiological requirements
for target cell death (2-4). Although lymphocytes were the prime suspects, the identity of the
effector cells involved in these cytotoxic reactions was not fully understood. Subsequently, T
lymphocytes were shown to play a major role in cell-mediated cytotoxicity (5,6). Cytotoxic
lymphocytes are the professional killer cells that form conjugates with target cells prior to killing
them. The target cells that are recognized by killer cells have been infected with cellular
pathogens or are phenotypically transformed, as in the case of tumor cells. This group of
cytotoxic effector cells includes cytotoxic T lymphocytes (CTL), natural killer (NK) cells,
gamma delta T cells and natural killer T (NKT) cells (7). CTL cytotoxicity is triggered by TCR
recognition of target cells bearing specific peptides on particular allotypes of MHC class I
molecules. By contrast, NK cell cytotoxicity is regulated by a delicate balance between the
engagement of inhibitory and activating germ line-encoded receptors. In spite of many
differences, both NK cells and CTL use the same basic mechanisms for destroying their targets.
(8). In NK cells, many of the effector molecules are pre-formed during development and they
can initiate their cytotoxic function without undergoing proliferation. Unlike NK cells, CTL
precursors are not cytotoxic and upon activation, they induce proliferation and upregulate the
expression of effector molecules to become better killers (9,10).
Cell-mediated cytotoxicity is important in regulation and termination of an immune
response. The regulatory role is directed at inducing the death by apoptosis of activated immune
cells. Uncontrolled cytotoxicity can lead to undesirable conditions like in the case of graft versus
host disease (8). Target cell lysis by cytotoxic cells differs from the cytotoxicity induced by
8
antibody and complement in the fact that the former affects the nucleus resulting in internal
disintegration of the targets (11,12). Target cells play an active role in their own destruction,
which is obvious from the fact that the severity of nuclear destruction induced by the same killer
cell varies with the nature of the target cell (13). The differential contribution of various effector
molecules towards the killing is determined to a large extent by the quality of target cell used to
measure the cytotoxicity (14).
Nonspecific cytotoxic cells (NCC) are the first identified cytotoxic cell population in
teleosts. Since their initial description in the channel catfish (15-18), these cells have been well
characterized in a number of lower vertebrates (19-23). Cytotoxic cell populations with different
phenotype than that of NCC have also recently been identified from channel catfish (24,25). The
importance of the Fas ligand-mediated killing mechanism of mammalian target cells by fish
cytotoxic cells has been previously reported (26-28). Although first described as agranular (15),
NCC do have small granules and contact with target cells leads to granule exocytosis with
necrotic and apoptotic death pathways in trout, tilapia and catfish (17,21,29). Requirement for
calcium for the induction of cytotoxicity by NCC was an early indication that these cells use the
granule exocytosis killing pathway (30). Catfish cytotoxic T cell lines have also been shown to
kill target cells by the granzyme/perforin pathway, but the lack of molecular tools did not allow
the identification of these proteins (31). NCC kill a variety of transformed mammalian cell lines,
much like human lymphokine activated killer cells (LAK), which also kill xenogeneic targets.
Unlike LAK cells however, NCC do not require activation with lymphokines to kill a wide range
of targets. Target cell lysis occurs following conjugate formation of the target cell with an
antigen receptor on NCC (NCCRP-1) (18,19). Complete abrogation or decreased expression of
NCCRP-1 as well as its blocking with specific antibody results in inhibition of killing of target
9
cells (18,20). The phenotypic changes that follow cross-linking of the antigen receptor, NCCRP1 with its natural ligand on target cells or a monoclonal antibody (mAb 5C6) are signals for
activation of cytotoxicity and degranulation (32).
Effector mechanisms of cell-mediated cytotoxicity
NK cells and CTLs can kill their targets by the vectorial release of the contents of their
granules or by engaging death receptors on the surface of target cells with corresponding ligands
(for example, the ligands of TNF superfamily). This generalization does not reflect the
contribution of the multiple pathways involved, depending upon the type of effector molecule or
the target cell, all of which will eventually lead to the cell death (10). The main constituents of
cytotoxic granules are perforin and a family of serine proteases called granzymes (8). FasL and
other similar TNF superfamily members are type II transmembrane proteins expressed mainly on
T cells and NK cells (33). In non-hematopoietic cells FasL is directly sorted to the cell
membrane (34). In contrast, FasL is co-expressed with granzymes and perforin in the secretory
granules of NK cells and CTLs (35). The cytoplasmic tail of FasL contains sorting information,
which targets newly formed protein to the secretory granules in cytotoxic cells (35). Fully active
granule associated FasL is released along with granzyme and perforin to the immunological
synapse between effector and target cells. This initial degranulation process, changes the
expression of FasL to the cell membrane following target cell recognition (35). Although the
different effector mechanisms used by cytotoxic cells are still not fully understood, most studies
agree that there are two main killing pathways: the granule mediated pathways and the pathways
involving members of the TNF superfamily.
10
GRANULE-MEDIATED PATHWAYS OF CYTOTOXICITY
Cytotoxic Granules
Upon formation of a well-defined immunological synapse between the target and effector
cells, the contents of the cytotoxic granules are released in close proximity of the target cell
(36,37). Professional secretory cells utilize secretory granules for exocytosis of their products
while maintaining a separate lysosomal compartment. In contrast, hematopoietic cells have
merged those two organelles in to a single compartment called the secretory lysosome (38).
Secretory lysosomes resemble lysosomes in their low pH, similar structure and the marker
proteins they contain (38-40). However, unlike lysosomes, they can be distinguished by their
ability to fuse with the plasma membrane in a regulated fashion (41).
The granules in cytotoxic cells have specialized structures with a core surrounded by a
multivesicular region (39). Cytotoxic granules may play a dual role with a regulated secretory
compartment (the core) and a pre-lysosomal peripheral compartment (42). Granzymes, a group
of serine proteases comprise about 90% of the mass of these granules (43). Other components of
the granule are perforin (39), calreticulin, which chaperons perforin (44), chemokines (45,46),
chondroitin sulfate-containing proteoglycans called serglycins (47), granulysin (48) and some
members of the TNF superfamily (35). The peripheral compartment of cytotoxic granules has
several lysosomal hydrolases as well as unique components such as the mannose-6-phsophate
receptor complex, usually absent in lysosomes, but present in early endocytic compartments
(42). Among the lysosomal enzymes, cathepsin C (also called dipeptidyl peptidase I, DPPI) has
functional significance in the activation of serine proteases. Other proteins identified in lytic
granules include lysosomal transmembrane proteins (CD63), lysosome-associated membrane
11
proteins (Lamp1 and Lamp2) (39) and cytotoxic T lymphocyte antigen 4 (CTLA-4), which plays
an important role in T cell signaling (49).
Granzymes
A total of 14 granzymes have been reported from humans and rodents that have been
named A to M. Of these, granzymes A and B are the most abundant and the best studied to date
(39). Reports of granzyme-like molecules from lower vertebrates are now becoming available
(50). Structurally, granzymes are closely related to chymotrypsin, with conserved catalytic triad
residues: His, Asp, and Ser (43). Granzyme subfamilies have trypsin-like, chymotrypsin-like or
elastase-like specificities and their genes are mapped to tryptase, chymase or Met-ase loci,
respectively (51). Most of the earlier studies on granzymes concentrated on granzymes A and B,
while the importance of other granzymes (called orphan granzymes because of the lack of
information about their physiological substrates) is now being widely appreciated (52).
Like other serine proteases, the catalytic activity of granzymes depends on the Ser residue
at the active site (51). There is an oxyanion hole to stabilize the transition state of the enzymesubstrate complex and a substrate binding pocket, which determines the substrate specificity.
Few granzyme crystal structures have been resolved (50,53,54). The 3D structure data from other
serine proteases has been used to construct molecular models that can predict the substrate
specificity and structural features for many other granzymes. These models have helped in
understanding the functional association of granzymes with other molecules and their predicted
role in cell mediated cytotoxicity (55).
Granzymes are synthesized as zymogens and are processed at the time of packaging into
the granules. Cleavage of a leader peptide leaves two amino acids at the N-terminal and final
12
enzymatic activation is achieved when those two residues are clipped off by Cathepsin C (56).
Granzymes A and B are found to have mannose-6-phosphate moieties, which will allow the
packaging thorough the mannose-6-phosphate receptor pathway (57). The level of glycosylation
varies widely between the granzymes. Granzyme K has no glycosylation, granzyme C has few
and granzyme D is very heavily glycosylated (43,58). All granzymes do have three highly
conserved disulfide linkages, which are responsible for the correct folding of the protein.
Granzyme A, K and M have an additional disulfide bond bridging the active site serine, like in
chymotrypsin (43). Granzyme A is the only granzyme found to form dimers as a result of interchain disulfide bonding (59).
Cell specificity of granzyme gene expression has been the subject of many investigations
(52). Granzymes were believed to be expressed mainly in CTL and NK cells (60). Initial analysis
of granzyme expression in CTLs derived from mixed lymphocyte reactions revealed a
predominance of granzyme A and B, with minimal expression of orphan granzymes (61).
However, expression of orphan granzymes along with granzymes A and B was found to be high
in NK cells. High level expression of orphan granzymes is restricted to lymphokine activated
killer (LAK) cells, NK cells, large granular lymphocytes (LGL) and thymocytes. Both CD4+ and
CD8+ T cells express orphan granzymes in an activation dependant manner, but at a very low
level compared to granzymes A and B (52). Considering the high level of expression in
thymocytes, it is hypothesized that orphan granzymes may play an important role in T cell
development (62). Recent findings, using single-cell RT-PCR studies, have suggested that each
lymphocyte expresses different combinations of perforin and/or one or more granzymes,
indicating an additional level of control over lymphocyte mediated killing (63).
13
Recent reports suggest that granzymes could also be involved in non-immune functions
(64,65). For example, granzyme genes have been found to be upregulated in cells treated with
follicle stimulating hormone (64). The presence of perforin-independent expression of granzyme
B and its specific inhibitor in human testis and placenta (66) could indicate a role of granzymes
in reproduction. The release of granzymes during cytotoxicity causes an increase in the level of
granzyme in the biological fluids and serves as a marker for activation of CTL and NK cells.
Thus, soluble granzyme levels are found to be elevated in viral infections (67), parasitic
infections (68), and rheumatoid arthritis (69). An alternatively spliced variant of granzyme K is
expressed in certain neuronal cell types (70). Similarly, an alternatively spliced variant of
granzyme M is exclusively expressed in photoreceptor cells of the retina in the mouse (71). The
functional significance of these variants has not been fully investigated.
Granule Exocytosis
The granule exocytosis pathways of mammalian cells and the outcome of many of the
complex events that take place in the killing of target cells have been recently reviewed (72). NK
cells have high quantities of constitutive granules and thus are armed for immediate response
following target cell contact. Once NK cells have killed a target, they require cytokine activation
to recycle (73). In contrast, CTL require initial antigen stimulation for the up-regulation of
cytokine receptor expression and for the synthesis of granular proteins (43). Subsequent
encounters with specific antigen-bearing target cells provide the signals for the armed clones of
CTL to degranulate and proliferate, a process that may take up to 3 days. This difference
between NK cells and CTL may reflect the distinct roles of each one of these cells in the immune
14
system. NK cell activity is part of the innate immune system with rapid but limited cytotoxic
activity. CTL take longer to become lethal, but acquire a higher killing capacity.
Conjugate formation is initiated by the interaction of activation receptors with surface
ligands on the target cells, although adhesion molecules are required to maintain a tight junction
between the effector and the target cell. Redistribution of granules on the NK cell or CTL to the
contact area with the target leads to vectorial release of granular contents and provides specificity
of killing to the granzyme pathway. This is known as the "lethal hit" (74-76). The granules can
move along the microtubule in both directions, towards and away from the microtubuleorganizing center (MTOC). Upon conjugate formation, the MTOC polarize towards the
immunological synapse (77-79). The CTL and NK cell interactions with the target cells are very
rapid (minutes) and transient, compared to the cell to cell interactions of CD 4+ T cells with
antigen presenting cells (80). The degranulation required for cell-mediated cytotoxicity is
controlled by actin polymerization, a requirement for killing (81-83). The delineation of the
molecular mechanisms involved in the delivery of the lethal hit has been investigated extensively
but are still not completely understood (84-87). As a result of these investigations, several
important signaling molecules have been identified as critical for granule exocytosis such as Syk,
Rac, Vav and ERK (81). These molecules appear to act as messengers that deliver signals from
the receptor on the effector cell to downstream effectors that induce the cytoskeletal changes
(88). It is important to note that the exact nature of the receptor(s) that initiate actin
polymerization in cytotoxic cells to activate degranulation has not yet been confirmed.
15
Entry of granule components in to the target cells
Models describing the entry of granule components into target cells have been undergone
multiple revisions. Negatively charged sulfated GAG chains of proteoglycans act as a reservoir
for cationic proteins found within the cytotoxic granules (40). During granule exocytosis, the
contents of the granules experience a sudden change from highly acidic to physiological pH,
which results in the release of perforin from the macromolecular complexes, while granzymes
remain bound (40).
Membrane pores formed by polymerization of perforin was initially believed to be the
site of entry for granzymes into the target cells (89). The perforin pores fail to admit
unglycosylated proteins or carbohydrate polymers and are found to be highly selective for
granzymes (90). Other membrane pores, like the ones formed by complement, cannot effectively
deliver granzymes into the target cells, suggesting a specific interplay between perforin and
granzymes (90). However, fluorescein isothiocyanate (FITC) labeled granzyme B has been
shown to enter the target cells even in the absence of perforin. It was demonstrated that
granzyme B can bind to the target cell surface in a specific, saturable manner in the absence of
perforin and remain in endocytic vesicles without any cytotoxic consequences (91-93). These
findings suggested the existence of a specific receptor, which can initially internalize granzyme
B into endocytic compartments. Sublytic concentrations of perforin, or any other endosmolytic
agents like non-replicating adenovirus, listeriolysin O, or streptolysin O, can act to help the
granzyme B to be released into the cytoplasm of target cells (90,91). While the mannose 6phosphate receptor has been reported as a candidate for cell surface receptor for granzyme B
(94), recent reports highly contradict the significance of this receptor in induction of target cell
death (95-97).
16
Once granzymes are released from cytotoxic granules, they are found as complexes
bound in a charge dependent manner with the proteoglycan serglycin (98-100). Thus, new
questions have been raised on the interaction of this complex with the receptor. Granzymes do
have positive surface charge, which is responsible for the charge-dependant binding between
granzyme and serglycin. The masking of charges due to this binding is predicted to affect the
interaction between granule components and cell-surface receptors on target cells (53,101,102).
Several candidate proteins have been reported to be involved in the uptake of the granzymeserglycin complex. Dynamin (103) as well as cell surface-bound heat shock protein-70 have been
reported to bind to and uptake granzyme B, resulting in a perforin-independent target cell death
(104).
Role of perforin in cytotoxicity
Cytotoxic lymphocytes have been described to form conjugates with their targets and
deliver a “lethal hit”, resulting in target cell death (105). Perforin, a major component of the
cytotoxic granule, is synthesized as an inactive precursor, which will undergo proteolytic
processing to remove carboxy-terminal glycosylated peptide and become active (106). Active
perforin monomers undergo calcium-dependant polymerization and get inserted into the target
cell membrane, forming pores on cell surfaces like the C9 component of terminal complement
complex (107). Target cell destruction was believed to be a result of osmotic lysis due to
excessive influx of ions into the target cells (108,109). Although gene knock-out experiments
have shown that perforin is required for target cell death (110-113), the exact mechanism
remains unclear. The discrepancy is partly due to the fact that the hallmark of target cells
attacked by cytotoxic lymphocytes is apoptosis, but perforin alone induces membrane damage
17
leading to necrosis (114,115). These observations led the way to the hypothesis that granzymes
have an important role in target cell death (116). The exact function of perforin in delivering
granzyme to the target cells is still under debate. In some circumstances perforin alone might
bring about the target cell death (90,99). Some of the main problems associated with determining
the role of perforin in cytotoxicity is the failure to generate active recombinant perforin and
inability to track perforin movement inside target cells by fluorescent microscopy (8). In an
effort to clarify the respective roles of perforin and granzymes in target cells death, these
components have been added separately in transfection experiments. When the non-cytotoxic rat
basophilic leukemia (RBL) cell line was transfected with perforin, high level of cytolysis against
RBC was observed, while nucleated cells were not killed effectively (117). These results appear
to indicate the poor efficiency of perforin in forming pores in nucleated cells. A possible
explanation could be that nucleated cells have the capacity to repair membrane damage (118).
Transfection of both granular components, granzyme and perforin, produced apoptosis in
nucleated target cells (91).
Role of granzymes in cytotoxicity
The first evidence for the involvement of cellular proteases in cell-mediated cytotoxicity
came from the experiments involving inhibition of killing by T-cells with general protease
inhibitors like diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF)
(119-122). Other natural protease inhibitors like alpha1-antitrypsin and alpha1-antichymotrypsin
can also suppress target cell killing (123,124). Pre-treatment of effector cells with these protease
inhibitors had no effect on the target cell killing, but the presence of inhibitors during the
effector-target conjugate formation, even for a short period of time, was found to be crucial in
18
inhibition of cytotoxicity (125). Similarly, a short period of incubation with microfilament
inhibitors was crucial in blocking target cell death (125). These results suggested that secretion
of proteases to the extracellular environment in close proximity to the target cells was a
necessary step in cell-mediated cytotoxicity. Subsequently, a DFP sensitive protease was isolated
from cytotoxic T cells and was found to be lethal to T24 human bladder carcinoma cells,
suggesting the direct involvement of such proteases in cytotoxicity (126). The serine proteases
present in cytolytic granules of cytotoxic lymphocytes were named granzymes.
Granzyme B: Apoptosis induced by CTLs is associated with the proteolytic processing of
procaspase-3 (127). The most studied of all the granzyme family members, granzyme B, was
originally thought to act by cleaving caspases and inducing apoptosis. Subsequent studies on the
hierarchy of granzyme B-initiated caspase activation suggested a two-tiered activation cascade,
involving seven caspases (128). More recently, granzyme B has also been found to induce a
caspase-independent pathway of cell death (129,130). Indeed, multiple intracellular substrates
for granzyme B have been identified, which are associated with cell death pathways.
Two of the alternative mechanisms of action of granzyme B are the direct cleavage of
caspases to induce DNA fragmentation (131-133), or the cleavage of key structural proteins in
the nuclear membrane or cytoskeleton (134,135). One of the most important direct targets for
granzyme B is BH3-interacting domain death agonist (BID), which following truncation can
destroy the membrane integrity of mitochondria (136). Mitochondrial lysis leads to the release of
multiple pro-apoptotic molecules including cytochrome c, HtrA2/OMI (a serine protease that
blocks inhibitor of apoptosis proteins) and endonuclease G (137-140). However, there is also
evidence for the disruption of mitochondrial transmembrane potential by granzyme B, which is
independent of BID and caspase (137,141,142). Therefore, it can be hypothesized that there are
19
multiple caspase-independent mitochondrial effects of granzyme B, one of them requiring BID
and the other one involving direct action of granzyme B (8).
It has been demonstrated that the truncated BID generated by granzyme B proteolysis
uses Bak, a mitochodrial proapoptotic Bcl-2 family member, for the release of cytochrome c
from mitochondria. These results have been derived from the lack of granzyme B-mediated
cytotoxicity in Bak deficient target cells (143). Furthermore, these data appear to concur with the
fact that Bak deficiency has been hypothesized as an immune evasion mechanism used by certain
tumor cells and virally transformed cells.
Mitochondrial apoptotic events initiated by granzyme B can occur even in the absence of
BID, indicating alternative pathways (144). The alternative mechanism involves Bim, a member
of the BH3-only-protein family, which can mediate the release of cytochrome c. Mcl-1L, which
is a pro-survival protein, can bind to Bim and mask its pro-apoptotic effects. Mcl-1L localizes
mainly to the outer membrane of mitochondria and is found to be a physiological substrate for
granzyme B (144). These findings suggested a novel pro-apoptotic pathway induced by
granzyme B. Inhibition of Mcl-1L in cells can induce apoptosis even in the absence of any
apoptotic stimuli, indicating the significance of this pathway in cell death (145)
Additionally, other pro-apoptotic effects of granzyme B are believed to be due to the
cleavage of downstream caspase substrates like poly (ADP ribose) polymerase, catalytic subunit
of DNA-dependant protein kinase and nuclear mitotic apparatus protein (146,147). A recently
identified pathway involves the cleavage of Rho-associated coiled coil-containing protein kinase
(ROCK) II by granzyme B (148). ROCK I is a caspase substrate and its breakdown leads to the
membrane blebbing in apoptotic cell death. ROCK II is closely related and has similar functions
20
to ROCK I. Cleavage of this protein by granzyme B indicates yet another caspase-independent
pathway to induce apoptotic morphology in target cells (148).
Other substrates for granzyme B include the zeta chain of T cell receptor (TCR), which is
cleaved at a different aspartic acid residue than that of the cleavage mediated by caspase-3 (149).
It has been shown that viruses like HIV can use the TCR zeta chain to contribute towards the
pathogenesis as an evasion mechanism (150,151). Therefore, direct degradation of the TCR zeta
chain by granzyme B has been hypothesized as a cytotoxic mechanism directed against T cells
that have been infected with such viruses.
Granzyme A: Most of the initial studies on granzymes concentrated on granzyme B
because it was fast and easy to measure its action on target cells, inducing classical signs of
apoptosis. Until recently, granzyme A was believed to be an inert granzyme or a very slow killer
(8). This idea was due to the absence of apoptotic signs, such as oligonucleotide fragment
release, for up to 16 hours post-treatment. Transfection experiments done with the RBL cell line
have shown that granzyme A, in combination with perforin, induces apoptosis in a perforindependant manner (152).
It is now apparent that recombinant granzyme A can induce cell death as rapidly as
recombinant granzyme B, but in a caspase-independent way (153). Cell death induced by
granzyme A has many of the classical features of apoptosis, including chromatin condensation,
externalization of phosphatidyl serine on cell membrane, loss of mitochondrial transmembrane
potential and nuclear fragmentation (134,153). Curiously, granzyme A is not active on any of the
downstream caspases. In addition, the loss of mitochondrial membrane potential did not result in
release of cytochrome c and overexpression of Bcl-2 did not have any observable effects on the
granzyme A mediated killing (8).
21
A set of molecules targeted by granzyme A have been identified. The main granzyme A
target is an endoplasmic reticulum-associated complex called SET (154). The principal
components of this complex include two tumor suppressor proteins (pp32 and NM23-H1), the
nucleosome assembly protein SET, the DNA-binding protein high-mobility group protein-2
(HMG-2), and the rate-limiting base excision repair enzyme apurinic/apyrimidinic endonulease 1
(APE-1) (155-157). Although the exact role of the SET complex is still unknown, it has been
hypothesized that it acts to facilitate activation of transcription and translation-related DNA
repair in response to oxidative stress (157). Granzyme A cleaves SET, HMG-2 and APE-1 in a
perforin-dependant manner. The DNA damage in response to granzyme is different than that
induced by granzyme B. Granzyme A induces single stranded nicks while oligonucleosomal
DNA fragments of ~200 bp are generated by granzyme B. The SET complex is believed to be
the key factor in the induction of DNA nicks. NM23-H1 is a granzyme A-induced DNAse
(GADD), while SET is its inhibitor (IGADD) (158). NM23-H1 has been shown to nick DNA
(159). After the granzyme A attack, SET and NM23-H1 translocate to the nucleus. While SET
has been degraded by granzyme A it allows for the DNAse activity of NM23-H1 to nick DNA.
Granzyme A and B target some important nuclear proteins. Both granzymes A and B are
found to translocate to the nucleus soon after entering the cells (160,161). Lamins are important
structural components of the nuclear envelop and they form the targets for both granzyme A and
B (134). Disruption of the nuclear envelop is believed to be a key step in apoptosis. Granzyme A
also targets linker histone H1 (162). Degradation of histone H1 facilitates the opening up of the
chromatin, making it more accessible for granzyme B.
Orphan granzymes: Granzyme K is believed to be very similar in activity to granzyme A
and is thought to be responsible for the residual esterase activity (Z-Lys-SBzl) of cytotoxic cells
22
from granzyme A deficient mice (163). Generation of recombinant granzyme K and resolution of
its structure have been reported (54,58,164). However, there are no documented studies for the
cytotoxic effects of recombinant granzyme K. Purified native granzyme K induces caspaseindependent cell death, without classical apoptotic nuclear morphology. Disruption of
mitochondrial membrane potential was observed in target cells in response to granzyme K (142).
Recombinant granzyme H is reported to have chymase activity, but the cytotoxic effects of this
granzyme has not been reported (165).
Another member of orphan granzyme family is granzyme M, which is of particular
interest due to its preferential expression in cytotoxic cells of innate immunity including NK
cells and gamma delta T cells (166). Prediction for a role of granzyme M in target cell lysis with
sub-optimal level of perforin has not been successful (51), but other roles have been assigned to
granzyme M. One of the hypothetical roles for granzyme M that has been put forward suggests
that it could be responsible for damaging viral particles during the lysis of infected cells (166).
While that mechanism is not well understood, the role for granzyme M as a regulatory protease
has been demonstrated (167). It has been shown that proteinase inhibitor 9 (PI-9) is a major
inhibitor of granzyme B (168), and recent findings suggest that granzyme M is not only resistant
to PI-9, but that it can effectively hydrolyze the inhibitor (167). Over expression of PI-9 has been
reported as one of the evasion strategies used by certain tumors (169). Thus, combined delivery
of granzyme B and M in target cells expressing PI-9 could facilitate the breakdown of the
inhibitor by granzyme M, allowing the active granzyme B to induce target cell death through
multiple pathways.
In addition to the regulatory roles for granzyme M, a novel form of perforin-dependant
cell death mediated by granzyme M has been recently reported (170). The kinetics of
23
cytotoxicity induced by granzyme M was comparable to that induced by similar amounts of
granzyme B, indicating the importance of this novel pathway. In contrast to granzyme A and C,
cell death induced by granzyme M did not involve the mitochondrial pathways. Overexpression
of Bcl-2 had no effect on the killing. Unlike granzyme B, cell death mediated by granzyme M
lacked the DNA fragmentation and occurred independent of caspase activities. These results
suggest that granzyme M-mediated cell death is molecularly distinct from all known pathways
(170). It is logical to extrapolate these findings to other orphan granzymes also, which might be
using still unidentified cell death pathways.
As explained above, cell-mediated cytotoxicity is a tightly regulated process. An
additional major component of cytotoxic granules is calreticulin (171). Calreticulin is known as
an inactivator of perforin-mediated lysis (171,172). One of the orphan granzymes, chymase 1 is
reported to be the “inactivator of inactivator” because of its ability to cleave calreticulin and
release active perforin (173,174).
In vivo significance of granzyme-mediated killing
The role of granzymes in inducing target cell death has been investigated extensively in
vitro and it is better understood than the role of perforin in cytotoxicity at the molecular level
(175). However, advances in in vivo studies demonstrating the significance of individual
granzymes in cytotoxicity lag behind that of perforin. Mice deficient in one or more granzymes
have focal immune deficits unlike the case with perforin knock-out mice, which have a wide
spread inability to kill target cells (175). Since many of the genes encoding granzymes are
structurally related and highly linked, deletion of one granzyme can result in the disruption of the
expression of other granzymes (61).
24
Cytotoxic cells isolated from granzyme A deficient mice retain the potency to induce
apoptosis of target cells in vitro (176). The residual cytotoxicity could be partially attributed to
the intact serine esterase activity of granzyme K that remains in granzyme A-/- mice (163).
Cytotoxic cells from granzyme B deficient mice can still exhibit classical signs of apoptosis in
target cells, but at a slower rate than the wild type (177). As granzyme B cluster deficiency in
mice is characterized by the additional loss of granzyme C and other neighboring orphan
granzymes (61), these findings suggested that the in vivo effects of cytotoxic cells are not solely
dependent on the granzyme B cluster. However, simultaneous disruption of granzymes A and B
functions resulted in increase in susceptibility towards ectromelia virus infection (178).
Recent attempts to understand the individual effects of members of the granzyme B gene
cluster have revealed the significant role played by orphan granzymes present downstream of the
granzyme B gene in mice (179). Both lymphokine activated killer cells and CTLs from mice
deficient in the entire granzyme B cluster had more severe cytotoxic defects than those from
mice deficient for granzyme B only (179). The emerging picture from recent studies points
towards indispensable, simultaneous roles of multiple granzymes rather than in isolation in
controlling certain viral infections (111,178,180). However, this pattern of susceptibility is not
true in all viral infection models, suggesting that granzymes might have some additional
extracellular functions or they might have evolved specifically to restrict replication of specific
viruses (181).
Synthetic Substrates and Inhibitors
Granzymes are broadly classified by their primary enzyme specificity. This specificity is
determined by the recognition of substrate residues immediately N-terminal to the sessile bond
25
(P1 residue). Based on the three dimensional structures of the members of serine proteases, it has
been proposed that three residues of the S1 substrate binding pocket confer the primary
specificity for all proteases. These residues are located at positions 189, 216 and 226
(chymotrypsin numbering) of the proteases (182). Four different enzymatic activities have been
described for all the known granzymes and they are: Tryptase, Met-ase, Asp-ase and Chymase.
Granzymes A and K have Asp at position 189, which permits ionic interaction with Arg
or Lys at position P1. This arrangement indicates that granzymes A and K have tryptase activity
(183). In granzyme B, the arginine residue at position 226 at the back of the primary binding
pocket (S1) interacts with Asp or Glu at the P1 position, facilitating highly specific Aspase
activity (184,185). The metase activity of granzyme M is determined by two residues, Lys-179
and Ser-201. Mutations of these key residues significantly reduced the Metase activity of the
enzyme, but had chymase activity towards Phe at P1 site (186). Although granzyme C has Gln at
position 226, which would predict preference for polar residues like Asn or Ser at P1 position, its
actual enzymatic activity remains unknown. Furthermore, while granzymes D-H have similar
residues around the S1 pocket predicting preference for Phe/Leu residues at P1 site (183), only
granzyme H has been shown to have chymase activity.
Synthetic peptide substrates are commonly used to detect enzyme activity during
isolation and to determine the specificity. The three commonly used synthetic substrates for
serine proteases are peptide thioesters, peptide p-nitroanilides (pNA) and peptide derivatives of
7-amino-4-methylcoumarin (AMC) (183). Compared to pNA and AMC, thioester substrates are
more sensitive and thus more useful in characterizing new and unreactive proteases (187).
Hydrolysis of thioester substrates can be monitored spectrophotometrically in the presence of
5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) to determine enzyme kinetics (188). A library of
26
Boc-Ala-Ala-X-Sbzl (X = Various P1 residues) along with Z-Lys-Sbzl, Z-Arg-Sbzl and Suc-PheLeu-Phe-Sbzl have been used to study the activities of various granzymes (173,189-193).
Synthetic inhibitors are powerful tools for identification of specificity and determination
of in vivo and in vitro functions of proteases. There are five major classes of inhibitors used to
study granzyme functions: simple substrate analogs, transition state analogs, alkylating agents
which react with active site histidine, acylating agents which react with active site serine and
mechanism-based (suicide) inhibitors (183). The first two classes of inhibitors are reversible
while others are irreversible. While general serine protease inhibitors like PMSF and DFP have
been used to identify granzymes as serine proteases (194,195), more specific inhibitors have
been developed to study specificity and function of granzymes. Irreversible inhibitors, such as
substituted isocoumarins, peptide chloromethyl ketones, peptide phosphonates, and
guanidinobenzoate derivatives have been shown to differentially inhibit granzymes A, B, K and
chymase I (173,192,195-197).
Evolution of granzymes
Functional redundancy of granzymes suggests the need for multiple molecular pathways
to compete with viral and tumor strategies to evade cell death. These pathogens have coexisted
and coevolved with immune systems of organism for millions of years (175). For example,
adenoviruses resist killing by granzyme B by producing pseudosubstrates (198). Discovery of
nonredundant mechanisms of action of granzyme A, B and C indicates importance of multiple
granzymes in providing a “fail-safe” mechanism for anti-viral and anti-tumor immunity
(8,199,200). This is exemplified by the ATP-independent cell death induced by granzyme A, that
could be the result of a race between the host and the virus for energy sources (201). When the
27
energy resources for the cell are depleted, the caspase-dependant apoptosis is blocked (202) and
viruses could have used this as an evasion strategy. The immune system appears to have
developed alternative pathways, like the one used by granzyme A to trigger cell death under such
conditions (203).
Evolution of serine proteases is believed to be a result of mutations in the S1 pocket
residues of the gene. However, attempts to interchange the primary specificity between the major
superfamily members through appropriate chimeric replacement of S1 pocket residues were
found to result in complete abolition of activity (204,205). It is believed that the ancestral serine
protease family members were resistant to inactivation due to mutational changes to S1 pocket
residues because of the structural plasticity of the S1 subsite, as evident in invertebrate and
prokaryotic serine proteases (206,207). However, these proteases have broad multivalent primary
specificities, suggesting progressive narrowing of primary specificities for proteases during
vertebrate evolution. Once a narrow specificity is reached, further generation of diversity is
achieved only by reversing to the original state (208).
Mammalian granzymes are separated into clusters on three chromosomes and there is
considerable sequence similarity among the members from the same group (52). There is ample
evidence suggesting a gene duplication of granzyme B leading to the evolution of granzyme C in
mice and granzyme H in human. Similarly, granzymes A, K and M seem to be related to each
other. Early gene duplication of granzyme A could have led to the evolution of granzyme K (52).
These two groups are believed to have evolved before the divergence of human and rodents.
However, a third group consisting of all other orphan granzymes is believed to have evolved in
rodents after such a divergence (52).
28
Role of granulysin in cytotoxicity
Granulysin is a member of the saposin-like protein family that includes amoebapores and
NK lysin (209). Expression of granulysin is found to be restricted to CD4+ and CD8+ T-cells, NK
cells and gamma-delta T cells (210-212). Granulysin was found to be upregulated in cytotoxic T
lymphocytes following activation and was shown to be cytotoxic against microbes and tumor
cells (48,213,214). The three dimensional structure of granulysin has provided new insights into
a possible mode of cytotoxicity for this molecule (215). The model predicts that the positive
charges on granulysin could help the molecule to orient towards negatively charged surface
molecules of target cells. Clusters of granulysin could cause lysis of the membrane by rolling in
the direction of its first, second and third helices, creating a scissoring motion. This would
further expose the lytic surface allowing granulysin to enter deeper into the target cell, bending
or tearing it (216).
CTL cytotoxicity against Cryptococcus neoformans was recently demonstrated to be
dependent on granulysin expression, but not on the expression of perforin (48). In contrast,
perforin was found to aid in the granulysin-mediated lysis of Mycobacterium tuberculosis,
indicating the need for a membrane perturbing agent to facilitate the access of granulysin to an
intracellular organism (48). The effective induction of apoptosis by granulysin of virally infected
cells, like in the case of varicella-zoster infection, has been reported (217). This protection
appears to be virus specific as granulysin had no effect on HIV replication (218). Another
important immune effector mechanism of granulysin is as an effective cytotoxic agent against a
variety of human tumors (219). Recent reports suggest that granulysin can be used as a marker
for the outbreak of certain human cancers. Progression of cancer was found to be associated with
decreased expression of granulysin in NK cells, while there was no correlation between tumor
29
progression and expression of perforin in these cells (220). In animal tumor models, efficacy of
granulysin as a therapeutic agent has been demonstrated. By expressing granulysin using
adenoviral vectors in a mouse lung cancer model, significant induction of apoptosis in tumor
cells was achieved (221).
Regulation of granule-mediated killing
Cytotoxic lymphocytes have developed mechanisms to protect themselves from the lethal
effects of granules they secrete (222). Surface expression of cathepsin B plays a significant role
in protecting the killer cells from accidental cell death. Cathepsin B can cleave perforin,
preventing the pore formation and protecting from membrane damage (223). Inactivation of
cathepsin B using membrane impermeable inhibitors produced perforin-dependant suicide of
CTLs and NK cells upon degranulation triggering (223).
Cytotoxic lymphocytes also express an abundance of endogenous serpins, such as
protease inhibitor-9 (PI-9), which would act as a pseudosubstrate for granzyme B and protect the
killer cells (168,224). Serpins were first discovered as blood plasma proteins regulating
fibrinolysis, blood coagulation, complement activation and tissue modeling (225). Plasma
serpins like alpha-1 protease inhibitor and antithrombin III can inhibit granzyme A and B
(226,227). Expression of PI-9 in immune privileged sites suggests its role in protection of bystander cells from destruction (228). Some types of tumor cells can escape the immune system
by upregulating PI-9 expression (169). Another known granzyme inhibitor that protects target
cells against granzyme B mediated killing is a viral inhibitor called cytokine response modifier
(Crm A). Cowpox Crm A has been shown to inhibit both cysteine and serine proteases very
30
effectively (168,229,230). Other serpins such as SPI-6, mBM2A and raPIT5a, expressed in
pituitary gland are also effective inhibitors of granzymes (231,232).
CYTOTOXIC PATHWAYS MEDIATED BY TNF SUPERFAMILY
Tumor necrosis factor
Tumor necrosis factor and lymphotoxin were first identified as products of lymphocytes
and macrophages that caused lysis of certain tumor cells (233,234). Later, more proteins with
similar structure were identified and all of them are now classified as members of the TNF
superfamily (235). The TNF-related ligands are type II transmembrane glycoproteins (Cterminus on the exterior, single transmembrane domain and short cytoplasmic N-terminus) with
conserved C-terminal domain called TNF homology domain (236). Members of TNF
superfamily have unique structural features that couple them directly to signaling pathways for
cell proliferation, survival and differentiation, enabling them to assume prominent roles in
coordinating proliferation and protective functions of pathogen-reactive immune cells (237).
The TNF homology domains are -sandwich structures containing two stacked
each formed by five anti-parallel
pleated sheets
strands that adopt a “jelly-roll” topology (238,239). The inner
sheets are responsible for the trimer contacts while the outer sheets are exposed to the surface.
The oligomer formation is crucial for TNF functions because the receptor binding sites on TNF
are located at the interfaces of adjacent subunits. The TNF homotrimer can aggregate up to three
receptors (240).
TNF is regarded as a major player in orchestrating differentiation and proliferation as
well as induction of cell death. The role of TNF in various pathological conditions, like cachexia,
septic shock, rheumatoid arthritis and autoimmunity has been well established (241).
31
TNF and cytotoxic cells
Antigen-specific CTLs have been shown to express members of the TNF ligand
superfamily on their cell surface and/or as a secreted form (35,242-248). The role of these
ligands in initiating cell death pathways in target cells were demonstrated with the use of specific
blocking reagents, which disrupt the interaction between the ligand and corresponding receptor
(245,247,249-251). Among various TNF superfamily members, Fas ligand is the one which is
most intensively studied for its role in induction of cytotoxicity. All activated immune cells are
found to express TNF alpha on their cell surface and secrete it as a soluble form
(242,243,245,252,253). Expression of TNF in T cells is rapidly inducible by of the T-cellactivating stimuli, but the expression is transient, lasting a few hours (242). Freshly isolated
(non-activated) and activated NK cells express a variety of TNF superfamily ligands, both at the
level of mRNA and protein (254). NK cells have been shown to express TNF both on the cell
surface and in the cytoplasm. Activation of these cells resulted in an increase in the expression of
TNF-mRNAs, but not membrane protein. (254). However, activated NK cells showed significant
increase in the secretion of soluble TNF (255).
TNF-induced target cell death
The two physiological forms of TNF (26 kDa membrane-bound form and 17 kDa
secreted form) are effective in killing tumor cells and virally infected target cells upon cell-tocell contact. An uncleavable mutant version of TNF was found to be very effective in inducing
cytotoxicity, suggesting that internalization of TNF is not a requirement for the cell death (256).
The secreted form of TNF is generated by proteolytic cleavage of the membrane-bound form by
a metalloprotease called TNF converting enzyme (TACE), which is expressed on the cell surface
32
(257). Both membrane-bound and secreted forms of TNF have been shown to be the major
cytotoxic effector mechanisms used by macrophages and CTL (243,258,259). The action of
membrane-bound TNF is considered to be more localized, requiring the cell-to-cell contact
between the effector and target cells, compared to the more systemic effects of the secreted form
of TNF (241).
The contribution of TNF towards the killing mediated by lymphocytes was not fully
appreciated before due to the dominant effects of perforin and Fas ligand in acute cytotoxicity
(260). After the derivation of null mice for perforin (110), TNF (261) and the description of FasL
mutant gld mice (262,263), the significance of TNF alpha in cell-mediated cytotoxicity was
better understood. All of the cell-mediated cytotoxicity can be described as the end result of one
or a combination of three mechanisms: pathways involving perforin/granzyme pathway,
Fas/FasL or TNF/other similar ligands (264). LAK cells generated from perforin and FasL
deficient mice were not cytotoxic in short term cytotoxicity assays, indicating the role of those
two molecules in acute cytotoxicity. However, upon prolonged incubation with TNF-sensitive
targets, these cells were effective in inducing cell death, which was blocked by addition of antiTNF antibodies. Both membrane-bound and soluble forms of TNF were effective in killing
(253).
T-cell receptor activated cloned CD8+ CTLs have been shown to express both the
membrane-bound and the secreted forms of TNF in high quantities. TNF was found to be
biologically active and the expression levels were highly upregulated upon activation of the cell,
indicating in vivo significance of TNF-alpha in a slower CTL-mediated cell death pathway (259).
Similarly, an ovalbumin peptide-specific CTL clone was found to use two distinct mechanisms
of cytotoxicity. One of them was obvious within four hours while the other took two days and
33
was neutralized by anti-TNF antibodies (265). Perforin-independent killing of certain targets by
CTLs was shown to be entirely due to TNF (266). These studies have demonstrated that the
lymphocytes choose from the three pathways to suit a particular situation according to the
susceptibility of the target and even effector to target ratios (267).
Antigen-specific target lysis by CTL also results in some degree of bystander cell death,
which is independent of class I molecules. Most of the bystander target lysis is attributed to the
Fas-FasL interactions between the activated effector and non-antigen presenting targets
(268,269). There was a significant reduction in the extent of bystander cell death by CTLs from
gld mice (270). However, after prolonged incubation (for 36-48 hours), delayed bystander lysis
was observed, which was shown to be mediated though the soluble form of TNF-alpha. There is
no evidence for the induction of bystander cell death by soluble FasL. These results indicate that
CTLs might use FasL or TNF in a kinetically and physically distinct fashion to mediate
bystander cell death (270). Role of TNF as a bystander in the clearance of certain kinds of
tumors also has been reported (271,272).
The importance of Fas-FasL interaction in mature T-cell death has long been established
(273). Many studies have suggested a Fas-independent mechanism, which also might play an
important role in activation-induced cell death (AICD) pathways (274,275). Studies using lpr
mice demonstrated that activation-induced cell death in response to a strong stimulus was
comparable to that of normal T cells (276). Treatment of T cells with anti-TNF antibody or
injection of antisense TNF oligodeoxynucleotides was shown to have an inhibitory effect on
AICD in those cells (277). Later, TNFR2 was found to be responsible for Fas-independent
activation-induced cell death of CD8+ CTLs (278). In addition to TNFR2, TNFR1 also is
believed to be involved in AICD (at least in a CD8+ population of transgenic mice) where Fas-
34
independent pathways were found to be important (279). AICD in CD4+ T cells was believed to
be entirely due to Fas-FasL interaction (280). However, TNF also was shown to play an
additional role in this pathway (281). A combination of lpr phenotype and deficiency of TNFR1
had an accelerated lymphoadenopathy and increased serum immunoglobulin levels (282). These
studies suggest that TNF-TNFR interactions can compensate for the loss of Fas-FasL signaling
pathway in inducing AICD in T cells.
Non-lytic functions of TNF
In addition to a role in cytotoxicity, TNF-TNFR interactions can result in cell activation
and proliferation. Induction of these biological responses appears to depend on TRAF-mediated
triggering of kinase-dependant signaling pathways, leading to activation of transcription factors
like NF-κB and AP-1 (264). Activation of these transcription factors can induce the expression
of genes like inhibitor of apoptosis proteins (IAPs), which can inhibit cell death induced by TNF
(283). It has been demonstrated that cIAP-1 and cIAP-2 can inhibit TNF-mediated activation of
downstream caspases (284,285).
Cells that produce TNF are generally responsive to TNF, suggesting autocrine feedback
loops. However, the role of the membrane bound form of TNF in such feedback loops is not
fully understood. Cells that permanently express membrane-integrated mutant version of TNF
are found to express TNF-TNFR complexes on their surfaces (286). These cells were shown to
have permanent activation of NF-κB, constitutive p38 mitogen-activated protein kinase activity
and increased expression of IL-6. These results suggest that cells that express both TNF and
TNFR on their surface can initiate autocrine signaling loops that might be important for their
optimal activation and survival. The apoptosis-inducting capacity of soluble forms of TNF-
35
superfamily ligands has been shown to be reduced significantly, compared to the membranebound form (287-290). Recombinant soluble TNF-α and lymphotoxin are capable of inducing
apoptotic cell death in mouse as well as human T lymphocyte blasts in vitro (291). Another study
showed that membrane-bound FasL was cytotoxic for human peripheral blood lymphocytes,
while the soluble form blocked the killing (292).
TNF superfamily ligands can enhance T cell growth though coactivation signaling along
with TCR signaling or other activation stimuli (293). Proliferation of gamma-delta T cells was
also reported in response to TNF (294). The proliferative response triggered by a mixture of antiCD2 monoclonal antibodies or LFA-3 was found to be enhanced by the addition of TNF
(295,296). Combination of TNF and IL-6 has been shown to have highly effective synergestic
signals for T cell proliferation (297).
TNF-induced signaling pathways leading to cytotoxicity or survival
Ligation of TNF ligands induces oligomerization of the receptors on the target cell
surface resulting in the aggregation of cytoplasmic domains. Formation of this complex is the
first step in a series of signaling events inside the target cell, leading towards cell death (298).
Based on binding of a variety of adapter molecules to the cytoplasmic tail of TNFR, a conserved
domain called death domain (DD) was identified, which is necessary to initiate apoptosis in
target cells. Another conserved domain identified is the one which binds to TNFR-associated
factors (TRAFs). Binding of TRAFs to the multimerized TNFRs activates signaling pathways
leading to cell death. A series of events have been characterized, which eventually lead to the
cleavage and activation of caspases and to apoptosis. Alternatively, activation of TNFR can
36
result in kinase-dependant signal transduction and the activation of transcription factors that are
crucial in regulation of cell activation and survival genes (298,299).
The first protein to be recruited to the multimerized TNFR1 is TNFR1-associated death
domain protein (TRADD), which serves as a platform for the recruitment of multiple proteins
like receptor-interacting protein (RIP), RIP-associated ICH-1 homologous protein with a death
domain (RAIDD), Fas-associated death domain protein (FADD) and TNF receptor-associated
factor-1 (TRAF2) (300-305). TRAF2 in turn can recruit TRAF1 to the complex (301,302). RIP is
necessary for the activation of NF-κB while FADD is important in induction of apoptosis.
Activation of cytoplasmic proteins by RIP will result in phosphorylation of IκB allowing
nuclear localization and activation of NF-κB (306). RAIDD has an N-terminal caspase
recruitment domain (CARD), which mediates interaction with similar domains on caspase 2
(305). Using C-terminal death domain, RAIDD can interact with RIP and TRADD mediating the
recruitment of caspase towards the oligomerized TNFR. TNFR1 needs TRADD as an
intermediary between FADD and the receptor. FADD has a death effector domain (DED), which
can interact with similar domains found at the N-terminus of caspase 8 (307). The multi-protein
complex consisting of TNFRs, TRADD, FADD and caspase 8 is known as death inducing
signaling complex (DISC) and it can induce the cleavage and activation of caspase 8 leading to
the activation of downstream capases or the Bcl-2 family member BID (308-310). Ligation of
TNF to its receptors can also lead to generation of reactive oxygen species in target cells, which
can lead to apoptosis or necrosis (311).
TNFR2 lacks a cytoplasmic death domain and has been shown to induce pathways of cell
activation and survival. However, TNFR2 mediated cytotoxicity of target cells has been
demonstrated (312). It has been proposed that the ligation of TNFR2 leads to the activation of
37
NF-κB and AP-1, which in turn will induce the transcriptional upregulation of membrane-bound
TNF and FasL leading to apoptosis in an autotropic or paratropic manner (312).
TRAF2 has been shown to signal for activation of JNK leading to phosphorylation of
AP1 (313). Among the target genes upregulated by the TNF pathway, IAP proteins are found to
play a major role in inhibiting downstream caspases (284).
REGULATION OF CELL DEATH IN CYTOTOXIC CELLS
Apoptotic cell death pathways play a critical role in shaping the repertoire of mature
lymphocytes, which is crucial for the proper functioning of the immune system. Lymphocytes
with developmental defects are removed early in lymphocyte development. During the peripheral
immune response, activated lymphocytes have to be deleted after antigen clearance to assure that
the highly active cytotoxic cells do not pose a threat to the host. Lymphocytes that suffer
irreparable DNA damage, or those which are neglected by removal from necessary survival
factors, undergo programmed cell death. The combined effects of all these events lead to the
establishment of homeostasis (314).
Peripheral T cells undergo apoptosis following high-intensity TCR signaling or absence
of growth factors like IL-2 (315,316). Activated T cells downregulate Fas inhibitor FLIP and
upregulate FasL to become more sensitive to cell death though Fas-FasL interactions (317,318).
The activation-induced cell death (AICD) in T cells is induced by FasL or TNF, either selfinflicted or killed by adjacent cells (319,320). T cells transiently express both TNF and FasL
during activation which can engage the corresponding receptors on themselves or on neighboring
cells. Cell death due to Fas-FasL interaction is shown to be much faster than that is induced by
TNF-TNFR2 (278). The reactive oxygen species pathway also has been implicated in T cell
38
AICD (321). Activation-induced NK cell death triggered by CD2, CD16 or CD94 was shown to
be extremely rapid compared to AICD in T cells (322,323). Moreover, the AICD in NK cells
were not inhibited by addition of antibodies against FasL or TNF. Granzyme B, which is leaked
out of NK cells during activation, was found to induce their death by cleaving BID, resulting in
mitochondrial tethering and apoptosis (324). NK cells express the protease inhibitor-9, which
make complexes with granzymes. However, the ratio between granzyme and the inhibitor
determines the outcome (324).
It has been reported previously that NK cells recycle 3-4 times during an in vitro
cytotoxicity assay enabling each cell to kill multiple targets (325). It can be hypothesized that
various pathways act together to counteract the signals for AICD in these cells upon the
activation. Several costimulatory signals have been demonstrated to save the T cells from AICD.
Interaction of CD28 with CD80 (B7.1) or CD86 (B7.2) has been shown to protect T cells (326).
Stimulation though CD28 was shown to enhance cytokine production, upregulate Bcl-xL and
FLIP and downregulate expression of FasL (326-329).
Inhibitor of apoptosis proteins (IAP) including hILP/XIAP, cIAP-1, cIAP-2, NAIP,
survivin and Bruce are characterized by the presence of a 70 amino acid BIR domain, which
shares similarity with similar domains in IAP proteins found in baculovirus (283,330). The role
of hILP in regulation of T cell apoptosis has been demonstrated (331). hILP, cIAP-1 and cIAP-2
can bind and inactivate the active forms of caspase 3 and 7, but not the pro-enzymes (332-334).
In addition, these proteins can bind, not only to the active caspase 9, but also to the procaspase 9,
preventing its processing and activation (332). In response to strong apoptosis stimuli, cellular
hILP can be cleaved by caspases leading to neutralization of its inhibitory effects on other
caspases and apoptotic death (331). The cleavage products of hILP remain associated with
39
caspases and can be detected in peripheral blood T lymphocytes isolated from healthy
individuals, which are stimulated to undergo AICD (335).
It has been reported that IAPs could be regulated during exposure to TNF alpha
(336,337). IAPs regulate TNF-mediated cytochrome c release and loss of mitochondrial
membrane potential by inhibiting the signaling pathways upstream of mitochondrial events and
apical caspase 8 (338). Members of the IAP family contain a C-terminal zinc-binding RING
finger motif that is commonly found in ubiquitin ligases (339). RING finger containing IAP
proteins were found to use their ubiquitinating properties to subject caspases to proteosomal
degradation (340,341). Several caspase-independent survival mechanisms of IAP proteins have
been documented in different tissues (342).
Cellular apoptosis susceptibility protein (CAS) was identified as a homolog of yeast
chromosome segregation protein (CSE1) (343-345), and it has been shown to be involved in both
apoptosis and cell proliferation (346). CAS functions as a nuclear transport factor and mediates
nuclear-to-cytosolic recycling of importin-alpha and probably other factors, which are important
in cell proliferation and apoptosis (347). CAS expression is upregulated in proliferating cells
while expression levels are low in quiescent cells and tissues (348,349). CAS contains a putative
recognition site for MEK-1 phosphorylation at its N-terminus, indicating that its functions are
regulated by phosphorylation (343,348). MEK plays an important role in proliferation as well as
in TNF mediated apoptosis (350).
Recent studies have demonstrated the regulation of apoptosis by proteins believed to be
involved in totally different cellular functions. For example, the alpha-chain of the nascent
polypeptide-associated complex has been found to bind and regulate the functions of FADD
40
(351). Inhibition of alpha NAC using anti-sense oligodeoxynucleotide resulted in an increased
cell proliferation of CD8+ CTLs (352).
In teleosts, several genes with homology to mammalian apoptosis regulators have been
identified by analyzing the expressed sequence tags (EST) from zebrafish (353). Important
among those are members of the Bcl-2 family, caspases, IAPs, DD containing receptors and
death ligands (353). However, the biological significance of these findings can be known only
after correlating the expression pattern to various tissues and cell types. Using cross-reacting
antibodies, expression of apoptosis regulatory proteins have been identified in activated NCC,
which might play an important role in protecting these cells from AICD (354,355). It has
previously been demonstrated, that NCC lack the capacity to recycle (356). Thus, the role played
by apoptosis regulatory proteins can be expected to be important for NCC functions (355).
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70
CHAPTER 3
EVIDENCE FOR THE EXISTENCE OF GRANZYME-LIKE SERINE PROTEASES IN
TELEOST CYTOTOXIC CELLS1
1
Praveen, K., D. L. Evans and L. Jaso-Friedmann. 2004. Journal of Molecular Evolution.
58:449-459. © Springer-Verlag New York, LLC 2004. Reprinted here with permission of
publisher. The original publication is available at
http://www.springerlink.com/app/home/contribution.asp?wasp=n2gwppwryr3bf3wn9h2m&refer
rer=parent&backto=issue,8,11;journal,9,109;linkingpublicationresults,1:100107,1
71
ABSTRACT
Granzymes are granule associated serine proteases, which are important effector
molecules in NK cell and CTL functions. The granzyme family poses a perplexing problem in
phylogenetics due to the lack of non-mammalian sequence information. We now report the
identification of a cDNA that codes for a granzyme homologue, channel catfish granzyme-1
(CFGR-1) from nonspecific cytotoxic cells (NCC) of a teleost. NCC are the first identified and
extensively studied cytotoxic cell population in teleosts. Ictalurus punctatus (channel catfish)
granzyme cDNA encodes a protein with ~50% similarity to granzymes A and K. Highly
conserved catalytic triad residues of serine proteases and other motifs common to granzymes
were also identified. Conserved amino acid sequences, structure-function data available for
serine protease family and the crystal structure of human granzyme K supported a model of
CFGR-1. It suggested an Arg/Lys primary substrate specificity that is shared with granzyme A
and K. Furthermore, CFGR-1 has the four conserved disulfide bonds of granzyme A, K, and M.
Phylogenetic analysis suggested that this molecule is a member of the granzyme family.
Expression of CFGR-1 in NCC was confirmed by RT-PCR analysis. Presence of a granzymelike molecule that might play an important role in the effector functions of NCC indicates that
cell-mediated immunity with granule exocytosis and Fas pathways have been conserved for more
than 300 millions of years.
Key words: NK cells, Cytotoxic T cells, Granzymes, Cytotoxicity, Granule exocytosis,
Comparative immunology/ Evolution.
72
INTRODUCTION
Mammalian cytotoxic T cells (CTL) and natural killer (NK) cells play an important role
in immunosurveillance against virus-infected and tumor cells. Although recognition of target
cells differs greatly, the mechanisms by which CTL and NK cells cause target cell death
following formation of conjugates appear to be the same. Two main cytotoxic pathways have
been described that are independent from each other (Waterhouse and Trapani 2002). One
pathway involves the crosslinking of death receptors on the target cell (members of the TNF
receptor family such as Fas) by death ligands present on effector cells (FasL, TRAIL). This
interaction leads to apoptotic target cell death (Trenn et al. 1987; Kagi et al. 1994). The other
pathway requires granule exocytosis with the resulting secretion of two abundant granular
proteins, perforin and granzymes, which appear to act in concert to induce target cell death
(Henkart 1985; Podack 1985; Young and Cohn 1986; Peters et al. 1990; Podack and Kupfer
1991; Doherty 1993). Both pathways act synergistically on many target cells, but the exocytosis
pathway alone is sufficient to kill target cells, which do not express death receptors.
The mechanisms of action of perforin and granzymes have been the subject of intense
studies (Henkart 1985; Podack 1985; Young and Cohn 1986; Peters et al. 1990; Podack and
Kupfer 1991; Doherty 1993). Perforin is a pore-forming molecule that in addition to inducing the
lysis of cells by osmotic leakage acts synergistically with granzymes to facilitate their entry into
target cells. Granzymes are members of a granule-associated serine proteases family expressed in
CTL and NK cells that induce apoptosis of target cells either through caspase activation or
through caspase-independent pathways. Recent evidence suggests that even in the absence of
perforin, granzymes can enter target cells by receptor-mediated endocytosis via the cationindependent mannose 6-phosphate receptor. However, internalization of granzyme in the absence
73
of perforin is without toxic consequences (Froelich et al. 1996; Jans et al. 1998; Browne et al.
1999; Motyka et al. 2000). Four orthologous members of the granzyme family with different
substrate specificities have been identified in humans and rodents (A, B, K, M) and additional
non-orthologous genes exist in the human (H), rat (J) and mouse (C, D, E, F, G, N) genome
(Trapani 2001; Barry and Bleackley 2002). The granzyme loci have been placed into three
groups by their chromosomal location and structural similarities. They have four primary
specificities including "aspase", "chymase", "tryptase" and "metase" referring to the type of
activities of the proteases encoded for in their genes. Granzymes A and B have different
substrate specificities (tryptase and aspase, respectively) but they are both pro-apoptotic.
Although the function of Granzyme M, a metase, has not been determined, it is believed to play a
role in NK cell function as it is only expressed in those cytotoxic cells. The substrate specificity
of other family members remains unknown.
The diversity of enzymatic activities of the granzyme family members has prompted
speculation that these proteases may play a role in other immune functions such as lymphocyte
migration and extravasation as well as amplification of cytokine function (Simon et al. 1991;
Sayers et al. 1992; Vettel et al. 1993; Smyth et al. 2001). It is apparent that in spite of their
biological importance, many questions remain unanswered about granzymes. At present, only
murine, rat and human sequence information for granzymes is available and the identification of
evolutionary precursors would provide an important step in the elucidation of their biological
activities. This is the first report of the characterization of a non-mammalian granzyme
sequenced from lower vertebrates.
Nonspecific cytotoxic cells (NCC) are the teleost equivalent to mammalian NK cells.
Since their initial description in the channel catfish (Graves et al. 1985; Evans et al. 1988; Jaso-
74
Friedmann et al. 1990; Evans et al. 1998; Evans and Jaso-Friedmann 1999), these cells have
been well characterized in a number of lower vertebrates (Faisal et al. 1989; Greenlee et al. 1991;
McKinney and Schmale 1994; Suzumura et al. 1994; Jaso-Friedmann and Evans 1999; JasoFriedmann et al. 2002). Although originally described as agranular lymphocytes (Graves et al.
1985), NCC from catfish, tilapia and trout have subsequently been shown to kill target cells by
granule exocytosis and use both necrotic and apoptotic pathways (Faisal et al. 1989; JasoFriedmann et al. 1990; Greenlee et al. 1991). We have previously reported the importance of the
Fas ligand-mediated killing mechanism of mammalian target cells by NCC (Bishop et al. 2000;
Jaso-Friedmann et al. 2000). While others have also reported that catfish cytotoxic T cell lines
kill target cells by the granzyme-perforin pathway, the lack of molecular tools did not allow the
authors to identify these proteins (Zhou et al. 2001). In the present study we have identified a
granzyme from a cDNA library constructed from cytotoxic cells of the lower vertebrate Ictalurus
punctatus. Sequence analysis showed that this novel teleost protein shares highest similarity
with the human and murine granzymes K and A. Primers designed from the sequenced enzyme
in catfish were used to identify another granzyme homologue from NCC of tilapia (Oreocromus
niloticus). Based on the phylogenetic sequence analysis we conclude that granzyme-like
homologs already exist in teleost fishes. Characterization of granzyme precursors from lower
vertebrates in turn will advance our understanding of the multiple roles of granzymes in the
mammalian immune system.
75
MATERIALS AND METHODS
Experimental animals and isolation of NCC.
Outbred channel catfish (Ictalurus punctatus) of both sexes weighing 10-25 g were
obtained from local commercial farms and outbred tilapia (Oreochromis niloticus) of both sexes
weighing 60-100 g were obtained from Americulture, Inc. Animas, NM. Fish were maintained in
fiberglass aquaria equipped with a constant flow through system at ambient water temperatures
for channel catfish and 23-25o C for tilapia. Tilapia and catfish were fed a commercial diet of
pelleted fish food (Southern States Co-operative Inc, VA). All animals were acclimatized for a
minimum of 3 months prior to experimentation and were free from any active infections. NCC
were purified from anterior kidney (bone marrow equivalent in fish) of channel catfish and
peripheral blood of tilapia as described before (Jaso-Friedmann et al. 1990; Bishop et al. 2000).
Purity of cell preparation was verified by flow cytometric analysis using 5C6 (a monoclonal
antibody detecting NCCRP-1, which is an activation marker found exclusively on NCC, Evans et
al. 1998, Jaso-Friedmann et al. 2002).
Construction of cDNA library
Total RNA was isolated from purified NCC with TRIzol® (Invitrogen) and mRNA was
purified by the PolyATtract® mRNA isolation system (Promega). The purified mRNA was used
to construct an expression cDNA library using ZAP-cDNA® Gigapack® III Gold Cloning kit
(Stratagene) according to manufacture's instructions. The library was amplified and recombinant
phages were converted into pBluescript plamids by in vivo excision according to manufacture's
instructions. This was used as template for the PCR reactions in the initial screening for
granzyme cDNA.
76
Screening for granzyme cDNA
The strategy used in sequencing granzyme mRNA of channel catfish is illustrated in
Figure 1. Multiple sequence alignments were done using Clustal W provided with vector NTI
package, version 6 (InforMax Inc) to identify conserved domains in the known mammalian
granzymes. Primers were designed (Table 1) and polymerase chain reactions were done to
amplify specific regions of the cDNA. YT-INDY, a human natural killer-like leukemic cell line
(Montel et al. 1995) cDNA was used as a positive control. Amplicons were TA cloned in to
pDrive cloning vector using a PCR cloning kit (Qiagen). Inserts were sequenced in two
directions and compared with the known sequences in DDBJ/EMBL/GenBank databases using
BLAST version 2.2.5 (Altschul et al. 1997).
Rapid amplification of cDNA ends
Sequence from one of the amplicon which had closest similarity to the known granzyme
sequences was used to design primers to amplify the entire 5' and 3' ends of the catfish granzyme
cDNA. Fresh RNA was purified from catfish anterior kidney NCC for the RACE. For 5' RACE,
purified mRNA (500 ng) was reverse transcribed using granzyme specific primers using
Generacer Superscript II RT module (Invitrogen). First strand cDNA was purified using PCR
purification kit (Qiagen) followed by homopolymeric tailing with Cytosine using terminal
transferace (Roche). Tailed cDNA was purified using PCR purification kit (Qiagen). Later dCtailed cDNA was amplified using abridged anchor primer and gene specific nested primer
followed by a re-amplification using a nested abridged universal amplification primer (AUAP)
and another nested gene-specific primer. The amplicons were purified by gel extraction kit
(Qiagen) and TA cloned for sequencing. For 3' RACE, catfish anterior kidney RNA was reverse
77
transcribed using an anchor primer, EPB-18T (Evans et al. 1998) to generate first strand cDNA.
Using gene specific nested primers and EPB the 3' end of the mRNA was amplified and TA
cloned for sequencing. The sequences were edited and assembled to obtain the complete
sequence of catfish granzyme. Later forward and reverse primers were designed at the two ends
of the open reading frame to amplify the coding region, which was TA cloned to verify the
sequences.
Phylogenetic analysis
Similar analyses as those previously done with NCCRP-1 of zebrafish were performed
(Jaso-Friedmann et al. 2002). Briefly, multiple sequence alignments were done using Clustal W,
using only the mature portion of various proteases. Phylogenetic analysis was done using Mega
version 2.1 (Kumar et al. 2001). The aligned data set was analyzed by the criteria of maximum
parsimony using the branch-and-bound algorithm. The reliability of the trees was tested using
1000 bootstrap replicates. The alignment was also analyzed by the neighbor joining method as
implemented by Mega with 1000 bootstrap replications. For neighbor joining method, Poisson
correction was used with the complete deletion option. The tree was rooted on a sub tree
consisting of trypsin and chymotrypsin sequences to resolve the differences between highly
similar granzyme sequences.
Protein modeling
The three dimensional structure of catfish granzyme was modeled using SWISS-MODEL
in the first approach mode accessible via the internet (http://www.expasy.org/swissmod). The
known crystal structures of serine proteases whose crystal structure has been resolved and had
78
high sequence identity upon pairwise alignments (protein databank entries 1MZA (Hink-Schauer
et al. 2002): human granzyme K, 1DST (Jing et al. 1998): complement factor D) were used as
template for the modeling. The co-ordinate files were imported to RasWin software version 2.6
for analyzing bond lengths and other conformational features of the molecule.
RT-PCR
Total RNA was extracted from channel catfish NCC (from anterior kidney, spleen and
peripheral blood), unfractionated head kidney, spleen, blood, gill, heart, muscle, and trunk
kidney using TRIizol® (Life Technologies). The purified RNA (3 µg) was reverse transcribed in
to cDNA using Generacer Superscript II RT module (Invitrogen). The final volume of cDNA
synthesis reaction was 50 µl and 2 µl of the same was used in a 50 µl PCR reaction. Beta-actin
was used as a normalization control for RT-PCR. PCR was done with initial denaturation at 94o
C for 2 minutes followed by 30 cycles as follows: 30 s denaturation at 94o C, 30 s annealing at
57o C, and 30 s extension at 72o C. The products were resolved on a 1.5% agarose gel and
visualized by ethidium bromide staining.
RESULTS
Nucleotide and deduced amino acid sequence of catfish granzyme
An expression cDNA library generated from catfish anterior kidney NCC was used as
template with different primers designed based on conserved (functional) domains in human and
murine granzymes. YT-INDY cDNA was used in the PCR reactions as a positive control. The
amplicons were analyzed on 1.5% agarose gel. Among the different products, one with identical
size to a product generated by the same primer set from YT cDNA was selected for sequencing.
79
The sequence was compared and verified for the similarity to known granzymes. Based on the
obtained product, additional non - degenerate primers were synthesized to obtain the sequence
for the complete transcript by 5'- and 3'-RACE. The transcript of the catfish granzyme cDNA
was 978 base pairs (bp) in length, with a single open reading frame yielding 768 bp (Fig. 2). One
putative start codon was identified near the 5' end of the full-length cDNA determined by 5'
RACE. The catfish granzyme cDNA encodes a putative mature protein with 231 amino acids.
The predicated molecular mass of the unglycosylated protein was calculated as 25,526 daltons,
with an isoelectric point of 9.56, suggesting highly basic nature of the enzyme.
Initial analysis of the translated cDNA sequence by comparison to other known
granzymes in the database suggested that the product was a member of the granzyme family of
serine proteases (Figure 2). The catfish sequence was subsequently submitted to GenBank
(Accession number: AY286012). The three key amino acid residues representing the catalytic
triad (charge relay system) of serine proteases as well as their neighboring residues are well
conserved in catfish granzyme (Figure 2, boxes). The three residues, His at position 57, Asp at
102 and Ser at 195 (chymotrypsinogen numbering system) are in similar locations as those of
other serine proteases. A summary of these results showed that the mature granzyme sequence
from catfish had 29-43% identity and 39-52% similarity to other granzymes (Table 2).
Primary sequence analysis for identification of signature motifs of known granzymes
A signal sequence of 20 amino acids was identified at the N-terminal end indicating that
this enzyme is sorted to the secretory pathway (Figure 3). The mature protein begins at +1 with a
highly conserved Ile-Ile-Gly-Gly motif found in the majority of the granzymes. The most likely
cleavage site for the signal sequence was predicted between residues at -5 (Cys) and -4 (Ser) as
80
determined by weight matrix analysis of von Heijne (von Heijne 1986). There is a single putative
glycosylation site at the N-terminus of the mature protein (Asn20-Asn-Ser).
Prediction of tertiary structure and substrate specificity
The three dimensional structure of catfish granzyme was modeled using SWIS-MODEL.
The templates used for modeling were based on known crystal structures of serine proteases.
Disulfide linkages are crucial in correct folding of serine proteases and their catalytic ability. The
mature catfish granzyme has an unusually high number of Cys residues (11 in total). Out of
these, 6 are highly conserved in all serine proteases (positions 42, 58, 136, 168, 182 and 201:
chymotrypsinogen numbering). By analogy and based on results from other granzymes, the
formation of disulfide bonds can be predicted as follows: 42 with 58; 136 with 168; 182 with
201). Cys191 and Cys220 (chymotrypsinogen numbering) could be analogous to a fourth
disulfide linkage which bridges active site serine, like in chymotrypsin. Additional comparisons
with other granzymes yielded more similarities like the formation of an internal salt bridge
between the characteristic N-terminal motif (IIGG) and Aspchymotrypsinogen # 194 . The overall
structure of the predicted model for catfish granzyme was similar to other granzymes, especially
granzyme K (data not shown). The model was also instrumental to predict the active site
conformation of catfish granzyme. Based on the structure of trypsin (Rypniewski et al. 1994), the
residues which determine the configuration of active site were identified in the model (Gly43,
Gly44, Gly140, Trp141, Gly142 and Leu155; chymotrypsinogen numbering). Those residues are
in close proximity of the catalytic triad suggesting a highly conserved trypsin like active site
structure. Four different enzymatic activities (tryptase, chymase, aspase and metase) have been
assigned to the granzymes identified so far. The combination of amino acid residues that are
81
found to determine the primary substrate specificity of granzymes (-6, +16, +17 and +18 relative
to active site Serine) were analyzed in catfish granzyme by examining the primary sequence and
predicted model. The most critical residues determining the S1 subsite are found at positions
189, 216 and 226 (chymotrypsin numbering). Comparing these residues to that e.g. in granzyme
A, B, cathepsin G and leukocyte elastase clearly indicates a Lys/Arg specificity (like granzyme
A and K) for CFGR-1 (Jackson et al. 1998).
Phylogenetic analysis
Initial multiple alignments and visual examination of the catfish granzyme exposed many
conserved domains common to the mammalian granzymes. The aligned data set was analyzed
by different methods to obtain reliable phylogenetic data. Similar results were obtained by the
methods used and a representative tree is shown in Figure 4. The similarity to multiple
granzymes at different regions of the molecule posed problems for the grouping with the known
enzymes. In order to resolve the differences between highly similar granzyme sequences, the tree
was rooted on a sub-tree consisting of trypsin and chymotrypsin sequences. Catfish granzyme
did not cluster with any of the granzyme families, but branched out as a separate group,
indicating that the three major granzyme branches (granzyme AK, granzyme BCDEFGH,
granzyme M/elastase-2/proteinase 3/factor D) started to diverge just before or after the
emergence of teleost fishes.
Expression pattern of the transcript for the catfish granzyme
The presence of granzyme transcripts were analyzed in different tissue. RT-PCR was
performed with primers designed based on the least conserved sequences between the teleost
82
granzymes and other known granzymes in order to selectively obtain the predicted amplicon.
Figure 5A shows a representative experiment suggesting that tissue with highest concentration of
NCC (head kidney, spleen and liver) had a higher level of granzyme mRNA than whole blood,
muscle and heart tissue. However, there was significant increase in the expression levels of
granzymes when the NCC were enriched from the whole blood (Figure 5B), indicating that NCC
are the main source of CFGR-1 in catfish.
Expression of granzymes in other fish species
An expression cDNA library was constructed from tilapia NCC purified from peripheral
blood. An initial primer pair, which amplified a 330 bp amplicon from catfish library, was used
to screen the tilapia library by PCR. An amplicon of similar size as obtained from catfish and
YT-INDY cDNA was sequenced. The sequence obtained was compared with known sequences
in DDBJ/EMBL/GenBank databases using BLAST version 2.2.5. The PCR product had high
similarity to known granzyme sequences. The tilapia granzyme PCR product was translated and
aligned with catfish granzyme using Clustal W (Figure 6, panel A). There are considerable
differences between the corresponding regions of the two molecules (36% identity and 46%
similarity between the corresponding regions), but at the same time tilapia sequence retains the
signature motifs common to all granzymes. This is suggestive evidence for the presence of
different members of the granzyme family in other fish species. In order to identify potential
representatives of granzyme family in other fish species, the catfish cDNA was compared with
sequences in the EST database of NCBI. A Salmo salar EST (Accession number CB516537)
was found to be similar to the catfish granzyme sequence. The EST sequence was edited and
translated to obtain the predicted protein sequence. Although this EST is lacking a few residues
83
at both the ends and also the two untranslated regions, considerable similarity (53% identity and
63% similarity) between the catfish granzyme molecule and the translation of the EST was found
(Figure 6, panel B). Additional searches showed that NCBI database has listed a partial sequence
of a granzyme-like molecule from Salmo salar (accession number AF434669), similar to human
granzyme M. The N-terminal portion of the mature protein from that sequence was compared
with catfish granzyme. The N-terminal 43 amino acid domain of that protein has 56% identity
and 65% similarity to the corresponding region of catfish granzyme (Figure 6, panel C).
DISCUSSION
Although several laboratories have functionally shown the existence of the
granzyme/perforin pathway in the cytotoxic activity of teleost CTL and NK-like cells (Greenlee
et al. 1991; Evans and Jaso-Friedmann 1999; Zhou et al. 2001), this is the first report of the
identification of a non-mammalian granzyme in catfish and tilapia NCC. The primary sequence
characteristics that place this protein in the granzyme family include the presence of signature
motifs shown in Figures 2 and 3: the catalytic triad amino acids each of them with surrounding
conserved residues; the presence of a propeptide that must be removed to convert the inactive
zymogen in to an active protease, the phylogenetically conserved N-terminus amino acids; the
cysteine residues important in disulphide bond formation; and the necessary leader sequence to
place the nascent protein into the endoplasmic reticulum. The novel enzyme's predicted tertiary
structure is consistent with serine proteases and phylogenetic analysis indicates that CFGR-1
may be a member of granzyme family (Figure 4). Furthermore, we show that the expression of
the catfish granzyme is restricted to lymphoid tissue (Figures 5).
84
Homology comparisons with known mammalian granzymes alone were not enough to
determine the substrate specificity of catfish granzyme because of the lack of predominant
similarities to a particular member of the family. However, based on residue identities and
residue similarities (table 2), CFGR-1 appears to be most similar to the mammalian granzymes A
and K. Both of these proteases are the only granzymes with tryptase activity sequenced to date.
Support for the idea that the teleost granzyme cleavage sites may be after arginine and lysine was
suggested by analyzing the putative substrate specificity pocket amino acids, which were
determined by comparison to similar residues in other granzymes. The catalytic triad residues
(histidine, aspartic acid and serine) are conserved across different granzyme species, suggesting
similar active site structure. VLTAAHC around His 57 and GDSGGPL around Ser 195 are
highly conserved, while DIML is conserved in many granzymes. Interestingly, the tilapia
product had closer similarity to granzymes from the metase family. Although, it is still early to
conclusively assign substrate specificity to this tilapia granzyme, the significance of this finding
is that there may be a fish ortholog to granzyme M. Granzyme M is uniquely expressed by NK
cells, which suggests that the NCC are the NK-like cells (or their precursors) in teleost. Other
indication that these two teleost granzymes may belong to different families are that RT-PCR
using catfish granzyme specific primers failed to amplify the tilapia cDNA.
In addition to the charge relay system, other granzyme signature motifs are present in the
catfish homologue. As has been shown in all mammalian granzymes (Kam et al. 2000; Smyth et
al. 1996), the catfish enzyme is produced as a propeptide. The leader sequence is a necessary
requirement for the translated message of these enzymes to be compartmentalized to the rough
endoplasmic reticulum and their final destination in the exocytic granules (Jenne and Tschopp
1988; Bleackley et al. 1988). Application of von Heijne's algorithm would leave a tetrapeptide as
85
the inactivating portion of the enzyme. Although the propetide portion of most granzymes is a
dipeptide, the only mammalian granzyme that is exclusively expressed in NK cells, granzyme M,
has a hexapeptide at this position (Smyth et al. 1993).
Mammalian granzymes vary significantly in the degree of glycosylation from none
(granzyme C) to accounting as much as 50% of their total mass (granzyme D). The possibility of
glycosylation for the catfish granzyme is interesting in that it could have functional significance.
It has been shown that granzyme A and B are glycosylated with phosphorylated mannose rich
residues, which recognize the mannose-6-phosphate receptor. This is an important pathway for
targeting of these enzymes to secretory granules. Moreover, glycosylation could allow target
cell entry of the catfish granzymes in the absence of perforin by binding to the mannose-6-P
receptor followed by endocytosis, an important point since the presence of teleost perforin has
not been reported. Although the actual glycosylation status of the catfish granzyme is not
presently known, it is noteworthy that the predicted protein has at least one putative
glycosylation site at position 36 (chymotrypsinogen numbering). It has been demonstrated that
glycosylation of granzymes occurs preferentially at Asn residues rather than Ser-Thr residues
(Griffiths and Isaaz 1993).
Based on the sequence information and expression pattern, this novel molecule can be
predicted as a new member of granzyme family. This is the first report of a non-mammalian
granzyme and there is convincing evidence for the presence of more granzyme-like molecules in
fish cytotoxic cells. By definition, granzymes are found within the granules of cytotoxic cells.
Characterization of granzymes from lower vertebrates sheds more light in to the evolutionary
patterns of serine proteases.
86
ACKNOWLEDGEMENTS
We thank Dr. David Peterson for critical review of the manuscript. This work was funded with
support from USDA (98-35205-6701).
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90
Figure 3.1. Cloning strategy used to identify and characterize the cDNA for catfish granzyme.
The primer sequences are listed in table 1.
91
92
Figure 3.2. Compiled full-length catfish granzyme cDNA sequence. Predicted signal sequence at
the N-terminus is underlined. Putative tetrapeptide propeptide is double underlined. The amino
acid numbering starts at the amino terminal of predicted mature protein as determined by
sequence homology to other granzymes. Highly conserved IIXG motif at the N-terminal is
boxed, while a region similar to the PHSRPYMA motif in other granzymes is highlighted.
Putative glycosylation site is circled. Cysteine residues in the mature protein are marked with
(•), while catalytic triad residues are marked with (♦). ATTTA motif in the 3' UTR is italicized
with underline and poly A tail is underlined.
93
M
1
E
R
N
T
L
K
V
K
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A
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-2
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16
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32
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49
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66
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82
H
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99
N
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116
A
G
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W
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T
H
V
132
CCCAGCAGGGACAAAATGTGAAGTAAGAGGGTGGGGAACCACTCATGTAA
P
K
A
C
D
T
L
Q
E
L
E
V
T
V
V
149
AAAATCCTAAGGCGTGTGACACCTTGCAAGAGCTGGAGGTGACGGTGGTG
R
E
L
C
N
C
Y
Y
N
S
K
P
T
I
T
A
166
GACAGGGAACTGTGTAACTGCTACTACAACAGCAAACCTACCATCACTGC
M
L
C
A
G
N
K
Q
R
D
K
D
A
C
W
182
CAACATGCTGTGTGCAGGAAACAAGCAGAGGGACAAAGATGCATGCTGGG
D
S
G
G
P
L
E
C
K
K
N
I
V
G
V
V
199
GGGATTCTGGCGGACCTCTGGAGTGTAAGAAAAACATTGTGGGTGTGGTG
S
701
F
AAGAAAAACAAGGTAGACGTTAAGAAGATTCCCAAATCAGGCAAAGACAT
G
651
L
CTAGGGTTCATGACATCATGCTCCTGAAGCTCCAAGACAAAGTCCAGCTT
N
601
T
GCGAGTGAAAGTCCTATGCTTTCACATCTCACCAAAGTTCAGTGCTACAA
D
551
G
N
L
V
V
K N
501
L
ATCGAAGTGCTTCTCGGTGCTCACTCTCTGACGAAGGACAAAAATGCTAT
P
451
-19
AGTGGGTTCTGACAGCTGCACACTGCAAAACTTTTTTACAATTTAAACCA
K
401
I
G
S
V
T R
351
L
I
Q
W
R
301
R
AGTCCAGTCCAATAACTCGCACATCTGTGGAGGAACTCTTATTCATCAGC
I
251
Q
TTCATCATAGGAGGCCGAGAGGTGAAAAAACCTAAACCATGGATGGCCTC
Q
201
L
I
V
151
Q
CCCTTCTTCTGATTCTCACCTTGTTTCAAGCAACAGCTTGCTCAGGCAGT
F
101
V
CAGTCCTCATTTCTGCACTCAAGAGCAGACCATGCACGTGCAACAACGTT
S L
51
H
G
G
S
G
C
G
N
P
K
K
P
G
V
Y
T
L
TCAGGAGGAAGTGGCTGTGGCAATCCCAAAAAACCTGGTGTCTATACTCT
L
S
K
E
H
I
D
W
I
N
K
I
I
K
K
*
751
TCTCTCAAAGGAACACATTGACTGGATCAACAAGATAATTAAAAAGTAAT
801
CCAACAGCACAGAGGCTTAGAAACACAACATTTATCAGTGCTTGCAGTAT
851
ATATTGTGTCATATACAGTATCTGTCCGCATATACAAGAGGTATATTTGC
901
TTGCTTGTTGGTTTTATCTGATCTTTTACTTCACACATATAGCTGAAATT
951
GAACAGTACAGCAAAAAAAAAAAAAAAAn
94
216
Figure 3.3. Multiple sequence alignment of predicted catfish granzyme sequence (mature
protein) with known granzymes and related proteins using CLUSTAL W. Highly conserved
regions around the catalytic triad residues are indicated as boxes above the alignment. Cysteine
residues marked (♦) are highly conserved among all known granzymes, while those marked (•)
are conserved among granzymes A, K and M. Residues marked with (*) are predicted to
contribute towards the primary substrate specificity of the protease. Mature portion of the
following proteases were used in this comparison: human granzyme K (accession No.
AAH35802), mouse granzyme K (accession No. AAC17930), rat granzyme K (accession No.
NP_058815), human granzyme A (accession No. AAH15739), mouse granzyme A (accession
No. NP_034500), rat granzyme M (accession No. Q03238), human granzyme M (accession No.
AAH25701), mouse granzyme M (accession No. NP_032530), human granzyme B (accession
No. AAH30195), mouse granzyme B (accession No. NP_038570), rat granzyme B (accession
No. NP_612526), human granzyme H (accession No. A32692), mouse granzyme C (accession
No. NP_034501), mouse granzyme D (accession No. NP_034502), mouse granzyme E
(accession No. NP_034503), mouse granzyme F (accession No. AAA37741), mouse granzyme
G (accession No. NP_034505), rat granzyme J (accession No. AAC53168), human mast cell
chymase (accession No. KYHUCM), and human cathepsin G (NP_001902). Darker shading
represents identical or residues with similar properties in 100% of the sequences and lighter
shading represents identical or residues with similar properties in 80% or more of all the
sequences.
95
CFGR-1
hum grnzK
mus grnzK
rat grnzK
hum grnzA
mus grnzA
rat grnzM
hum grnzM
mus grnzM
hum grnzB
mus grnzB
rat grnzB
hum grnzH
mus grnzC
mus grnzD
mus grnzE
mus grnzF
mus grnzG
rat grnzJ
hu mast chymase
hu cathepsinG
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
IIGGREVK-KPKPWMASVQSNN----SHICGGTLIHQQWVLTAAHCKTFLQ-FKPIEVLL
IIGGKEVSPHSRPFMASIQYGG----HHVCGGVLIDPQWVLTAAHCQYRFTKGQSPTVVL
IIGGREVQPHSRPFMASIQYRS----KHICGGVLIHPQWVLTAAHCYSWFPRGHSPTVVL
IIGGREVQPHSRPFMASIQYRG----KHICGGVLIHPQWVLTAAHCYS---RGHSPTVVL
IIGGNEVTPHSRPYMVLLSLDR----KTICAGALIAKDWVLTAAHCNLN----KRSQVIL
IIGGDTVVPHSRPYMALLKLSS----NTICAGALIEKNWVLTAAHCNVG----KRSKFIL
IIGGREAVPHSRPYMVSLQNTK----SHMCGGVLVHQKWVLTAAHCLSEP--LQQLKLVF
IIGGREVIPHSRPYMASLQRNG----SHLCGGVLVHPKWVLTAAHCLAQR--MAQLRLVL
IIGGREAVPHSRPYMASLQKAK----SHVCGGVLVHRKWVLTAAHCLSEP--LQNLKLVL
IIGGHEAKPHSRPYMAYLMIWDQKS-LKRCGGFLIQDDFVLTAAHCWGS-----SINVTL
IIGGHEVKPHSRPYMALLSIKDQQP-EAICGGFLIREDFVLTAAHCEGS-----IINVTL
IIGGHEADPHSRPYMAYLQYKNEDSRDTICGGFLIREDFVLTAAHCSGS-----KINVTL
IIGGHEAKPHSRPYMAFVQFLQEKS-RKRCGGILVRKDFVLTAAHCQGS-----SINVTL
IIGGNEISPHSRPYMAYYEFLKVGGKKMFCGGFLVRDKFVLTAAHCKGR-----SMTVTL
IIGGHVVKPHSRPYMAFVMSVDIKGNRIYCGGFLIQDDFVLTAAHCKNS-----SMTVTL
IIGGHVVKPHSRPYMAFVKSVDIEGNRRYCGGFLVQDDFVLTAAHCRNR-----TMTVTL
IIGGHEVKPHSRPYMARVRFVKDNGKRHSCGGFLVQDYFVLTAAHCTGS-----SMRVIL
IIGGHEVKPHSRPYMAFIKSVDIEGKKKYCGGFLVQDDFVLTAAHCRNR-----SMTVTL
IIWGTESKPHSRPYMAFINFYDSNSDLNRCGGFLVAKDIVMTAAHCNGR-----NIKVIL
IIGGTECKPHSRPYMAYLEIVTSNGPSKFCGGFLIRRNFVLTAAHCAGR-----SITVTL
IIGGRESRPHSRPYMAYLQIQSPAGQS-RCGGFLVREDFVLTAAHCWGS-----NINVTL
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
54
56
56
53
52
52
54
54
54
54
54
55
54
55
55
55
55
55
55
55
54
CFGR-1
hum grnzK
mus grnzK
rat grnzK
hum grnzA
mus grnzA
rat grnzM
hum grnzM
mus grnzM
hum grnzB
mus grnzB
rat grnzB
hum grnzH
mus grnzC
mus grnzD
mus grnzE
mus grnzF
mus grnzG
rat grnzJ
hu mast chymase
hu cathepsinG
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
GAHSLTKDKNAMRVKVLCFHISPKFSAT--TRVHDIMLLKLQDKVQLKKNKVDVKKIPKS
GAHSLSKNEASKQTLEIKKFIPFSRVTS-DPQSNDIMLVKLQTAAKLNKH-VKMLHIRSK
GAHSLSKNEPMKQTFEIKKFIPFSRLQS-GSASHDIMLIKLRTAAELNKN-VQLLHLGSK
GAHSLSKNEPMKQTFEIKEFIPFSGFKS-G--TNDIMLIKLRTAAELNKH-VQLLHLRSK
GAHSITREEPTKQIMLVKKEFPYPCYDP-ATREGDLKLLQLTEKAKINKY-VTILHLPKK
GAHSINKE-PEQQILTVKKAFPYPCYDE-YTREGDLQLVRLKKKATVNRN-VAILHLPKK
GLHSLHDPQDPGLTFYIKQAIKHPGYN--LKYENDLALLKLDGRVKPSKN-VKPLALPRK
GLHTLDS---PGLTFHIKAAIQHPRYKPVPALENDLALLQLDGKVKPSRT-IRPLALPSK
GLHNLHDLQDPGLTFYIREAIKHPGYN--HKYENDLALLKLDRRVQPSKN-VKPLALPRK
GAHNIKEQEPTQQFIPVKRPIPHPAYNP-KNFSNDIMLLQLERKAKRTRA-VQPLRLPSN
GAHNIKEQEKTQQVIPMVKCIPHPDYNP-KTFSNDIMLLKLKSKAKRTRA-VRPLNLPRR
GAHNIKEQEKTQQVIPVVKIIPHPAYNA-KTISNDIMLLKLKSKAKRTRA-VKTLSLPRS
GAHNIKEQERTQQFIPVKRPIPHPAYNP-KNFSNDIMLLQLERKAKWTTA-VRPLRLPSS
GAHNIKAKEETQQIIPVAKAIPHPDYNP-DDRSNDIMLLKLVRNAKRTRA-VRPLNLPRR
GAHNITAKEETQQIIPVAKDIPHPDYNA-TIFYSDIMLLKLESKAKRTKA-VRPLKLPRS
GAHNIKAKEETQQIIPVAKAIPHPDYNA-TAFFSDIMLLKLESKAKRTKA-VRPLKLPRP
GAHNIRAKEETQQIIPVAKAIPHPAYDD-KDNTSDIMLLKLESKAKRTKA-VRPLKLPRP
GAHNIKAKEETQQIIPVAKAIPHPAFNR-KHGTNDIMLLKLESKAKRTKA-VRPLKLPRP
GAHNIKKRE-NTQVISVLKAKPHENFNS-DSLVNDIMLLKLERKAQLNGV-VKTIALPRS
GAHNITEEEDTWQKLEVIKQFRHPKYNT-STLHHDIMLLKLKEKASLTLA-VGTLPFPSQ
GAHNIQRRENTQQHITARRAIRHPQYNQ-RTIQNDIMLLQLSRRVRRNRN-VNPVALPRA
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
112
114
114
109
110
109
111
110
111
112
112
113
112
113
113
113
113
113
112
113
112
CFGR-1
hum grnzK
mus grnzK
rat grnzK
hum grnzA
mus grnzA
rat grnzM
hum grnzM
mus grnzM
hum grnzB
mus grnzB
rat grnzB
hum grnzH
mus grnzC
mus grnzD
mus grnzE
mus grnzF
mus grnzG
rat grnzJ
hu mast chymase
hu cathepsinG
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
-GKDIPAGTKCEVRGWGTTHVKNPKACDTLQELEVTVVDRELCNCYYN--SKPTITANML
--TSLRSGTKCKVTGWGATDPDSLRPSDTLREVTVTVLSRKLCNSQSYYNGDPFITKDMV
--NYLRDGTKCQVTGWGTTKPDLLTASDTLREVTVTIISRKRCNSQSYYNHKPVITKDMI
--NYIRDGTKCQVTGWGSTKPDVLTTSDTLQEVTVTIISRKRCNSQSYYNHKPVITKDMI
-GDDVKPGTMCQVAGWGRTH-NSASWSDTLREVNITIIDRKVCNDRNHYNFNPVIGMNMV
-GDDVKPGTRCRVAGWGRFG-NKSAPSETLREVNITVIDRKICNDEKHYNFHPVIGLNMI
PRDKPAEGSRCSTAGWGITHQ-RGQLAKSLQELDLRLLDTRMCNNSRFWNGVLTDS--ML
-RQVVAAGTRCSMAGWGLTHQ-GGRLSRVLRELDLQVLDTRMCNNSRFWNGSLSPS--MV
PRSKPAEGTWCSTAGWGMTHQ-GGPRARALQELDLRVLDTQMCNNSRFWNGVLIDS--ML
-KAQVKPGQTCSVAGWGQTAP-LGKHSHTLQEVKMTVQEDRKCESDLRHYYDSTI---EL
-NVNVKPGDVCYVAGWGRMAP-MGKYSNTLQEVELTVQKDRECESYFKNRYNKTN---QI
-NFKVKPGDVCYVAGWGKLGP-MGKFPDKLQEVELTVQEDQECETYLKNAYDKAN---QI
-KAQVKPGQLCSVAGWGYVS--MSTLATTLQEVLLTVQKDCQCERLFHGNYSRAT---EI
-NAHVKPGDECYVAGWGKVTP-DGEFPKTLHEVKLTVQKDQVCESQFQSSYNRAN---EI
-NARVKPGDVCSVAGWGSRSINDTKASARLREVQLVIQEDEECKKRFR-YYTETT---EI
-NARVKPGDVCSVAGWGPRSINDTKASARLREAQLVIQEDEECKKRFR-HYTETT---EI
-NARVKPGHVCSVAGWGRTSINATQRSSCLREAQLIIQKDKECKKYFY-KYFKTM---QI
-NARVKPGDVCSVAGWGKTSINATKASARLREAQLIIQEDEECKKLWY-TYSKTT---QI
-QDWVKPGQVCTVAGWGTLA--NCTLSNTLQEVNLEVQKGQKCQGMSR-NYNDSI---QL
-FNFVPPGRMCRVAGWGRTG-VLKPGSDTLQEVKLRLMDPQACSHFRD--FDHNL---QL
-QEGLRPGTLCTVAGWGRVS--MRRGTDTLREVQLRVQRDRQCLRIFG-SYDPRR---QI
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
169
172
172
167
168
167
168
166
168
167
167
168
166
168
168
168
168
168
165
166
165
96
*
***
CFGR-1
hum grnzK
mus grnzK
rat grnzK
hum grnzA
mus grnzA
rat grnzM
hum grnzM
mus grnzM
hum grnzB
mus grnzB
rat grnzB
hum grnzH
mus grnzC
mus grnzD
mus grnzE
mus grnzF
mus grnzG
rat grnzJ
hu mast chymase
hu cathepsinG
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
CAGNKQRDKDACWGDSGGPLECKKN--IVGVVSGGSG--CGNPKKPGVYTLLSKEHIDWI
CAGDAKGQKDSCKGDSGGPLICKGV--FHAIVSGGHE--CGVATKPGIYTLLTKKYQTWI
CAGDARGQKDSCKGDSGGPLICKGI--FHALVSQGYK--CGIAKKPGIYTLLTKKYQTWI
CAGDRRGEKDSCKGDSGGPLICKGV--FHALVSGGYK--CGISNKPGVYTLLTKKYQTWI
CAGSLRGGRDSCNGDSGSPLLCEGV--FRGVTSFGLENKCGDPRGPGVYILLSKKHLNWI
CAGDLRGGKDSCNGDSGSPLLCDGI--LRGITSFGGE-KCGDRRWPGVYTFLSDKHLNWI
CLKAGAKGQAPCKGDSGGPLVCGKG-KVDGILSFSSK-NCTD-IFKPTVATAVAPYSSWI
CLAADSKDQAPCKGDSGGPLVCGKGRVLAGVLSFSSR-VCTD-IFKPPVATAVAPYVSWI
CLKAGSKSQAPCKGDSGGPLVCGKG-QVDGILSFSSK-TCTD-IFKPPVATAVAPYSSWI
CVGDPEIKKTSFKGDSGGPLVCNKV--AQGIVSYGRN-----NGMPPRACTKVSSFVHWI
CAGDPKTKRASFRGDSGGPLVCKKV--AAGIVSYGYK-----DGSPPRAFTKVSSFLSWI
CAGDPKIKCASFQGDSGGPLVCKKV--AAGIVSYGRK-----DGSTPRAFTKVSTFLSWI
CVGDPKKTQTGFKGDSGGPLVCKDV--AQGILSYGNK-----KGTPPGVYIKVSHFLPWI
CVGDSKIKGASFEEDSGGPLVCKRA--AAGIVSYGQT-----DGSAPQVFTRVLSFVSWI
CAGDLKKIKTPFKGDSGGPLVCHNQ--AYGLFAYAKN-----GTISSGIFTKVVHFLPWI
CAGDLKKIKTPFKGDSGGPLVCDNK--AYGLLAYAKN-----RTISSGVFTKIVHFLPWI
CAGDPKKIQSTYSGDSGGPLVCNNK--AYGVLTYGLN-----RTIGPGVFTKVVHYLPWI
CAGDPKKVQAPYEGESGGPLVCDNL--AYGVVSYGIN-----RTITPGVFTKVVHFLPWI
CVGNPNERKATAGGDSGGPFVCNGV--AQGIVSYRLC-----TWTPPRVFTRISSFIPWI
CVGNPRKTKSAFKGDSGGPLLCAGV--AQGIVSYGRS-----DAKPPAVFTRISHYRPWI
CVGDRRERKAAFKGDSGGPLLCNNV--AHGIVSYGKS-----SGVPPEVFTRVSSFLPWI
CFGR-1
hum grnzK
mus grnzK
rat grnzK
hum grnzA
mus grnzA
rat grnzM
hum grnzM
mus grnzM
hum grnzB
mus grnzB
rat grnzB
hum grnzH
mus grnzC
mus grnzD
mus grnzE
mus grnzF
mus grnzG
rat grnzJ
hu mast chymase
hu cathepsinG
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
NKIIKK----------KSNLVPPHTN------KSKLAPSRAH------KSKLAPSSAH------IMTIKGAV--------KKIMKGSV--------RKVIGRWSPQPLT---RKVTGRSA--------RKVIGRWSPQSLV---KKTMKRH---------KKTMKSS---------EETMKKS---------KRTMKRL---------KKTMKHS---------SWNMKLL---------SRNMKLL---------SRNMKLL---------STNMKLL---------QKTMKLLQQP------NQILQAN---------RTTMRSFKLLDQMETPL
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
231
238
238
233
234
232
238
232
238
227
227
228
226
228
228
228
228
228
228
226
235
97
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
225
228
228
223
226
224
225
224
225
220
220
221
219
221
221
221
221
221
218
219
218
Figure 3.4. Phylogenetic analysis of catfish granzyme: Phylogram showing relationships of
catfish granzyme to other known serine proteases. The tree was derived by parsimony analysis,
with Mega version 2. Numbers shown above the branches are bootstrap values based upon 1000
replicates for parsimony. A separate analysis using maximum likelihood and neighbor joining
methods produced a tree with similar topology. The tree was rooted on a sub-tree containing
trypsin and chymotrypsin of fish and murine origin to determine the clustering of catfish
granzyme with very similar known granzyme sequences. In addition to the previously described
granzyme sequences, mature sequences of the following proteases were used to obtain the tree
human adpsin (accession No. NP_001919), rat chymotrypsin B (accession No. AAA98732), rat
pancreatic protease (accession No. NP_036861), lampray (Petromyzon marinus) trypsin
(accession No. AAB69656), eel (Anguilla japonica) trypsin (accession No. BAB85634), and
salmon (Salmo salar) trypsin (accession No. CAA49679).
98
99
Figure. 3.5. Analysis of tissue expression of catfish granzyme. (A) RT-PCR analysis was
performed with catfish granzyme specific primers using cDNA from various tissues. Lane 1,
Anterior Kidney; lane 2, Spleen; lane 3, Total Blood; lane 4, Liver; lane 5, Gill; lane 6, Muscle;
lane 7, Trunk Kidney; lane 8, Heart; lane 9, NCC cDNA library (Positive Control). (B) NCC
were enriched from three tissues to analyze the granzyme expression. Lane 1, NCC purified from
peripheral blood, Lane 2, NCC purified from anterior kidney, Lane 3, NCC purified from spleen.
100
101
Figure 3.6. Alignment of other fish granzyme sequences with catfish granzyme. A, A portion of
tilapia granzyme was amplified with primers used in cloning of catfish gene. The nucleotide
sequence was edited, translated and aligned with catfish granzyme. Residues marked (•) above
the alignment differ from catfish granzyme, but are highly similar to different mammalian
granzymes. B, Partial sequence of a Salmo salar granzyme derived from an EST (Accession no
CB516537) was edited and translated to compare with mature catfish granzyme. C, N-terminal
region of a partial sequence from Salmo salar (accession no AF434669) which resembles human
granzyme M was edited and translated to compare with the mature catfish granzyme.
102
(A)
ccf grnz (CFGR1) : MHVQQRSLLLILTLFQATACSGSFIIGGREVKKPKPWMASVQSNNSHICGGTLIHQQWVL :
ilapia grnz pcr : ------------------------------------------------------------ :
60
-
ccf grnz (CFGR1)
tilapia grnz pcr
: TAAHCKTFLQFKPIEVLLGAHSLTKDKNAMRVKVLCFHISPKFSATTRVHDIMLLKLQDK : 120
: ---------------------------------------------LNFSNDIMLLKLSRK : 15
ccf grnz (CFGR1)
tilapia grnz pcr
: VQLKKNKVDVKKIPKSGKDIPAGTKCEVRGWGTTHVKNPKACDTLQELEVTVVDRELCNC : 180
: LQLDK-KVKPIQLARKEIKAKDNVKCQVAGWGFTETSG-KTVDVLRWVDVPLIDLNVCKR : 73
ccf grnz (CFGR1)
tilapia grnz pcr
: YYNSKPTITANMLCAGNKQRDKDACWGDSGGPLECKKNIVGVVSGGSGCGNPKKPGVYTL : 240
: KLKAKVTLPKGVVCAGGSDTKNGFCQGDSGGPLVCNN----------------------- : 110
ccf grnz (CFGR1)
tilapia grnz pcr
: LSKEHIDWINKIIKK : 255
: --------------- :
-
(B)
CFGR-1
salmo est
: ---------MHVQQRSLLLILTLFQATACSGSFIIGGREVK-KPKPWMASVQSNNSHICG :
: ERFNRIIRFGGPLLQTALLIASVWKLAACSEVTIVGGHEVKPHSRPWMVSLQVKNNHVCG :
50
60
CFGR-1
salmo est
: GTLIHQQWVLTAAHCKTFLQFKP--IEVLLGAHSLTKDKNAMRVKVLCFHISPKFSATTR : 108
: GTLIRDQWVLTAAHCKGVFGESKKFVEAVLGAHSLTSSKNTQRVGIEEYYVPGTYSDRTK : 120
CFGR-1
salmo est
: VHDIMLLKLQDKVQLKKNKVDVKKIPKSGKDIPAGTKCEVRGWGTTHVKNPKACDTLQEL : 168
: EDDIMLIKLKMKVKINSKAVKVKEVSKSGKDLQVGTQCHVTGWGVVSATGSMPSDTLQGA : 180
CFGR-1
salmo est
: EVTVVDRELCNCYYNSKPTITANMLCAGNKQRDKDACWGDSGGPLECKKNIVGVVSGGSG : 228
: EVDILDRKLCDCFYNRNPVITQDMLCASNNKRQADACKGDSGGPLECKKAFVGLVSGGLG : 240
CFGR-1
salmo est
: CGNPKKPGVYTLLSKEHIDWINKIIKK : 255
: --------------------------- :
-
(C)
CFGR-1
: MHVQQRSLLLILTLFQATACSGSFIIGGREVKKPKPWMASVQSNNSHICGGTLIHQQWVL :
salmo grnzM-like : ----------MILSTAASFWTSTMILLYLHSGRGVCVCLCYPTNIYYYFSNEVTVGVLSL :
60
50
CFGR-1
: TAAHCKTFLQFKPIEVLLGAHSLTKDKNAMRVKVLCFHISPKFSATTRVHDIMLLKLQDK : 120
salmo grnzM-like : LLGDCAEIIGGKEVTPHSLPYMALLEDNKGNKKCGGILSHQQWVLTGAHC---------- : 100
CFGR-1
: VQLKKNKVDVKKIPKSGKDIPAGTKCEVRGWGTTHVKNPKACDTLQELEVTVVDRELCNC : 180
salmo grnzM-like : ------------------------------------------------------------ :
CFGR-1
: YYNSKPTITANMLCAGNKQRDKDACWGDSGGPLECKKNIVGVVSGGSGCGNPKKPGVYTL : 240
salmo grnzM-like : ------------------------------------------------------------ :
CFGR-1
: LSKEHIDWINKIIKK : 255
salmo grnzM-like : --------------- :
-
103
Table 3.1. Oligonucleotide primers used in the cloning of catfish granzyme and for expression
studies by RT-PCR.
* AP (5' RACE Abridged Anchor Primer) and AUAP (Abridged Universal Amplification
Primer) were obtained as a component of 5' RACE system (Invitrogen, CA). AP and AUAP are
specifically engineered for the optimal amplification of dC-tailed cDNA. It contains strategically
positioned deoxyinosine (I) and U residues to maximize specific priming at the dC-tailed end.
Name
Sequence
Use
F1
5’-GCTGCAGTAGCATGATGTCA-3’
Initial Screening
R1
5’- GTTACACACAAGAGGGCCTCCA-3’
Initial Screening
5RC1
5’-GCATGATGTCATTGGAGAAGTTC-3’
5' RACE
5RC2
5'-GGGAATCTTCTTAACGTCTAC-3'
5' RACE
5RC3
5'- GTTCCCCACCCTCTTACTTCAC-3'
5' RACE
3RC1
5'- CAAGCAGAGGGACAAAGATGCA-3'
3' RACE
3RC2
5'- TGGAGGCCCTCTTGTGTGTAAC-3'
3' RACE
CDF1
5'-CCCGGATCCATGCACGTGCAACAACGTTC-3'
ORF amplification
CDR1
5'-GGGGTTCTAGATGCTTTTTAATTATCTTGTTGATCCAGTCA-3'
ORF amplification
EPB-18T
5'-GCGATTTCTGCAGGATCCAAACTT(17)-3'
3' RACE RT
EPB
5'-GCGATTTCTGCAGGATCCAAACT-3'
3' RACE
AP (*)
5'-CUACUACUACUACACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3'
5' RACE
AUAP (*)
5'-CUACUACUACUACACGCGTCGACTAGTAC-3'
5' RACE
GRRTF1
5'-GTTTCAAGCAACAGCTTGCTCAGGCA-3'
RT PCR
GRRTR1
5'-GCATAGGACTTTCACTCGCATAGCA-3'
RT PCR
ACTINF
5'-GACAATGGTTCCGGTATGTGCA-3'
RT PCR
ACTINR
5'-GGTGCCAGATCTTCTCCATGTCA-3'
RT PCR
104
Table 3.2. Amino acid identity and similarity of mature catfish granzyme to other known
granzymes and related proteins.
Human GRNZ A
Mouse GRNZ A
Human GRNZ B
Mouse GRNZ B
Rat GRNZ B
Mouse GRNZ C
Mouse GRNZ D
Mouse GRNZ E
Mouse GRNZ F
Mouse GRNZ G
Human GRNZ H
Rat GRNZ J
Human GRNZ K
Mouse GRNZ K
Rat GRNZ K
Human GRNZ M
Mouse GRNZ M
Rat GRNZ M
Human Cathepsin G
Human Mast Cell Chymase
% Identity
41.0
39.7
32.1
35.9
37.0
30.3
31.6
30.3
31.1
32.4
32.9
34.6
43.3
45.8
45.4
33.1
32.5
33.9
31.2
37.4
105
% Similarity
53.1
53.2
40.9
46.8
46.6
41.2
43.0
42.0
42.4
43.7
43.9
45.4
55.4
55.8
55.0
47.5
42.9
47.1
44.5
49.2
CHAPTER 4
NONSPECIFIC CYTOTOXIC CELLS OF TELEOSTS ARE ARMED WITH
MULTIPLE GRANZYMES AND OTHER COMPONENTS OF THE GRANULE
EXOCYTOSIS PATHWAY1
1
Praveen, K., J. H. Leary, D. L. Evans and L. Jaso-Friedmann. To be submitted to Molecular
Immunology after revision
106
ABSTRACT
Granzymes are members of the serine protease family and major components of cytotoxic
granules of professional killer cells. Multiple granzymes have been identified from human and
rodents with different substrate specificities. Although the significance of granzymes A and B in
cell-mediated cytotoxicity has been extensively investigated, recent reports suggest that other
granzymes may have either equal or greater importance in mediating cell death. Studies on the
evolution of these closely related proteases were hindered by the lack of sequence and
biochemical information of granzymes from “lower vertebrates.” Here we report the generation
of a catalytically active recombinant granzyme identified in the cytotoxic cells of an ectothermic
vertebrate. Fully active, soluble recombinant catfish granzyme-1 (CFGR-1) was generated using
a yeast-based expression system. In vitro enzyme kinetic assays using various thiobenzyl ester
substrates verified its tryptase activity in full agreement with previous observations by sequence
comparison and molecular modeling. The tryptase activity that was secreted from catfish NCC
during an in vitro cytotoxicity assay strongly correlated with the cytotoxicity induced by these
cells. Evidence for additional granzymes with different substrate specificities in NCC was
obtained by analysis of the protease activity of supernatants collected from in vitro cytotoxicity
assays. Searches of the catfish EST database further confirmed the presence of teleost
granzymes with different substrate specificities. Granzyme activity measurements suggested a
predominance of chymase and tryptase activities in NCC. Further proof that the granule
exocytosis pathway is one of the cytotoxic mechanisms in NCC was provided by the expression
of granule components perforin, granulysin and serglycin detected by RT-PCR analysis. These
results demonstrate the evidence for a parallel evolution of effector molecules of cell-mediated
cytotoxicity in teleosts.
107
Key words: Granzymes, perforin, granulysin, serglycin, thiobenzyl ester substrates, nonspecific
cytotoxic cells
INTRODUCTION
Cell-mediated cytotoxicity against virally transformed and tumor cells is mediated
through highly specialized lymphocytes. Cytotoxic T lyphocytes (CTLs) and natural killer cells
(NK cells) are the most important among them (Lieberman, 2003). Although these two cells use
distinct mechanisms to recognize their targets, following receptor engagement and activation
they use the same effector pathways to mediate target cell death. One of the major effector
mechanisms used by these cells, which would eventually lead to target cell death, involves
release of perforin and a family of serine proteases called granzymes into the immunological
synapse formed between an effector and its target cell (Podack, 1995, Wowk and Trapani, 2004).
In addition to granzymes and perforin, other proteins are also present in the cytotoxic granules in
smaller quantities, which appear to have supportive roles in killing by granule exocytosis (Smyth
et al., 2001, Clark and Griffiths, 2003).
Cytotoxic granules are considered secretory lysosomes because of the presence of
lysosomal proteins along with lytic molecules, which can induce target cell death (Burkhardt et
al., 1990, Peters et al., 1991). The granules have a morphologically distinct core consisting
mainly of perforin and granzymes. The association of these lytic proteins with proteoglycans in a
pH-dependant fashion, helps to maintain their enzymatic activity in an inactive state and thus,
avoids damaging the effector cells (Masson et al., 1990). In fact, unlike conventional lysosomes
which lack proteoglycans, the presence of serglycin in these granules, serves as a good indicator
of the role of a cell in cell-mediated cytotoxicity (Clark and Griffiths, 2003).
108
The involvement of multiple granzymes in cell death pathways has been documented.
Experimental expression of granzymes along with perforin in non-cytotoxic cells can make them
efficient killers, suggesting that they are very important components of the granule exocytosis
pathway of cytotoxicity (Hayes et al., 1989, Shiver et al., 1992). Experimental animals lacking
select granzymes have been shown to suffer significant immunodeficiencies, although the
phenotypic characteristics of the effects were focal. These results suggest that multiple
granzymes, whose functions may not be clear at present, may be major players in death pathways
(Trapani and Smyth, 2002, Ebnet et al., 1995, Wilharm et al., 1999b, Heusel et al., 1994, Revell
et al., 2005).
Granzymes are classified based on the four primary substrate specificities that have been
described to date: tryptase (trypsin-like, cleaving after Arg/Lys), Asp-ase (similar to caspases,
cleaving after Asp), Met-ase (cleaving after Met/Leu) and chymase (chymotrypsin-like, cleaving
after Phe/Trp/Tyr) (Kam et al., 2000). The most extensively studied mammalian granzymes fall
under three of the above specificities. Granzymes A and K have tryptase activity, granzyme B
has Asp-ase activity, while granzyme M has Met-ase activity (Kam et al., 2000). The majority of
the remaining granzymes, whose functions are not fully understood, have chymase specificity.
Some of them have been implicated in the regulation of perforin activity (Hudig et al., 1993,
Woodard et al., 1998). The significant roles that these chymases may be playing in novel cell
death pathways are now widely appreciated.
We have previously shown the presence of granzyme-like serine proteases in teleost
nonspecific cytotoxic cells, NCC (Praveen et al., 2004). The first granzyme gene identified in
catfish NCC was predicted to have a tryptase activity due to the presence of conserved triplet
residues, which are important in the formation of the substrate specificity pocket (Wouters et al.,
109
2003, Praveen et al., 2004). We now report on the expression of CFGR-1 as a recombinant
protein and we show its specificity for Lys/Arg residues, in agreement with the previously
predicted activity profile. The presence of other granzymes with different substrate specificities
as well as additional components of cytotoxic granules is also reported. These results
demonstrate the significance of NCC as a major cytotoxic cell population in channel catfish
(Ictalurus punctatus).
2.
MATERIALS AND METHODS
2.1.
Production of mature and pro-CFGR-1 in Pichia pastoris
The cDNAs coding for the pro and mature forms of CFGR-1 were PCR amplified and
cloned into pPICZ-alpha vector for expression in GS115 strain of Pichia pastoris (Invitrogen,
CA). Forward primers CFGRYSTF1 (5’-aactctcgagaaaagaatcataggaggccga-3’) and
CFGRYSTF2 (5’-aactctcgagaaaagaagtttcatcataggaggccga-3’) were designed to engineer an Xho I
site upstream of the mature and pro CFGR-1, respectively. A reverse primer CFGRYSTR1 (5’ggggttctagatgtgttcctttgagagaagagtatagac-3’) was designed to incorporate an Xba I site.
Combinations of these primers were used to PCR amplify the insert using a full-length CFGR-1
clone, which was previously sequenced in both directions (Praveen et al., 2004). The PCR
products were purified, digested with restriction enzymes (Xho I and Xba I) and ligated into the
similarly cut pPICZ-alpha vector. The insert was introduced into this vector downstream of the
alpha factor signal sequence and Kex2 protease cleavage site. The insert’s position facilitates the
production of recombinant proteins with a native N-terminus and a polyhistidine tag at the Cterminus as well as secretion to the medium with minimum contamination from yeast proteins.
As is the case with other yeast expression systems, the vector was grown in E. coli before
110
expression in yeast cells. Thus, the ligation reaction was first used to transform competent
JM109 E. coli cells and positive clones were selected on a low-salt LB plate with 25 µg/ml
Zeocin (Invitrogen, CA). The clones were verified for the presence of insert by restriction
digestion and colony PCR methods. The two chosen clones (pPICZ-alpha-CFGR-1 and pPICZalpha-proCFGR-1) were sequenced in both directions using 5’ and 3’ AOX1 primers (Invitrogen,
CA) to verify the correct reading frame. The recombinant plasmids were linearized by digesting
with Sac I before electroporation into GS115 strain of Pichia pastoris according manufacturer’s
instructions. Positive clones were selected on YPD agar plates containing 100 µg/ml Zeocin.
Positive clones were tested for insert by PCR and expression of recombinant proteins were
assessed by small scale expression trial followed by Western blot analysis of TCA precipitated
supernatants. Clones with high level of expression were selected for scale-up.
In order to obtain enough recombinant protein for purification, the selected clones were
grown in baffled flasks for optimum aeration. Using a single colony, 25 ml of buffered glycerolcomplex medium [BMGY: 1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH =
6.0), 1.34% yeast nitrogen base, 4 x 10-5 % biotin, 1% glycerol] in a 250 ml baffled flask was
incubated at 30o C for 18 hours until the OD600 reached 2-6. Cells were harvested by
centrifugation and pellet was resuspended in buffered methanol-complex medium [BMMY:
BMGY with glycerol replaced with 0.5% methanol] to an OD600 of 1.0 in a 1 liter baffled flask.
The flasks were incubated at 30o C for various time points, till the optimum production was
verified by Western blotting of supernatants collected at different time points. The supernatants
were collected and stored at -80o C until ready for purification.
His-tagged recombinant protein purification was accomplished by concentration of the
supernatants with ammonium sulfate precipitation, dialysis in phosphate buffered saline (PBS,
111
pH = 7.2), followed by metal affinity chromatography using Ni-NTA agarose (Qiagen, CA)
according to manufacturer’s instructions. The positive fractions were pooled, desalted and
concentrated using Centricon YM-3 centrifugal filters (Millipore, MA). Aliquots were stored at 80o C. Protein concentrations were estimated using Biorad Protein Assay Kit (Biorad, CA).
2.2.
Western blotting
Proteins were resolved on 12.5% gel, transferred to nitrocellulose membrane and probed
with INDIA His probe-HRP (Pierce, IL) or anti-His monoclonal antibody (Qiagen, CA). Proteins
were detected with SuperSignal West Pico Chemiluminescent Substrate (Pierce, IL).
2.3.
In vitro enzyme assays
Activity assays for proteases were performed at 37o C in 50 mM Tris-HCl, 0.15 M NaCl,
0.01% Triton X-100 (pH = 7.6) containing 0.2 mM Ellman’s reagent [5,5’-dithiobis(2nitrobenzoic acid), Sigma, MO] in 96-well microtiter plates with a reaction volume of 200 µl.
Various thiobenzyl ester substrates at a concentration of 0.2 mM were used to determine the
enzyme kinetics by measuring increase of absorbance at 405 nm wavelength over time with a
Spectra Max Plus microplate reader (Molecular Devices, CA). Results for controls and samples
are expressed as change in mOD/min. Stock solutions of Z-Lys-SBzl (Calbiochem, CA) were
made in ethanol, while Z-Arg-SBzl, Boc-Ala-Ala-Asp-SBzl and Boc-Ala-Ala-Met-SBzl (MP
Biomedicals, OH) were dissolved in dimethyl sulfoxide (DMSO) and stored at -20o C. Stock
solution of substrates were added to the reaction buffer prior to the assay.
112
2.4.
Granule exocytosis
NCC were prepared from anterior kidney of channel catfish as described before (Praveen
et al., 2004). The target cells (HL-60) were obtained from American Type Culture Collection
(ATCC, VA) and were grown as suspension cultures in RPMI-1640 supplemented with 10%
heat-inactivated FBS. Cells were washed three times in PBS containing 5% BSA and diluted to
1×105 cells/ml. Targets (10,000 cells in 100 µl volume) were delivered into 96-well round
bottom microtiter plates. NCC were added (in 100 µl) at different concentrations to attain several
E:T ratios and the plates were centrifuged (500 x g) to allow the formation of effector-target cells
soft pellets. After incubation for 4 hours, the plates were centrifuged again at 1200 x g and
supernatants were collected for measurement of protease activity.
2.5.
Cell-mediated Cytotoxicity
Target cells (2×106 HL-60 cells/ml) were labeled with 20 µCi of sodium chromate
(Amersham, IL) for 3 h at 37°C. Cells were washed three times in medium and diluted to 1×105
cells/ml. Labeled targets were mixed with effector cells (catfish NCC) to attain different E:T
ratios and the plates were centrifuged at 500 x g. After a 4 hour incubation, 100 µl of each
supernatant was removed to determine radioactivity (Cobra II Autogamma counter; Packard,
CT). The results are expressed as percentage specific release (SR).
Percent SR = (Test Release – Spontaneous Release/ Total Release – Spontaneous release) ×100.
2.6
Identification of cytotoxic granule components in catfish NCC
Comparisons of full-length mammalian perforin sequences with the EST sequences for
channel catfish in NCBI identified three overlapping ESTs (CB939050, CB937333 and
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CB937672) with considerable sequence similarity to N-terminal, central and C-terminal regions
of mammalian perforins. These EST sequences were edited, and joined together to obtain the full
open reading frame for catfish perforin. Primers were designed based on this sequence and used
in nested RT-PCR of cDNA from catfish NCC isolated from anterior kidney. The primers used
were: outer primers CFPERFF1: 5’-cctggctcaagaaggacaagtcct-3’ and CFPERFR1: 5’ccacccagagtcacctttgagatat-3’, nested primers CFPERFF2: 5’-gcgaccaaatcagaagtgcagaac-3’ and
CFPERFR2: 5’-gcgaaaccctgtatctgtagtatcca-3’. Similarly, ESTs with similarity to known
granulysin (CB938093) and serglycin (CK411555) sequences were identified and used to design
primers (CFGRYF1: 5’-gctcgttgcttctttcttcataggc-3’, CFGRYR1: 5’-gcgaattgtgctttaatcatgtcca-3’
and CFSRGF1: 5’-cggataatgtactcggagcctca-3’, CFSRGR1: 5’-gggttacgttccttccggctta-3’) to
amplify those molecules by RT-PCR.
3.
RESULTS
3.1.
Expression of recombinant CFGR-1 in Pichia pastoris
We previously identified the first non-mammalian cDNA sequence from a granzyme
(CFGR-1) from the cytotoxic cells of a teleost fish (Praveen et al., 2004). Comparison of this
serine protease with other granzymes from human and rodents suggested a tryptase activity for
CFGR-1. In order to assess the primary substrate specificity for this protease, we expressed it as
a recombinant protein in Pichia pastoris. Selection of the expression system was based on the
assumption that this protease could be expressed in eukaryotic cells in its native form without the
need for refolding. The coding region of CFGR-1 was amplified and cloned in between Xho I
and Xba I sites of the expression vector pPICZ-alpha. For pro-CFGR-1, two amino acids
upstream of the N-terminus of the mature protein (Ser and Phe) were retained by making the
114
appropriate changes in the forward primer used to amplify the insert. Introduction of these
amplicons at this position resulted in an in-frame fusion between Sacharomyces cervisiae alpha
factor mating signal sequence and pro/mature CFGR-1 (Fig 1a). PCR primers were engineered to
incorporate a Kex2 cleavage site in between the signal sequence and CFGR-1. This facilitated
the processing of the signal sequence by Pichia so that mature/pro CFGR-1 with native Nterminus was released into the culture supernatants. At the C-terminus of the recombinant
protein, a c-myc epitope and poly histidine tag were incorporated to enable efficient purification
and detection of the proteins (Fig. 1a). Previous studies have demonstrated that small C-terminal
tags can be incorporated in proteases without affecting the proper folding and activity (Beresford
et al., 1997, Huang et al., 1997, Huang et al., 1998, Xia et al., 1998). In pPICZ-alpha, the
expression cassette is under the influence of highly-inducible AOX1 promoter, which can drive
the expression of the recombinant protein with methanol induction.
Several clones were selected from YPD-Zeocin plates and expressions of recombinant
proteins were assessed by growing in 10 ml BMGY medium in 50 ml conical tubes. Clones with
highest level of expression were selected for further studies. Selected clones were grown and
induced to secrete the recombinant proteins in 300 ml culture volume in a 1000 ml baffled flasks
and culture supernatants were collected at various time points for TCA precipitation. Later, the
proteins were resolved on a 12.5% SDS-PAGE and transferred to nitrocellulose. Expression
levels of recombinant proteins were assessed by staining for His-probe. A protein of
approximately 33 kDa was found to be upregulated by methanol in both mature and pro CFGR-1
culture supernatants. For mature CFGR-1, significant level of expression was noticed only after
72 hours of induction. In case of pro-CFGR-1, high level of expression was observed as early as
24 hours. Prolonged incubation resulted in degradation of the proteins in both instances.
115
Optimum time of induction for large scale production was selected based on these results (Fig.
1b).
3.2.
Large scale production and purification of recombinant CFGR-1
Ni-NTA agarose beads were mixed with a small volume (20 ml) of the culture
supernatants and His-tagged proteins were purified to assess the success of metal affinity
chromatography. Almost all of the recombinant proteins were eluted in the first three elutions,
detected by Western blotting and protease activity assays (data not shown). Based on these
results, large volumes (1-2 liters) of the culture supernatants were subjected to ammonium
sulfate precipitation to concentrate the proteins. Nickel affinity chromatography was used to
purify the His-tagged proteins (Fig. 2).
3.3.
In vitro proteolytic activity of CFGR-1
Comparison of CFGR-1 amino acid sequences with other mammalian granzymes
suggested the primary specificity determining triplet residues as DGG, similar to that in
granzymes A and K. Therefore, Z-lys-SBzl was selected to check the proteolytic activity of the
purified CFGR-1. As expected from the sequence comparisons and molecular modeling studies
(Praveen et al., 2004), CFGR-1 hydrolyzed the thiobenzyl ester designed to study tryptase-like
proteases. The pro-CFGR-1 had no activity suggesting the retaining the two pro-peptides at the
N-terminus of the protease can affect the proper folding and activity of CFGR-1 (Fig 3a). The
most preferred P1 residues for CFGR-1 was determined to be Arg and Lys, similar to that of
granzyme A and K (Fig 3b). Mature CFGR-1 lacked the Met-ase or Asp-ase activity as
determined by the enzyme kinetics assays using Boc-Ala-Ala-Met-SBzl and Boc-Ala-Ala-Asp-
116
SBzl substrates, respectively (Fig 3b). In all these cases, pro-CFGR-1 did not exhibit proteolytic
activities.
3.4.
Correlation between the NCC cytotoxicity and exocytosis of tryptases
The enzymatic activity of supernatants from NCC and target cells were collected for
protease activity measurement and correlated with Cr release cytotoxicity assays. Catfish NCC
were mixed with target cells (HL-60) at different E:T ratios in duplicate experiments. In one set
of experiments, the targets were labeled with 20 µCi of sodium chromate in RPMI with 10%
fetal bovine serum. In the other set of experiments, nonlabeled target cells were incubated with
NCC in PBS with 10% BSA, in order to minimize the effects of anti-protease activity of serum
on the protease assays. Cytotoxicity was determined by measuring the radioactivity in the
supernatants of the first set of experiments and granule exocytosis was determined by measuring
the hydrolysis of Z-Lys-SBzl by the supernatants collected from the second set of experiments.
There was a strong correlation between the cytotoxicity and residual tryptase activity in the
supernatants (Fig 4). These results suggested the presence of preformed tryptases like CFGR-1 in
the cytotoxic granules of NCC.
3.5.
Granzyme-like protease activities in catfish NCC granules
The presence of other serine proteases with activities similar to mammalian granzyme
was determined by measuring the Asp-ase, Met-ase, tryptase and chymase activities in the
supernatants after incubating NCC with target cells for four hours. The highest enzymatic
activity in the supernatants was chymase, followed by tryptase, while little Asp-ase and Met-ase
117
activities were detected (Fig 5). The possibility of the contribution to enzymatic activity from
target cells was ruled out by measuring the protease activities of total cell lysates of HL-60.
3.6.
Expression of additional granzyme-like proteases in NCC.
Although the proteolytic activities in supernatants from NCC contained at least two
substrate specificities, this alone was not sufficient evidence for the presence of other granzymes
in NCC. Other cDNA sequences containing signature granzyme motifs were searched in the EST
databases. Partial sequences for various proteases expressed in various hematopoietic tissues
were identified. Among them, three sequences had motifs characteristic of all granzymes
identified so far. The comparison of deduced amino acid sequences for these proteins with
CFGR-1 suggested conservation of residues around the catalytic triad and also the N-terminal
region (Fig 6a). However, the triplet residues, which determine the primary substrate specificity
varied considerably from CFGR-1, suggesting deviation from tryptase activity for these
proteases (Fig 6a). The three proteins were arbitrarily named as CFGR-2, CFGR-3 and CFGR-4
for identification. CFGR-3 and 4 are peculiar due to the fact that they have Gly at position 186
(chymotrypsin numbering), which is not seen in any other granzyme sequences. Another teleost
granzyme identified from tilapia (Praveen et al., 2004) also had similar triplet residues and was
shown to have a chymase activity (Praveen et al. Manuscript in preparation). Based on these
findings, at least two of the catfish granzymes identified can be predicted to have chymase
activity.
Expression of these granzyme sequences in catfish NCC was analyzed by RT-PCR.
Catfish NCC express all the four granzymes identified so far (Fig 6b). This might explain the
118
high level of chymase activity observed in the supernatants when NCC were mixed with target
cells.
3.7.
Expression of cytotoxic granule-associated proteins in NCC.
Expression of granule-associated proteins like perforin, granulysin, serglycin etc was
analyzed by RT-PCR. By searching catfish EST database using sequences for similar proteins
from mammalian sources as query, partial sequences spanning various regions of such genes
were obtained. Using that sequence information, primers were designed to amplify portions of
those genes. Intron spanning regions (by comparing the mammalian gene sequences for the
corresponding proteins) were selected for primer design to make sure a different size product is
amplified while cDNA or genomic DNA is used as template. Catfish NCC was shown to express
perforin, granulysin and serglycin cDNAs (Fig 7), indicating the presence of all major granule
components. The identity of the amplicons was verified by sequencing and it was found to
correspond to the catfish homologs of the indicated proteins.
4.
DISCUSSION
The ability to express purified granzymes in recombinant form is crucial in understanding
their individual role in various physiological processes. Purification of catalytically active
mammalian granzyme B, using yeast expression system has been previously reported (Pham et
al., 1998, Sun et al., 1999). Similarly, other expression systems like mammalian cells (Smyth et
al., 1995) and insect cells with recombinant baculovirus (Xia et al., 1998) were successfully used
to generate active granzyme B. Attempts to express granzyme B in E. coli as a fusion protein
with maltose binding protein was not successful because of the inability of the protein to fold
119
correctly and regain solubility (Sun et al., 1999). However, catalytically active recombinant
granzyme A and K could be expressed in E. coli (Beresford et al., 1997, Wilharm et al., 1999a).
The granzymes were first generated as a pro-enzyme and processed with enterokinase or bovine
cathepsin C after refolding of the precursor to regain the activity. Successful expression of
recombinant human granzyme A zymogen in many mammalian cell lines was reported after
infection with recombinant vaccinia virus (Kummer et al., 1996). Expressing the protease as an
inactive zymogen allowed elimination of proteolytic damage to the expression system. However,
the need for further processing of these pro-enzymes necessitated extra steps in the purification
process. Granzyme M also was expressed successfully in mammalian cells using CDM8
mammalian expression vector (Kelly et al., 1996). Other similar expression trials for related
proteases include the use of Pichia pastoris for human mast cell tryptase (Niles et al., 1998)
human mast cell chymase (Lockhart et al., 2005), insect cells for mouse mast cell protease 6 and
7 (Huang et al., 1998, Huang et al., 1997).
Properly engineered amino terminus starting with intact I (I/V)GG motif has been shown
to be essential for the granzymes to adopt an active conformation. Here, expression cassette for
the mature CFGR-1 was engineered by amplifying the portion of the gene coding for amino
acids staring at Ile-25 of the pre-pro enzyme (CFGR with propeptides and signal sequence
intact). Similarly, pro-CFGR-1 was generated by retaining two amino acids at the N-terminus
(Ser-23 and Phe-24). The presence of this pro-peptide was intended for preventing the proper
folding of the enzyme, providing a negative control for the activity assays, as it has been
demonstrated for many other proteases (Pham et al., 1998, Sun et al., 1999). The use of pPICZalpha vector facilitated the secretion of the recombinant proteins in to the supernatants due to the
presence of an alpha mating signal sequence upstream of the gene of interest. Presence of Kex2
120
cleavage site between the signal sequence and CFGR-1 insert allowed the generation of the
recombinant proteins with a desired N-terminal end, as compared to the addition of vectorencoded residues at the N-terminus of recombinant proteins expressed with other vectors (Fig
1a). The supernatants were collected at various time points to check the expression level of the
recombinant proteins by Western blot analysis. Addition of two small tags at the C-terminus
provided means of easy detection either with antibodies against c-myc/poly-histidine (data not
shown) or with INDIA-His probe (Fig 1b).
Once the conditions were optimized, the production of recombinant proteins was scaledup and supernatants were collected for protein purification by nickel affinity chromatography.
(Fig. 2). In vitro protease assays using thiobenzyl esters demonstrated that the mature CFGR-1
was active even in the presence of imidazole used in the elution of the Ni-NTA agarose columns
(data not shown). As it had been demonstrated for other proteases, the presence of two amino
acids at the N-terminus prevented the proper folding of the recombinant CFGR-1, which is
evident from the lack of activity for pro-CFGR-1 (Fig 3a). Comparison of crystal structures of
granzymes and other serine proteases has lead to the theory that three residues of S1 substrate
binding pocket confer primary specificity to these proteases. These residues are located at
position 189, 216 and 226 (chymotrypsin numbering) (Shotton and Hartley, 1970, Perona and
Craik, 1997, Tsu et al., 1997, Wouters et al., 2003). By comparing the CFGR-1 sequence with
other proteases, this granzyme had been previously predicted to have a tryptase activity due to
the S1 specificity pocket residues (DGG) similar to granzyme A and K (Praveen et al., 2004).
Using recombinant CFGR-1 and thiobenzyl ester substrates designed for tryptase, Asp-ase and
Met-ase, the fine specificity of this granzyme towards basic residues (Arg or Lys) at P1 was
identified (Fig 3b).
121
The initial description of catfish NCC was as agranular lymphocytes because of the lack
of large cytotoxic granules in these small cells by Giemsa staining and light microscopy (Evans
et al., 1984). However, their dependence on the presence of calcium for cytotoxic activity was an
indication that these cells might utilize perforin and granzymes to induce cell death (Carlson et
al., 1985). Subsequently, the molecular evidence for the expression of granzyme-like proteases
in these cells indicated that NCC could contain very small granules, difficult to visualize by
normal microscopic techniques (Praveen et al., 2004). CFGR-1 was identified as one of the main
tryptase like granzymes found in catfish NCC. Correlation between the exocytosis of proteases
and the cytotoxicity induced by these cells was determined by measuring target cell death by
chromium release assay along with the tryptase activity of supernatants. The strong correlation
between the cytotoxicity and the tryptase activity suggested CFGR-1 is released on target cells
by NCC (Fig. 4). However, these observations cannot provide the exact contribution of CFGR-1
towards the cytotoxicity.
Conjugate formation between NCC and target cells can be expected to result in release of
effector molecules in to the immunological synapse. Some of it will be taken up by the target cell
and the rest will remain in the supernatants. Measuring the protease activities of the supernatants
using the thibenzyl ester substrates designed for common granzymes will give biochemical
evidence for the types of granzymes exocytosed by the effector cells during conjugate formation.
Catfish NCC had pre-formed granzymes stored inside, which was released towards the target
cells upon conjugate formation. The main activity observed was chymase and tryptase, but very
little Met-ase and Asp-ase activity (Fig. 5). However, this would not guarantee the existence of
granzymes with novel substrate specificities than the existing mammalian granzymes.
122
Molecular evidence for the expression of multiple granzymes in catfish NCC came from
the RT-PCR studies using primers designed based on EST sequences (Fig. 6b). Comparison of
predicted amino acid sequences for these granzymes supported the findings from the granule
exocytosis assay (Fig 6a). Only CFGR-1 had a specificity pocket triplet similar to granzyme A
and K (DGG). All the remaining granzymes do have the triplet similar to known chymases. The
closest similarity of these sequences was with another teleost granzyme identified in tilapia
cytotoxic cells (Praveen et al., 2004). This granzyme has been expressed and its substrate
specificity has been verified (Praveen et al. Manuscript in preparation).
Following a similar strategy, expression of other granule-associated proteins was also
assessed in catfish NCC (Fig 7). Of particular interest was to determine if NCC had
proteoglycans (such as serglycin), known to be specific for cytotoxic cells. Our observations
suggest that cytotoxic cells of teleosts have multiple effector molecules of the granule exocytosis
pathway, similar to mammalian cytotoxic lymphocytes. The localization of these molecules in
cytoplasmic granules can only be verified by immuno-electron microscopic studies using
antibodies specific for fish granular proteins. These reagents are presently not available.
In conclusion, catfish NCC were found to express components of the granule exocytosis
pathway of cell-mediated cytotoxicity. Based on the mammalian serine protease evolution,
CFGR-1, which has tryptase activity, can be predicted to have co-evolved with other granzymes
with more specific substrate specificities. These studies predict a parallel evolution of cellmediated cytotoxicity in ectothermic vertebrates. Future studies are aimed at determining the
physiological role played by individual granzymes in induction of cell death or regulation of
NCC functions.
123
ACKNOWLEDGEMENTS
We acknowledge the help of Dr. Dorothy Hudig and Dr. Dieter Jenne for their valuable
suggestions and providing reagents and expertise. This work was funded with support from
USDA (98-35205-6701).
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126
Figure 4.1. Production of active and pro-CFGR-1 in Pichia pastoris. (a) Schematic
representation of the expression cassettes constructed to express the recombinant proteins using
plasmid pPICZ-alpha. The recombinant proteins were expressed as fusion proteins starting with
the yeast alpha factor at the N-terminus and poly-histidine and c-myc tagged granzyme at the Cterminus. Presence of a Kex2 site in between the alpha factor and the granzyme allowed the
expression of granzyme with a native N-terminus, which can be secreted in to the culture
medium with minimum contaminating yeast proteins. (b) Optimization of recombinant protein
expression. The culture supernatants were collected at the time points indicated and subjected to
TCA precipitation followed by Western blotting. His-tagged proteins were detected with INDIAHis probe conjugated to HRP followed by chemiluminescence. Yeast trsansfected with empty
vector alone (E) also were checked for the expression of proteins (only 72 hour time point is
shown).
127
A
B
128
Figure 4.2. Purification of recombinant proteins. Supernatants were collected after methanol
induction for 72 hours and total proteins were concentrated by ammonium sulfate precipitation.
His-tagged proteins were purified by metal affinity chromatography. Purification success was
verified by Western blotting and detection with INDIA-His probe. Lane V: vector alone, lane F:
flow though from the column, lane E1, E2, and E3: three consecutive elusions of the column
using buffer containing 250 mM imidazole.
129
130
Figure 4.3. Enzymatic activity of recombinant CFGR-1. (a) Plot depicting the change in optical
density vs. time from an in vitro enzyme kinetic assay using Z-lys-SBzl as a substrate. (b)
Substrate specificity of CFGR-1. The enzymatic activity of mature and pro-CFGR-1 was
measured using the indicated thiobenzyl ester substrates in final concentrations of 0.3 mM.
Enzyme concentration was approximately 1 nM for both mature and pro-CFGR-1.
131
A
B
Boc-Ala-Ala-Asp-Sbzl
Boc-Ala-Ala-Met-Sbzl
CFGR-1
Pro-CFGR-1
Z-Arg-SBzl
Empty vector
Z-Lys-SBzl
0
1
2
3
4
Change in mOD/min
132
5
6
Figure 4.4. Correlation between cytotoxicity and exocytosis of granzymes with tryptase
activity in catfish NCC. Radioisotope labeled HL-60 targets were incubated with catfish
NCC at various effector:target ratios for four hours and cytotoxicity was measured by
determining the residual radioactivity in the supernatants. In a parallel experiment, nonlabeled targets were incubated with effector cells and the supernatants were collected to
measure the residual tryptase activity by hydrolysis of Z-Lys-SBzl.
133
7
35
6
30
5
25
4
20
3
15
2
10
5
1
0
0
160
80
40
E:T Ratio
134
20
Change in mOD/min
% Specific Release
40
Cytotoxicity
Protease Activity
Figure 4.5. Expression of other proteases in the cytotoxic granules of catfish NCC. HL-60 target
cells were incubated with NCC for four hours at various E:T ratios and supernatants were
collected to measure the residual protease activity using different thiobenzyl esters to determine
various protease activities associated with known granzymes (Z-Lys-SBzl for tryptase, NSuccinyl-Phe-Leu-Phe-SBzl for chymase, Boc-Ala-Ala-Asp-SBzl for Asp-ase and Boc-Ala-AlaMet-SBzl for Met-ase). The spontaneous release of any granzymes from HL-60 was also
determined for each kind of activity. Also, the target cells were lysed to determine the presence
of any granzyme activity from those cells.
135
Metase
Aspase
HL-60 lysate
HL-60 Only
Tryptase
NCC + HL-60
Chymase
0
10
20
30
Change in mOD/min
136
40
50
Figure 4.6. Identification of multiple granzymes from catfish NCC. (a) Three additional
granzyme-like proteases sequences from channel catfish were identified by searching the EST
database. Multiple sequence alignment of predicted amino acid sequences for those proteases
with CFGR-1 shows conserved motifs, characteristic of known granzymes. The residues at the
relevant positions in the specificity pocket triplet, are represented with small circles above the
alignment. (b) RT-PCR analysis of cDNA from catfish head kidney NCC for the expression of
multiple granzymes. Control reactions were run using genomic DNA as a template with primers
that would result in a product with bigger size. This ensured that the source of the amplicons was
cDNA and not contaminating genomic DNA.
137
A
CGRR-1
CFGR-2
CFGR-3
CFGR-4
:
:
:
:
IIGGREVKK-PKPWMASVQSNNSHICGGTLIHQQWVLTAAHCKTFLQFKP---IE
IIGGNEVDRHSRPYMASVQFKKAHMCGGFLIRKDYVLTAAHCVDNIDHSGKDKLE
IINGKKAKKNSFQYMASVQSKGKHICGGFLISPSYVLTAAHCFQSN-------LS
IVNGSVVTANSRPYMVSVQKDGKHLCGGFLMSENFVMTAAHCWEAG-----VKLT
:
:
:
:
51
55
48
50
CGRR-1
CFGR-2
CFGR-3
CFGR-4
:
:
:
:
VLLGAHSLTKDKNAMR-VKVLCFHISPKFSAT-TRVHDIMLLKLQDKVQLKKNKV
VLLGAHNINQKESQQQRIQVQKYILHPCYERG-ERPNDIMLLKLKSKAKENKF-V
VVLGTQNIDAKRNELRRYAVKSMHIHPSYKENPRYGSDIMLLKFSGKVNLNKD-L
VVVGSHELKKSKSAISHMEVKLYHIHPKFDSE-NLLNDIMLLQLKKTIKKSKN-I
:
:
:
:
104
108
102
103
CGRR-1
CFGR-2
CFGR-3
CFGR-4
:
:
:
:
DVKKIPKSG-KDIPAGTKCEVRGWGTTHVKNPKACDTLQELEVTVVDRELCNCYY
KVIALPKKD-ENLPARQECSIAGWGKTKQ-NSAESSVLREVKLKLENNSQCKKFW
KVIKISSNH-KRVKPNTKCQVAGWGKT-E-TQKTVNDLMVTDVSTIDITVCKKQW
NWISIPKKMNQDVKAKQVCSIAGWGKKSE-NGGTSDRLMEVNVTVIDTKVCEKSW
:
:
:
:
158
161
154
157
CGRR-1
CFGR-2
CFGR-3
CFGR-4
:
:
:
:
NS-KPTITANMLCAGNKQRDKDACWGDSGGPLECKKNIVGVVSGGS--GCGNPKK
Q--NYFDTDNMICTVSD-GKKAFCQGDSGSPLICGNEPQGIAAYTHPHDCLNPTY
NKENVELPAKILCAGGYGTKSGACQGDSGGPLVCSGLAVGIVSFNLHDNCSYPNV
G--TPFSVSSLVCTK---GRGGFCQGDSGGPLVCQNTAVGIVSFTD-ADCKYPKN
:
:
:
:
210
213
209
206
CGRR-1
CFGR-2
CFGR-3
CFGR-4
:
:
:
:
PGVYTLLSKEHIDWINKIIKK--PGVYMKIS-YFLPWIKQVIH---PNVYTEIS-AYADWINKVIKRDAPYVYTKIS-TFSALD---------
:
:
:
:
231
232
231
220
B
138
Figure 4.7. Expression of other granule components in catfish NCC. RT-PCR analysis of cDNA
isolated from catfish NCC using primers designed based on EST sequences similar to
mammalian granule-associated proteins. (a) Expression of perforin in catfish NCC. Lane Lib:
cDNA library, cDNA: fresh cDNA, gDNA: catfish genomic DNA, NTC: no template control (b)
Nested PCR for perforin, using cDNA as a template (c) Expression of granulysin in catfish NCC.
(d) Expression of serglycin in catfish NCC.
139
A
B
C
D
140
CHAPTER 5
MOLECULAR CHARACTERIZATION AND EXPRESSION OF A GRANZYME WITH
CHYMASE ACTIVITY FROM CYTOTOXIC CELLS OF AN ECTOTHERMIC
VERTEBRATE1
1
Praveen, K., J. H. Leary, D. L. Evans and L. Jaso-Friedmann. To be submitted to Journal of
Immunology after revisions
141
ABSTRACT
We have identified the gene coding for a novel serine protease with close similarities to
mammalian granzymes from nonspecific cytotoxic cells of a teleost fish Oreochromis niloticus.
The genomic organization of tilapia granzyme-1 (TLGR-1) has the signature five exon-four
intron structure shared by all granzymes. Several sequence motifs are shared among the teleost
and mammalian granzymes, including the N-terminal tetrapeptides, catalytic triad residues
constituting the charge relay system and residues surrounding the triad. Molecular modeling
studies suggested a granzyme-like structure for this protein with four disulfide linkages and two
additional Cys residues. The expression of this gene is found to be restricted to cytotoxic cell
populations with a low level of constitutive expression when compared to similar granzymes in
other teleost species. High levels of transcriptional activation of TLGR-1 with different stimuli
suggested that this gene is highly induced during immune reactions. Triplet residues around the
active site Ser of TLGR, which determine the primary substrate specificity of granzymes, differ
significantly from that of other granzymes. Recombinant TLGR-1 was expressed in mature or
pro-enzyme forms using pPICZ-alpha vector in Pichia pastoris expression system. Recombinant
TLGR-1 was used to determine the primary substrate specificity of this protease using various
synthetic thiobenzyl ester substrates. In vitro enzyme kinetics assays suggested a preference for
residues with bulky side chains at the P1 site, indicating a chymase-like activity for this protease.
These results indicate the presence of novel granzymes in cytotoxic cells from ectothermic
vertebrates.
Key words: Granzyme, chymase, thiobenzyl ester, nonspecific cytotoxic cells, lymphocytes,
serine proteases, cell-mediated cytotoxicity
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INTRODUCTION
Granzymes belong to a family of serine proteases, which constitute the major
components of granules of professional killer cells such as cytotoxic T lymphocytes (CTLs) and
natural killer cells (NK cells). Along with perforin, a pore forming protein that is also found in
these granules, granzymes play a major role in inducing target cell death (1-3). All the
mammalian granzymes described so far are highly conserved and are located in three clusters on
three chromosomes (4). Granzymes A and B are the most extensively studied granzymes. The
different pathways used by granzymes A and B to initiate cytotoxicity have been described (5,6).
However, novel mechanisms of cytotoxicity are being discovered for these granzymes,
expanding our knowledge about complex pathways involved in cell death and proliferation (712). The function of many of the other granzymes is not well understood, thus they are
collectively called as orphan granzymes (4).
Non-redundant pathways of cell-mediated cytotoxicity have been proposed for
granzymes, suggesting a ‘fail-safe’ mechanism provided by multiple granzymes (2,4). The role
of granzymes in inducing target cell death has been investigated extensively in vitro (13).
However, advances in in vivo studies demonstrating the significance of individual granzymes in
cytotoxicity lag behind. Targeted gene disruption studies of granzymes have resulted in focal
immune deficiencies (13-16). Recent attempts to understand the individual effects of members of
the granzyme B gene cluster has revealed the significant role played by orphan granzymes
present downstream of the granzyme B gene in mice (17). Such results indicate the relative
significance of other granzymes that have not been fully investigated.
Granzymes are classified based on their primary substrate specificities and four
enzymatic activities have been described: tryptase , Asp-ase, Met-ase and chymase. The most
143
extensively studied mammalian granzymes fall under three of the above specificities. Granzyme
A and K have tryptase activity, granzyme B has Asp-ase activity, while granzyme M has Met-ase
activity (18). Many of the orphan granzymes have chymase activity.
The presence of multiple granzymes can be interpreted as a result of molecular strategies
developed by the immune system to compete with pathogens. These pathogens have coexisted
and coevolved with immune systems of organisms for millions of years (13). Genetic
comparisons and chromosomal clustering of granzymes suggest gene duplication events as a
means of generating newer granzymes (4). Almost all the assumptions on granzyme evolution
are based on the gene sequence information from human and rodents, due to the lack of sequence
information in other organisms. The availability of non-mammalian granzyme sequences would
add new insights into the evolution of these proteases with very narrow substrate specificity and
it would also aid in the discovery of newer killing pathways (19,20).
Nonspecific cytotoxic cells (NCC) are the first identified cytotoxic cell population in
teleosts. First described in the channel catfish (21), these cells have been well characterized in a
number of lower vertebrates (22-25). Although first described as agranular (21), NCC in trout,
tilapia and catfish do have small granules and contact with target cells leads to granule
exocytosis with necrotic and apoptotic death pathways (26-28). The requirement for calcium for
the induction of cytotoxicity by NCC was an early indication that these cells use the granule
exocytosis killing pathway (29). We have previously reported the existence of granzyme-like
serine proteases in NCC of channel catfish (Ictalurus punctatus) and tilapia (Oreochromis
niloticus) (20). Here, evidence is presented of the full-length sequence of the cDNA and gene for
TLGR-1, its expression pattern and the substrate specificity of the recombinant protein. These
144
results demonstrate for the first time, the molecular characterization of granzymes from a
cytotoxic cell of non-mammalian vertebrates with novel substrate specificities.
MATERIALS AND METHODS
Cloning of full-length TLGR-1
The identification and partial sequence of a granzyme-like serine protease from a tilapia
NCC cDNA library has been previously reported (20). Full-length sequence of that tilapia
granzyme (TLGR-1) was obtained by rapid amplification of cDNA ends (RACE). Fresh RNA
was purified from tilapia NCC from peripheral blood for the RACE, as previously described
(20). Briefly, mRNA (500 ng) was reverse transcribed with granzyme specific primers using
Generacer Superscript II RT module (Invitrogen, CA). First strand cDNA was subjected to
homopolymeric tailing with cytosine using terminal transferase (Roche). Later, dC-tailed cDNA
was amplified using abridged anchor primers and gene specific nested primers followed by a reamplification using a nested abridged universal amplification primer (AUAP) and another nested
gene-specific primer. The amplicons were purified and TA cloned for sequencing. For 3'RACE,
RNA was reverse transcribed using an anchor primer, EPB-18T (30) to generate first strand
cDNA. Using gene specific nested primers and EPB, the 3'end of the mRNA was amplified and
TA cloned for sequencing. The sequences were edited and assembled to obtain the complete
sequence of TLGR-1. Forward and reverse primers were designed at the two ends to amplify the
full-length cDNA, which was TA cloned and sequenced in both directions using standard
protocols.
145
TLGR-1 gene and promoter sequences
The complete genomic sequence of TLGR-1 gene and its promoter elements were
obtained by sequencing a cosmid clone containing the TLGR-1 gene. The tilapia genomic DNA
library was constructed in SuperCos-1, as reported for channel catfish (31). A directed PCRbased iterative screening protocol was used to identify clones with TLGR-1 gene (32). Specific
PCR primers (TLGRCOSF1 5’-gcacccacgatatgacaaagttga-3’ and TLGRCOSR1 5’ggcctttcaattttctcttacagaca-3’) were designed based on the cDNA sequence. After several rounds
of screening, several TLGR-1 positive clones were isolated, expanded and frozen in glycerol
stocks for further analysis. The complete TLGR-1 gene sequence was obtained by sequencing
the cosmid clone in a 373 A DNA sequencer (Applied Biosystems, CA) at the Integrated Biotech
Laboratories (University of Georgia, Athens). The upstream region of the TLGR-1 gene
(promoter region: 1200 bp) was directly sequenced from the cosmid. Primers were synthesized
as needed for “primer walking" (Genosys, TX). The TRANSFAC database (33) and
MatInspector web based software (34) were used to locate transcription factor binding sites.
Phylogenetic analysis and molecular modeling
Phylogenetic analysis and molecular modeling of TLGR-1 were done as described before
(20). Briefly, all the known granzymes and similar serine proteases were aligned and
phylogenetic analysis was done with Mega version 2.1 (35). Analysis using different methods
resulted in similar trees and reliability of the trees was tested using 1000 bootstrap replications.
The tree was rooted on a subtree consisting of adipsin-like proteases from various sources.
Molecular modeling of TLGR-1 was done using SWISS-MODEL in the first approach mode
146
accessible via the internet (http://www.expasy.org/swissmod). RasWin version 2.6 was used to
visualize the coordinate data.
Real-time PCR analysis
After exposing NCC activation treatments, total RNA was isolated using RNeasy mini-kit
(Qiagen, CA) following the manufacturer’s protocol, quantified spectrophotometrically and
treated with RNAse free DNAse (Promega, WI). The first strand cDNA was synthesized from
3 µg of total RNA using First Strand cDNA synthesis kit (Invitrogen, CA). A cDNA stock for
constructing a relative standard curve was synthesized using the same method. Primers were
designed based on available gene sequences to amplify ~150 bp amplicons for TLGR-1 and betaactin. Forward and reverse primers were selected at positions to include an intron so that
genomic DNA contamination in the samples could be easily detected. A relative standard curve
was constructed for target gene and house-keeping gene ( -actin) using either cDNA stock or full
length cDNA clone for TLGR-1 and -actin. Briefly, reactions were set-up with different
concentrations ranging from 10 to 3000 pg per reaction for the standard curve. The reactions
were carried out using Brilliant® SYBR Green QPCR master mix (Stratagene, CA) according to
manufacturer’s instructions. The efficiencies of the primer pairs were determined and appropriate
adjustments were made to optimize reaction conditions. Diluted cDNA samples were used as a
template for every 25 µl reaction. Each sample was set-up in triplicates for the PCR. The
following PCR protocol was used for gene amplification: 95 °C, 3 min; 40 cycles: denature
95 °C, 30 s; anneal 60 °C, 30 s; extend 72 °C, 30 s, followed by 4 °C hold using Mx3000P realtime PCR system (Stratagene, CA).
147
Production of mature and pro-TLGR-1 in Pichia pastoris
The cDNAs coding for pro and mature forms of TLGR-1 were PCR amplified and cloned
in to pPICZ-alpha vector for expression in GS115 strain of Pichia pastoris (Invitrogen, CA).
Forward primers TLGRYSTF1 (5’-aactgtcgacaaaagaatcataaatggca-3’) and TLGRYSTF2 (5’aactgtcgacaaaagaagtgaaatcataaatggca-3’) were designed to engineer an Xho I site upstream of
mature and pro TLGR-1 respectively. The coding region of TLGR-1 contained a recognition site
for Xba I. Therefore, in order to clone the insert between Xho I and Xba I sites of pPICZ-alpha, a
reverse primer TLGRYSTR1 (5’-ggggttctagatggcattgctttttattgagaatgt-3’) was designed to
incorporate a Sal I site. Digestion of DNA with Sal I has been shown to generate cohesive ends
compatible with that made with Xba I digestion. Combination of these primers were used to PCR
amplify the insert using a full-length TLGR-1 clone, which was previously sequenced in both
directions. The PCR products were purified and digested with a combination of restriction
enzymes (Xho I and Sal I) and ligated in to the pPICZ-alpha vector digested with Xho I and Xba
I. The insert was introduced into this vector downstream of alpha factor signal sequence and
Kex2 protease cleavage site. This position facilitated the production of recombinant proteins
with native N-terminus and a polyhistidine tag at the C-terminus, which would be secreted to the
medium with minimum contamination from yeast proteins. The ligation reaction was used to
initially transform competent JM109 E. coli cells and positive clones were selected on a low-salt
LB plate with 25 µg/ml Zeocin (Invitrogen, CA). The clones were verified for the presence of
insert by restriction digestion and colony PCR methods. Recombinant plasmids (pPICZ-alphaTLGR-1 and pPICZ-alpha-proTLGR-1) were sequenced in both directions using 5’ and 3’
AOX1 primers (Invitrogen, CA) to verify the correct reading frame. Subsequently, the
recombinant plasmids were linearized by digesting with Sac I before electroporating in to GS115
148
strain of Pichia pastoris according manufacturer’s instructions. Positive clones were selected on
YPD agar plates containing 100 µg/ml Zeocin. Positive clones were tested for insert by PCR.
Expression of recombinant proteins was assessed by a small scale expression trial followed by
Western blot analysis of the TCA precipitated supernatants. Clones with high level of expression
were selected to scale-up production.
In order to obtain enough recombinant protein for purification, the selected clones were
grown in baffled flasks for optimum aeration. Using a single colony, 25 ml of buffered glycerolcomplex medium [BMGY: 1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH =
6.0), 1.34% yeast nitrogen base, 4 x 10-5 % biotin, 1% glycerol] in a 250 ml baffled flask was
incubated at 30o C for 18 hours until the OD600 = 2-6. Cells were harvested by centrifugation and
the pellet was resuspended in buffered methanol-complex medium [BMMY: BMGY with
glycerol replaced with 0.5% methanol] to an OD600 of 1.0 in a 1 liter baffled flask. The flasks
were incubated at 30o C for various time points, till the optimum production was verified by
Western blotting of supernatants collected at different time points. The supernatants were
collected and stored at -80o C until ready for purification.
For purification of the His-tagged recombinant proteins, the supernatants were
concentrated by ammonium sulfate precipitation followed by dialysis in phosphate buffered
saline (PBS, pH = 7.2). The recombinant proteins were purified by metal affinity
chromatography using Ni-NTA agarose (Qiagen, CA) according to manufacturer’s instructions.
The positive fractions were pooled, desalted and concentrated using Centricon YM-3 centrifugal
filters (Millipore, MA). Aliquots were stored at -80o C. Protein concentrations were estimated
using Biorad Protein Assay Kit (Biorad, CA).
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Western blot analysis
Proteins were resolved on 12.5% gel, transferred to nitrocellulose membrane and probed
with INDIA His probe-HRP (Pierce, IL) or anti-His monoclonal antibody (Qiagen, CA). Proteins
were detected with SuperSignal West Pico Chemiluminescent Substrate (Pierce, IL).
In vitro enzyme assays
Activity assays for proteases were performed at 37o C in 50 mM Tris-HCl, 0.15 M NaCl,
0.01% Triton X-100 (pH = 7.6) containing 0.2 mM Ellman’s reagent [5,5’-dithiobis(2nitrobenzoic acid), Sigma, MO] in 96-well microtiter plates with a reaction volume of 200 µl.
Various thiobenzyl ester substrates at a concentration of 0.2 mM were used to determine the
enzyme kinetics by measuring increase in absorbance at 405 nm wavelength over time with a
Spectra Max Plus microplate reader (Molecular Devices, CA). Results for controls and samples
are expressed as change in mOD/min. Stock solutions of Z-Lys-SBzl (Calbiochem, CA) were
made in ethanol, while Z-Arg-SBzl, Boc-Ala-Ala-Asp-SBzl, Boc-Ala-Ala-Met-SBzl (MP
Biomedicals, OH), N-Succinyl-Phe-Leu-Phe-SBzl and N-Succinyl-Ala-Ala-Pro-Phe-pNA
(Sigma, MO) were dissolved dimethyl sulfoxide (DMSO) and stored at -20o C and added to the
reaction buffer prior to the assay.
RESULTS
Identification and analysis of full-length cDNA and gene for TLGR-1
For identification of genes involved in cytotoxic functions of NCC, a cDNA library was
constructed using activated cytotoxic cells. The identification of a partial sequence of a serine
protease with novel predicted substrate specificity from tilapia NCC library has been reported
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(20). Later, full-length cDNA for that gene was identified and sequenced. Primers were designed
based on this sequence to screen a tilapia genomic library and a cosmid clone containing the
gene and promoter elements was identified (Fig. 1). Surprisingly, the TLGR-1 gene had an
approximate size of 1.32 kb (starting from the 5’ UTR and ending at the polyadenylation signal),
which is much smaller than mammalian granzyme genes. However, comparison of the cDNA
and genomic sequences of TLGR-1 gene, revealed a five exon and four intron organization,
shared among many members of the granzyme family (Table 1). The teleost granzyme gene has
shorter introns than the mammalian granzyme genes. The promoter region of TLGR-1 has
transcription factor binding sites, which are similar to other granzymes. All of the intron/exon
boundaries of TLGR-1 gene fulfill the GT-AG rule (36).
The proximal promoter region of TLGR-1 was analyzed for the putative transcription
factor binding sites. Similar to other granzyme gene promoters, TLGR-1 has many binding sites
for transcription factors specific for the immune system (Fig. 1). There are two AP-1 and NFAT
binding sites along with sites for binding of interferon response factors and STATs. This would
appear to suggest that there may be a significant correlation between the transcriptional
upregulation of TLGR-1 and activated immune response reactions.
TLGR-1 cDNA sequence was initially obtained by RT-PCR using specific primers at the
5’ and 3’ end of the gene and the sequence was verified by comparing it with the genomic
sequence. The cDNA and genomic sequences were submitted to GenBank (accession numbers
AY918866 and AY918867, respectively). This cDNA has a total length of 999 bp with an open
reading frame of 765 bp that encodes a putative protein with 254 amino acids (Fig. 1). Using
algorithms to predict signal sequences, a stretch of 23 residues at the N-terminal end was
identified as a signal sequence, indicating that this protein is sorted to the secretory pathway.
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Signal sequence cleavage sites were predicted using weight matrix analysis of von Heijne (37).
TLGR-1 is predicted to have a dipeptide (Ser-Glu) as pro-peptide, much similar to the majority
of mammalian granzymes. There is a single putative glycosylation site (NGTA) located towards
the C-terminus of the protein (Fig. 1). The predicted molecular mass of the non-glycosylated
mature TLGR-1 was calculated as 25,339 daltons (229 amino acids) with an isoelectric point of
9.57. Other granzymes also have a highly basic charge.
Disulfide linkages are crucial for the the proper folding of fully active granzymes.
Molecular modeling revealed that the overall structure of tilapia granzyme had a close similarity
to available crystal structures of mammalian granzymes. TLGR-1 can be predicted to have four
disulfide linkages (Fig. 2), similar to what was shown for catfish granzyme (20). Formation of
disulfide bonds between Cys residues can be predicted as follows: 42 with 58; 136 with 168; 182
with 201. Cys191 and Cys220 (chymotrypsinogen numbering) could be analogous to a fourth
disulfide linkage which bridges active site serine, like in chymotrypsin. Although the tilapia
granzyme has additional Cys residues (Fig. 2), these are not present at corresponding positions to
that of granzyme A, which is crucial in the dimerization (19).
Multiple sequence comparisons of TLGR-1 with related granzyme sequences enabled the
identification of signature motifs shared by members of the granzyme family. While the Nterminal IIXG motif is conserved in TLGR-1, the PHSRPYMA motif is not entirely conserved.
However, the residues in corresponding position share close similarity to those in the mammalian
sequences. The three key amino acid residues representing the catalytic triad (charge relay
system) of serine proteases (His57, Asp102 and Ser 195: chymotrypsinogen numbering system)
as well as their neighboring residues are well conserved in TLGR-1 (Fig. 3). The combination of
amino acid residues, which are crucial in the formation of the substrate specificity pocket, can be
152
used as a prediction tool for primary specificity for a protease (38). Based on comparison of the
residues in TLGR-1, alignment with other granzyme sequences and manually editing the
alignment based on crystallography structure profile (39), a novel substrate specificity pocket
triplet (GNN) can be predicted for this teleost granzyme. None of the other granzymes described
to date share a specificity pocket triplet with TLGR-1. TLGR-1 has overall sequence similarity
with almost all of the mammalian granzymes, with highest sequence similarity to granzyme B
and G sequences (Table 2).
Phylogenetic analysis of TLGR-1
TLGR-1 sequence was compared with known serine proteases and the data was analyzed
by different methods to obtain clues on evolutionary position of this granzyme. Phylogenetic
analysis using different methods yielded trees with similar topology and a representative tree is
depicted in Fig 4. In order to resolve the difference between closely related granzyme sequences,
the tree was rooted on a subtree comprising of complement factor D, which has been shown to be
evolutionarily distinct from granzymes while sharing many sequence features. TLGR-1 didn’t
cluster with any of the mammalian granzymes. However, it grouped with other teleost
granzymes identified by EST database searches. Comparison of substrate specificity pocket
triplet residues in the teleost sequences revealed a close similarity to TLGR-1 suggesting that
they may share similar primary specificities.
Expression pattern of TLGR-1
Expression of TLGR-1 gene in various tissues was assessed by RT-PCR analysis using
specific primers. Purified NCC were shown to be a major tissue for expression of this gene (Fig.
153
5). Tissues rich in NCC were the only places where the expression could be detected. Compared
to the granzymes from catfish NCC, constitutive expression of TLGR-1 was found to be very
low, based on the intensity of the amplified bands.
Transcriptional regulation of TLGR-1
Inducibility of the TLGR-1 message in tilapia NCC was assessed by gene quantification
with real-time RT-PCR. NCC were treated with various stimuli and cDNA were prepared for
analysis. Recombinant tilapia TNF was found to have a profound effect on the transcription of
TLGR-1 (Fig. 6a). Within two hours of treatment with 100 ng/ml recombinant tilapia TNF, there
was a 29 fold increase in TLGR-1 expression. The expression was reduced after two hours, but
remained higher than base levels even after four hours. Similarly, treatment of NCC with
PMA/calcium ionophores resulted in more than two fold increase in TLGR-1 expression (Fig
6b). More than six fold increase in expression could be observed by treating the cells with LPS
for four hours (Fig 6c).
Expression of recombinant TLGR-1
In order to express the pro and mature forms of TLGR-1, expression cassettes were
engineered so that it could be inserted between Xho I and Xba I sites of yeast expression vector,
pPICZ-alpha. Recombinant vectors were introduced in to GS115 strain of Pichia pastoris so that
the recombinant proteins were expressed as a fusion protein with an N-terminal alpha signal
sequence and C-terminal granzyme. By inserting a Kex2 recognition sequence in between the
signal sequence and granzyme, active (cleaved) TLGR-1 could be secreted in to the culture
supernatant. Incorporation of small C-terminal tags without affecting the protease activity has
154
been demonstrated for many proteases before (40-43). Both pro and mature recombinant TLGR1 had a His-tag followed by a c-myc tag at the C-terminus (Fig. 7a). Expression of pro-TLGR-1
was achieved by retaining the activation dipeptide at the N-terminus, which is known to yield
inactive enzyme due to improper protein folding.
Several expression clones were identified by plating the transformed cells on YPD plates
with Zeocin. Small scale expression trials were done to assess the level of expression of selected
clones upon induction with methanol (data not shown). Clones with highest expression levels
were selected for further studies. Single yeast colony was grown and induced to secrete the
recombinant proteins in 300 ml culture volume in a 1000 ml baffled flasks and culture
supernatants were collected at various time points for TCA precipitation. The proteins were
resolved on a 12.5% SDS-PAGE and transferred to nitrocellulose. Expression levels of
recombinant proteins were assessed by staining with His-probe. A protein of approximately 33
kDa was found to be upregulated in both mature and pro TLGR-1 culture supernatants (Fig. 7b).
For mature TLGR-1 high level of expression of recombinant proteins were observed from 24
hours lasting till 72 hours. From that point time on, there was significant degradation of the
proteins. Similarly, expression levels were high for pro-TLGR-1 from 48 hours and remained
high for 96 hours (Fig. 7b).
High level expression and purification of recombinant TLGR-1
In order to assess the success of metal affinity chromatography, Ni-NTA agarose beads
were mixed with a small volume (20 ml) of the culture supernatants and His-tagged proteins
were purified eluting the bound proteins in a buffer containing 250 mM imidazole. Almost all of
the recombinant proteins were eluted in the first elutions, detected by Western blotting and
155
protease activity assays (data not shown). Later, large volumes (1-2 liters) of the culture
supernatants were subjected to ammonium sulfate precipitation to concentrate the proteins. This
was followed by nickel affinity chromatography to purify His-tagged proteins (Fig. 8).
In vitro proteolytic activity of recombinant TLGR-1
Due to the novel specificity pocket triplet residues in TLGR-1, it was expected not to
hydrolyze thiobenzyl esters specially designed for granzymes with tryptase, Met-ase or Asp-ase
activity. The hydrolysis of synthetic substrates used to study proteases with chymase specificity
was tried to analyze the activity of TLGR-1. Effective hydrolysis of N-Succinyl-Phe-Leu-PheSBzl (Fig 9a) and N-Succinyl-Ala-Ala-Pro-Phe-pNA (Fig 9b) by TLGR-1 suggested its strong
preference for bulky residues at P1 site. Later, a comparison of hydrolysis of various thiobenzyl
ester substrates revealed in fact, that TLGR-1 had chymase activity TLGR-1 (Fig 10). The proTLGR-1 failed to cleave any of the substrates, indicating that the presence of pro-peptide at the
N-terminus prevented proper folding of the protease which is crucial for its activity.
DISCUSSION
We have previously reported the expression of granzyme-like serine proteases in NCC
purified from peripheral blood of tilapia (20). The full-length cDNA coding for TLGR-1 was
identified and sequenced in two directions to obtain the putative amino acid sequence of the
protein and to identify sequence motifs shared with other granzymes. Later, a genomic library
was screened using specific primers to obtain a cosmid clone containing full-length TLGR-1
gene. We sequenced genomic DNA comprising the full gene and the promoter region (Fig. 1).
Compared to other granzyme sequences, TLGR-1 gene had a smaller size. However, the intron-
156
exon structure of the gene had closest similarity to other mammalian granzymes (1,44).
Additionally, the number and position of introns of TLGR-1 suggests that this could be a
member of the sixth class of serine proteases (45), like NK cell specific granzyme M (44,46).
The first one of the substrate specificity triplet residues (Gly 173; -6 with reference to active site
Ser) is encoded within the fourth exon (Fig 1 and Table 1) as in the case of other chymotrypsinlike proteases (47).
Studies on proximal promoter elements of mammalian granzymes had revealed the
conserved sequences enabling cell-specific expression of these proteases. Granzyme B promoter
has binding sites for T cell specific transcription factors such as Ikaros and core-binding factor
as well as for more ubiquitous transcription factors like AP-1 (48-50). These sequences are found
to be sufficient to drive reporter gene expression in T cell lines where these factors are
constitutively active (51,52). Promoter region of granzyme M gene has been shown to have
binding sites for factors like AP-2, AP-3 and glucagons-G3A, influencing its bias for expression
in NK cells (44,53). The presence of multiple transcription factors in the promoter region of the
TLGR-1 gene in NCC is an indirect indication that this gene is highly regulated during the
immune response.
Highly conserved Cys residues at similar positions in all granzyme sequences give rise to
three pairs of disulfide bonds, which are crucial in proper folding and activation of granzyme
during processing. Among these disulfide bonds, the ones between Cys26 and Cys42 as well as
between Cys151 and Cys167 link the primary and secondary substrate-binding sites, forming S1
subsites in granzymes A, K and M (54). A fourth pair of Cys residues form an additional
disulfide bond, which bridges the active site Ser residue is found in granzymes M and K (44,55).
Granzyme A is the only mammalian granzyme shown to have one additional Cys residue, which
157
is responsible for intermolecular disulfide bonds (19,56). TLGR-1 was shown to have two
additional Cys residues (Fig 2). At this time their function is unknown as their positions do not
match with those corresponding to the dimer-forming Cys residue of granzyme A (19).
Multiple sequence alignment of TLGR-1 with other granzymes revealed highly
conserved regions shared by all known granzymes (Fig. 3). The N-terminal IIXG motif and
catalytic triad residues that make up the charge relay system were highly conserved. Moreover,
residues around the triad residues were highly conserved among all the sequences. The presence
of Gly residue at position -6 upstream of the active site Ser indicated a substrate specificity that
was different from that of granzymes A/K, B and M. Other residues at position +15 to +17 and
+28 relative to the active site Ser are found to be important in determining the primary substrate
specificity of Ser protease (57). TLGR-1 has Ser-Phe-Asn (+15 to +17) and Asn (+28) at these
positions, which is quite different from all other known granzymes. A comparison of crystal
structures of Ser proteases has led to a theory that three residues of the S1 substrate binding
pocket confer primary specificity. These specificity-conferring residues are located at positions
189, 216, and 226 (chymotrypsin numbering) (38). Alignment of the TLGR-1 sequence with
other granzymes and matching the secondary structure elements, the substrate specificity pocket
residues for this protease were determined as Gly-Asn-Asn. These triplet amino acid residues
have not been previously described in mammalian granzymes.
Multiple sequence comparisons alone are not sufficient to draw conclusions on
evolutionary positions of closely related proteins like granzymes. Phylogenetic analysis of
mammalian granzymes has revealed the clustering of granzymes with similar substrate
specificity, giving rise to the classification of granzymes in to three major groups (4,38). Similar
analysis of teleost granzymes along with mammalian serine protease placed TLGR-1 in to a
158
separate cluster, indicating a parallel evolution for teleost granzymes. Catfish granzymes with
similar predicted substrate specificity were the other members of that cluster. The experimental
verification of the enzymatic activity for the catfish granzymes would be crucial to make the
correlation between the substrate specificity and sequence similarity.
The pattern of tissue expression of TLGR-1 suggests that this protease is associated with
cytotoxic cell populations (Fig. 5). Tissues with appreciable TLGR-1 expression were the ones
with high NCC density. Use of cDNA derived from total tissues for expression studies could be
misleading due to the interference from different cell populations that might be present in these
tissues. The cell line (TMB-8), which was derived from heart tissue, was used to assess the
specificity of expression with more confidence. TMB-8 cells have been used as nonhematopoietic and non-cytotoxic controls in previous experiments (58). Compared to the CFGR1, which had a high level of constitutive expression in NCC from different tissues (20), TLGR-1
expression levels were very low in resting NCC (Fig. 5). Transcriptional upregulation of TLGR1 with various stimuli indicated the high degree of inducibility for this tilapia granzyme (Fig. 6).
Transcriptional activation of granzyme A in response to TNF has been previously reported in
lymphokine-activated killer cells and CTLs (59,60).
Purification of catalytically active recombinant granzymes is critical in understanding
their individual physiological roles, compared to combined effects of multiple granzymes in
purified cytotoxic granules. Generation of mammalian granzymes in recombinant forms have
been reported (40,43,61-65). Use of Pichia pastoris expression system with pPICZ-alpha
expression vector allowed the generation of recombinant granzymes with desired N-terminal
end. By inserting the tilapia granzyme expression sequence downstream of an alpha mating
signal sequence, secretion of the protein to the expression medium could be achieved. The pro-
159
TLGR-1 was generated by retaining two residues at the N-terminus of the mature protease (Fig
7a). This has been shown to prevent the proper folding and activation of mammalian
recombinant granzymes (62,65,66). The supernatants were collected at various time points and
expression levels were determined by Western blotting (Fig. 7b)
Purification of recombinant TLGR-1 from culture supernatants was carried out after
concentrating the proteins by ammonium sulfate precipitation. This method had been previously
used without affecting the proteolytic activities of human mast cell chymase (67). After the
ammonium sulfate precipitation, the pellet was dissolved in PBS and concentrated to 50 ml
followed by dialyzing against PBS to remove traces of ammonium sulfate. His-tagged proteins
were purified by nickel affinity chromatography (Fig. 8). The Western blot analysis of TLGR-1
suggests that the protein might be glycosylated. The predicated molecular mass of mature
TLGR-1 is 25.3 kDa, while the protein appeared to have an approximate mass of 33 kDa,
suggesting the presence of sugar residues in the predicted glycosylation site on the polypeptide
chain. Glycosylation of granzymes is critical because they have been shown to be targeted to the
lytic granules by the mannose-6-phosphate receptor (68).
Granzymes are characterized by their very narrow substrate specificity. The requirement
for P1 residue varies significantly between different classes of mammalian granzymes (18). In
most cases the substrate specificity of individual granzymes can be predicted based on the
architecture of its substrate binding pocket (38). Such prediction algorithms suggested that
TLGR-1 had a preference for bulkier P1 residues than that for tryptases, Asp-ases or Met-ases.
Using synthetic substrates designed with a bulky Phe residue at P1 site, the activity of TLGR-1
was shown to be similar to that of mammalian chymases (Fig. 9 & 10).
160
In conclusion, the novel serine protease identified from tilapia NCC was shown to have
chymase activity. It is not clear whether these cells do express other granzymes and other
components of granule exocytosis pathway. The TLGR-1 gene is highly inducible with various
stimuli. Compared to the expression pattern of similar granzymes in channel catfish NCC, tilapia
granzymes require longer activation for the transcription and translation of cytotoxic molecules,
as determined by the protease activity of supernatants from granule exocytosis assay (data not
shown). Studies are underway to uncover the physiological roles of this protease in these
cytotoxic cells and also to identify other granule components in tilapia NCC.
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48. Haddad, P., A. Wargnier, J. F. Bourge, M. Sasportes, and P. Paul. 1993. A promoter
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166
Figure 5.1. Genomic organization of TLGR-1 gene and promoter region. Exon sequences are
represented in upper case letters, while introns and promoter region are represented as lower case
letters. Putative transcription factor binding sites in the promoter region are represented as
underlined bold letters with the name of the factor above the sequence. The predicted signal
sequence at the N-terminus is highlighted while the dipeptide is represented with double
underline. A putative glycosylation site is boxed and the polyadenylation signal is represented by
underlined bold letters in the 3’ UTR. Triad residues constituting the active site are represented
as bold letters in the sequence of amino acid chain.
167
1
gtctgtttttttaaacatccttcactgccaccttaaattcatttgatgtg
51
gctacatatttggtggtatgcatgaagagagaacacaagagagaaccgtc
101
aatagcaaaacctctacatgcccaccccctttgacagcagaagttcttat
151
ttccacagcttcacttatcagaagacaccaatcagattgtgatctgatta
201
tgatcatgctgagtcattttgtgtggttgtgtttgttcagggcttcattt
AP-1
NFAT
BRIGHT
251 taaatgctgctctcaccttttgtcatgtttccagttacacacttattaat
301 ttagcttttgtttcccatgttttgtccaaaaagcacacaaaagaaaaaag
351
caaaaggacattgccgattttttcttttttacattgctttaaaagacaaa
401
tgagcttattgaacatgtagatagttacactgattttaaaaaatccctat
GATA-3
TATA-Box
451 aaacataaaaaatgtccaaatgtacatattaataacagactggttaagaa
501
cgtttgactgcaattcttttgtaattattttggtaaatgctactttcatg
551
catcatttattactgtattccacagtattccaacttccttatgtgttctt
c-ETS-1
GATA-1
IRF-7
601 attatctgaaaattatttgcaaatacccgacttcattttcacatttcata
IRF-2
651 aacaagcaaagtcttcgagccaacagccaataagatcactgcctcattcc
701 acttttcaacatgcagattacttactgaaataaataagcattgctgtttg
751
AP-1
tttatattgttgactgattcaatgacctttaagcgttttttcataacaga
801 agctgcaagagagcaaagagtcaaacctaaagcttgtatgtgtggttttt
851 cttactcatcatctactcatctagcattgggtttattgttgccgtgacag
IRF-1
901 catcccacaaacaggctgaaggtttaagtttttcattttgagagcctgga
NFAT
951 gaggcatttaaacaaaaaacactcgatagatgcaggaggaaaaaaatgtc
STAT-1
1001 acaagcgcctcataacatgctatttccaggtagcacaaccacaggcgttg
1051 tatttactgaaacattcgtatggtctgtgatgcctcacagtttgactgac
1101 cgcacaggctcatcatagcgatccttttaaggttgcactcatgcagaaac
TATA Box
1151 gatataagtaagaagtcaagggcaagcagatgaaaaccacagtctgaata
1201
1251
1301
1351
1401
M M H
taaGGGTCTGACTCAACACCTCAAAGACGACAGGCTGAAAAGATGATGCA
A V H D L M F V Y L L T C L G Q
TGCTGTGCACGATCTTATGTTTGTCTATCTTCTAACATGTCTGGGACAAC
H
ATGgtaagattacaaaatgtaattacttatctaatgaaaacatccaacat
G H G S E I I N
taacttttttcctctcactcttgcagGACATGGAAGTGAAATCATAAATG
G K N V P Q N S M Q Y M A S V Q I
GCAAAAATGTCCCACAGAACTCAATGCAGTATATGGCTTCTGTGCAGATC
168
3
19
28
45
1451
1501
1551
1601
1651
1701
1751
1801
1851
1901
1951
2001
2051
2101
2151
2201
2251
2301
2351
2401
2451
2501
2551
D G K H V C G G F L V S E D F V L
GATGGAAAACACGTATGTGGAGGATTTCTTGTCAGTGAAGACTTTGTGCT
T A A H C Y K N
CACGGCTGCACATTGTTACAAAAAgtaagtgaacaacctgtgtactttta
tttgtttttctcaaaaaaacccagctaacacctacttaatattcgctcgc
S P M E V V I G T H N L K K V
tcagTTCTCCTATGGAGGTTGTAATTGGAACCCACAATCTGAAGAAGGTT
N N N K M R Y S V K T C K H P R Y
AATAACAACAAAATGAGATACAGTGTAAAGACATGCAAGCACCCACGATA
D K V E S G N D I M L L K
TGACAAAGTTGAGTCTGGTAATGACATCATGCTCCTCAAAgtaagtaaat
atctttgaatgtgattcattacattacaataactccacttcgtacttcac
caaagacttgatataagatcagaaatgtacagagttggtttctatcagtc
L S R K L Q
tttttattttttttccttttttggttcttctagCTCTCGAGGAAACTTCA
L D K K V K P I Q L A R K E I K
ACTGGACAAGAAAGTGAAACCGATACAACTGGCAAGGAAGGAGATTAAAG
A K D N V K C Q V A G W G F T E T
CAAAAGACAATGTAAAGTGTCAGGTGGCTGGATGGGGTTTCACAGAAACC
N G K A V D V L R W V D V P L I D
AATGGCAAAGCTGTTGATGTGCTGAGATGGGTAGATGTGCCTCTTATTGA
L N V C K R K L K G K L P K G V
CCTTAATGTCTGTAAGAGAAAATTGAAAGGCAAACTTCCAAAAGGTGTTA
I C A G G S D T K N G F C Q
TCTGTGCAGGAGGATCTGACACAAAGAATGGATTCTGCCAGgtatgtcct
ctgttcttttaaagaaagtatcacttcacttgtagtattaaataaaagat
G D S G G P L
aaataaaacaagcttcttcccccttacagGGTGATTCTGGTGGCCCTCTG
V C N G T A V G V V S F N I N G N
GTGTGCAATGGAACAGCAGTTGGTGTTGTGTCTTTCAACATAAATGGAAA
C K Y P N Y P N V Y T D I S K H
TTGTAAATACCCAAATTACCCCAACGTCTATACAGATATATCAAAGCACC
L S W I K N I L N K K Q C *
TTTCCTGGATCAAAAACATTCTCAATAAAAAGCAATGCTAAGTATACACT
GTAACAATGTGCAATCTGCAATATCTGGCATTATAATACTGTATGTGAAC
TGCAGGATAAATGTAAAATAGCTTAAGACATCTCTCCTGTATTGCAAAGA
TATTCCTGCTTGCTCTTAAATTAAAGTAAAAAATATTGAAGCTAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
169
62
70
85
102
115
121
137
154
171
187
201
208
225
241
254
Figure 5.2. Predicted three dimensional model for TLGR-1. Four disulfide bonds which are
conserved in other granzyme sequences are represented by circles. The extra Cys residue is
marked with an arrow (One additional Cys residue at the C-terminus of the molecule is omitted
from the model and not shown).
170
171
Figure 5.3. Comparison of TLGR-1 with other granzyme sequences. Multiple sequence
alignments showing the conserved regions in TLGR-1 sequence, which represent the signature
motifs for granzyme-like proteases. Conserved residues at the catalytic triad are represented with
a black circle above the alignment. Darker shading represents identical or residues with similar
properties in 100% of the sequences and lighter shading represents identical or residues with
similar properties in 80% or more of all the sequences.
172
TLGR-1
CFGR1 AAQ54830
Human grnzA A31372
Human grnzB AAH30195
Human grnzK P49863
Human grnzM AAH25701
Human grnzH A32692
Mus grnzC NP_034501
Mus grnzD NP_034502
Mus grnzE NP_034503
Mus grnzF AAA37741
Mus grnzG NP_034505
:
:
:
:
:
:
:
:
:
:
:
:
IINGKNVPQNSMQYMASVQIDG----KHVCGGFLVSEDFVLTAAHCYKNSP-IIGGREVK-KPKPWMASVQSN--N--SHICGGTLIHQQWVLTAAHCKTFLQFK
IIGGNEVTPHSRPYMVLLSLD----RKTICAGALIAKDWVLTAAHCNLNKR-IIGGHEAKPHSRPYMAYLMIWDQKSL-KRCGGFLIQDDFVLTAAHCWGSS--IIGGKEVSPHSRPFMASIQYG--G--HHVCGGVLIDPQWVLTAAHCQYRFTKG
IIGGREVIPHSRPYMASLQRNG----SHLCGGVLVHPKWVLTAAHCLAQR--M
IIGGHEAKPHSRPYMAFVQFLQEKSR-KRCGGILVRKDFVLTAAHCQGSS--IIGGNEISPHSRPYMAYYEFLKVGGKKMFCGGFLVRDKFVLTAAHCKGRS--IIGGHVVKPHSRPYMAFVMSVDIKGNRIYCGGFLIQDDFVLTAAHCKNSS--IIGGHVVKPHSRPYMAFVKSVDIEGNRRYCGGFLVQDDFVLTAAHCRNRT--IIGGHEVKPHSRPYMARVRFVKDNGKRHSCGGFLVQDYFVLTAAHCTGSS--IIGGHEVKPHSRPYMAFIKSVDIEGKKKYCGGFLVQDDFVLTAAHCRNRS---
:
:
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:
47
48
47
49
49
47
49
50
50
50
50
50
TLGR-1
CFGR1 AAQ54830
Human grnzA A31372
Human grnzB AAH30195
Human grnzK P49863
Human grnzM AAH25701
Human grnzH A32692
Mus grnzC NP_034501
Mus grnzD NP_034502
Mus grnzE NP_034503
Mus grnzF AAA37741
Mus grnzG NP_034505
:
:
:
:
:
:
:
:
:
:
:
:
--MEVVIGTHNLK-KVNNNKMRYSVKTCKHPRYDKV-ESGNDIMLLKLSRKLQ
P-IEVLLGAHSLT-KDKNAMRVKVLCFHISPKFSAT-TRVHDIMLLKLQDKVQ
--SQVILGAHSITREEPTKQIMLVKKEFPYPCYDPA-TREGDLKLLQLTEKAK
--INVTLGAHNIKEQEPTQQFIPVKRPIPHPAYNPK-NFSNDIMLLQLERKAK
QSPTVVLGAHSLSKNEASKQTLEIKKFIPFSRVTSD-PQSNDIMLVKLQTAAK
AQLRLVLGLHTLD---SPGLTFHIKAAIQHPRYKPVPALENDLALLQLDGKVK
--INVTLGAHNIKEQERTQQFIPVKRPIPHPAYNPK-NFSNDIMLLQLERKAK
--MTVTLGAHNIKAKEETQQIIPVAKAIPHPDYNPD-DRSNDIMLLKLVRNAK
--MTVTLGAHNITAKEETQQIIPVAKDIPHPDYNAT-IFYSDIMLLKLESKAK
--MTVTLGAHNIKAKEETQQIIPVAKAIPHPDYNAT-AFFSDIMLLKLESKAK
--MRVILGAHNIRAKEETQQIIPVAKAIPHPAYDDK-DNTSDIMLLKLESKAK
--MTVTLGAHNIKAKEETQQIIPVAKAIPHPAFNRK-HGTNDIMLLKLESKAK
:
:
:
:
:
:
:
:
:
:
:
:
96
98
97
99
101
97
99
100
100
100
100
100
TLGR-1
CFGR1 AAQ54830
Human grnzA A31372
Human grnzB AAH30195
Human grnzK P49863
Human grnzM AAH25701
Human grnzH A32692
Mus grnzC NP_034501
Mus grnzD NP_034502
Mus grnzE NP_034503
Mus grnzF AAA37741
Mus grnzG NP_034505
:
:
:
:
:
:
:
:
:
:
:
:
LDK-KVKPIQLARKEIKAKDNVKCQVAGWGFTETN-GKAVDVLRWVDVPLIDL
LKKNKVDVKKIPKSGKDIPAGTKCEVRGWGTTHVKNPKACDTLQELEVTVVDR
INK-YVTILHLPKKGDDVKPGTMCQVAGWGRTHN-SASWSDTLREVNITIIDR
RTR-AVQPLRLPSNKAQVKPGQTCSVAGWGQTAP-LGKHSHTLQEVKMTVQED
LNK-HVKMLHIR-SKTSLRSGTKCKVTGWGATDPDSLRPSDTLREVTVTVLSR
PSR-TIRPLALPSKRQVVAAGTRCSMAGWGLTHQ-GGRLSRVLRELDLQVLDT
WTT-AVRPLRLPSSKAQVKPGQLCSVAGWGYVS--MSTLATTLQEVLLTVQKD
RTR-AVRPLNLPRRNAHVKPGDECYVAGWGKVTP-DGEFPKTLHEVKLTVQKD
RTK-AVRPLKLPRSNARVKPGDVCSVAGWGSRSINDTKASARLREVQLVIQED
RTK-AVRPLKLPRPNARVKPGDVCSVAGWGPRSINDTKASARLREAQLVIQED
RTK-AVRPLKLPRPNARVKPGHVCSVAGWGRTSINATQRSSCLREAQLIIQKD
RTK-AVRPLKLPRPNARVKPGDVCSVAGWGKTSINATKASARLREAQLIIQED
:
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:
:
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:
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:
147
151
148
150
152
148
149
151
152
152
152
152
TLGR-1
CFGR1 AAQ54830
Human grnzA A31372
Human grnzB AAH30195
Human grnzK P49863
Human grnzM AAH25701
Human grnzH A32692
Mus grnzC NP_034501
Mus grnzD NP_034502
Mus grnzE NP_034503
Mus grnzF AAA37741
Mus grnzG NP_034505
:
:
:
:
:
:
:
:
:
:
:
:
NVCKRKLKG----KLPKGVICAGGSDTKNGFCQGDSGGPLVCNG--TAVGVVS
ELCNCYYNSK--PTITANMLCAGNKQRDKDACWGDSGGPLECKK--NIVGVVS
KVCNDRNHYNFNPVIGMNMVCAGSLRGGRDSCNGDSGSPLLCEG--VFRGVTS
RKCESDLRH---YYDSTIELCVGDPEIKKTSFKGDSGGPLVCNK--VAQGIVS
KLCNSQSYYNGDPFITKDMVCAGDAKGQKDSCKGDSGGPLICKG--VFHAIVS
RMCNNSRFWN--GSLSPSMVCLAADSKDQAPCKGDSGGPLVCGKGRVLAGVLS
CQCERLFHG---NYSRATEICVGDPKKTQTGFKGDSGGPLVCKD--VAQGILS
QVCESQFQS---SYNRANEICVGDSKIKGASFEEDSGGPLVCKR--AAAGIVS
EECKKRFR----YYTETTEICAGDLKKIKTPFKGDSGGPLVCHN--QAYGLFA
EECKKRFR----HYTETTEICAGDLKKIKTPFKGDSGGPLVCDN--KAYGLLA
KECKKYFY----KYFKTMQICAGDPKKIQSTYSGDSGGPLVCNN--KAYGVLT
EECKKLWY----TYSKTTQICAGDPKKVQAPYEGESGGPLVCDN--LAYGVVS
:
:
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:
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:
194
200
199
198
203
199
197
199
199
199
199
199
TLGR-1
CFGR1 AAQ54830
Human grnzA A31372
Human grnzB AAH30195
Human grnzK P49863
Human grnzM AAH25701
Human grnzH A32692
Mus grnzC NP_034501
Mus grnzD NP_034502
Mus grnzE NP_034503
Mus grnzF AAA37741
Mus grnzG NP_034505
:
:
:
:
:
:
:
:
:
:
:
:
FNINGNCKYPNYPN-VYTDISKHLSWIKNILNKKQCGGSGCGNPKK--PGVYTLLSKEHIDWINKIIKK---FGLENKCGDPRGPGVYILLSKKHLNWIIMTIKGAV-YGRNNGMP----PR-ACTKVSSFVHWIKKTMKRH--GGHECGVATK--PGIYTLLTKKYQTWIKSNLVPPHTN
FSSRVCTDIFKPP--VATAVAPYVSWIRKVTGRSA-YGNKKGTPPG-----VYIKVSHFLPWIKRTMKRL--YGQTDGSAPQ-----VFTRVLSFVSWIKKTMKHS--YAKNGTISSG-----IFTKVVHFLPWISWNMKLL--YAKNRTISSG-----VFTKIVHFLPWISRNMKLL--YGLNRTIGPG-----VFTKVVHYLPWISRNMKLL--YGINRTITPG-----VFTKVVHFLPWISTNMKLL---
173
:
:
:
:
:
:
:
:
:
:
:
:
229
231
234
227
238
232
226
228
228
228
228
228
Figure 5.4. Phylogenetic analysis of TLGR-1. Phylogram represents the evolutionary position of
TLGR-1 compared to related serine proteases. The tree was derived by parsimony analysis, with
Mega version 2.1. Sequences of mature protease were used in the analysis and accession
numbers are provided after the name. Numbers shown above the branches are bootstrap values
based upon 1000 replicates for parsimony. The tree was rooted on a sub-tree containing
complement factor D to determine the clustering of TLGR-1 with similar granzyme.
174
98
Mus grnzB NP 038570
Rat grnzB NP 612526
Mus grnzC NP 034501
Human grnzB AAH30195
98
Human grnzH A32692
Mus grnzF AAA37741
83
Mus grnzG NP 034505
99
Mus grnzD NP 034502
90
87
Mus grnzE NP 034503
Cow granzB NP 776721
Mus cathepsin G CAA55290
99
100
Human cathepsin G AAA51919
Rat grnzJ AAC53168
100
100
Mus mast cell chymase AAA39492
Rat mast cell chymase NP 037224
Human mast cell chymase NP 001827
Mus mast cell chymase 2 P15119
97
Mus mast cell chymase 10 AAK51074
88
Mus mast cell chymase 4 NP 034909
76
100
99
Rat mast cell chymase 1 P09650
CFGR2 EST:CB940297
Danio hypothtical
CFGR4 EST:CB940725
TLGR-1
96
100
CFGR3 EST: CB940628
Pig Cap37 P80015
Human Cap37 P20160
Human myeloblasin elastase P24158
100
Mus myeloblasin elastase Q61096
99
Mus neut elastase NP 056594
100
Human neut elastase AAA36359
100
Mus grnzK AAC17930
100
Rat grnzK NP 058815
Human grnzK P49863
CFGR1 AAQ54830
Fugu hypothetical
Xenopus GrnzA CAD66429
Chick grnzA CAD66428
Human grnzA A31372
100
Mus grnzA NP 034500
Human grnzM AAH25701
Rat grnzM Q03238
100
100
Mus grnzM NP 032530
100
Rat comp factor D I55608
Mus comp factor D WMMS28
Pig comp factor D P51779
100
99
175
Human comp factor D AAH57807
Figure 5.5. Analysis of tissue expression of TLGR-1. RT-PCR analysis for TLGR-1 using cDNA
from various tilapia tissues. Lane 1, purified NCC from peripheral blood; lane 2, total peripheral
blood leukocytes; lane 3, total blood; lane 4, muscle; lane 5, liver; lane 6, gill; lane 7, spleen;
lane 8, kidney; lane 9, TMB8 (a tilapia continuous cell line TMB-8 was provided by Dr R.
Hedrick, University of California-Davis. These cells were originally established from
Oreochromis mossambicus cardiac tissue). Beta actin was used as a normalizer.
176
177
Figure 5.6. Transcriptional regulation of TLGR-1 in NCC. Real-time RT-PCR analysis of cDNA
from NCC purified from tilapia peripheral blood after subjecting to different treatments. (a) NCC
were treated with recombinant tilapia TNF-alpha and incubated for two or four hours and
analyzed for TLGR-1 expression. (b) NCC treated with PMA and calcium ionophore (A23187)
for two or four hours. (c) NCC treated with lipopolysaccharides (from E. coli).
178
A
30
Fold Increase
25
20
15
10
5
0
0H
2H
4H
Time
B
2.5
Fold Increase
2
1.5
1
0.5
0
0H
2H
4H
Time
C
7
Fold Increase
6
5
4
3
2
1
0
0H
2H
Time
179
4H
Figure 5.7. Production of recombinant TLGR-1 in Pichia pastoris. (a) Representation of
expression cassettes engineered to generate pro and mature TLGR-1 using pPICZ-alpha
expression vector. Pro and mature forms of TLGR-1 were expressed as fusion proteins starting
with the yeast alpha factor at the N-terminus and poly-histidine and c-myc tagged granzyme at
the C-terminus. Presence of a Kex2 site in between the alpha factor and the granzyme allowed
the expression of granzyme with a native N-terminus, which can be secreted into the culture
medium with minimum contaminating yeast proteins. (b) Optimization of recombinant protein
expression. The culture supernatants were collected at the time points indicated and subjected to
TCA precipitation followed by Western blotting. His-tagged proteins were detected with INDIAHis probe conjugated to HRP followed by chemiluminescence. Yeast trsansfected with empty
vector alone (E) also were checked for the expression of proteins and only 72 hour time point is
shown.
180
A
B
181
Figure 5.8. Purification of pro and mature TLGR-1 recombinant proteins. Supernatants were
collected after methanol induction for 72 hours and total proteins were concentrated by
ammonium sulfate precipitation. His-tagged proteins were purified by nickel affinity
chromatography. Purification success was verified by Western blotting and detection with
INDIA-His probe. Lane V: vector alone, lane F: flow though from the column, lane E1, E2, and
E3: three consecutive elutions of the column using buffer containing 250 mM imidazole.
182
183
Figure 5.9. Enzymatic activity of recombinant TLGR-1. (a) Plot depicting the change in optical
density against time from an in vitro enzyme kinetic assay using N-succinyl-Phe-Leu-Phe-SBzl
as a substrate. At a final concentration of 0.3 mM. Enzyme concentration was approximately 1
nM for both mature and pro-TLGR-1. (b) Hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-pNA by
mature and pro-TLGR-1.
184
A
B
185
Figure 5.10. Substrate specificity of TLGR-1. The enzymatic activity of mature and pro-TLGR-1
were measured using the indicated thiobenzyl ester substrates in final concentrations of 0.3 mM.
Enzyme concentration was approximately 1 nM for both mature and pro-TLGR-1. Rate of
hydrolysis is depicted as change in mOD per minute.
186
Boc-Ala-Ala-Asp-SBzl
Boc-Ala-Ala-Met-SBzl
N-Succinyl-Phe-LeuPhe-SBzl
Empty Vector
Pro-TLGR
TLGR
Z-Arg-SBzl
Z-Lys-SBzl
0
2
4
6
8
Change in mOD/min
187
10
12
Table 5.1: Organization of TLGR-1 gene. Amino acid numbering based on the mature
polypeptide chain.
Segment
Size
Amino acids
Features
Exon 1
100 bp
-25 to -5
5’ untranslated region, Signal
peptide
Exon 2
148 bp
-4 to 28
Activation dipeptide (Ser and
Glu) and N-terminus of mature
protein, Active site His residue
Exon 3
136 bp
29 to 73
Active site Asp residue
Exon 4
258 bp
74 to 159
First of the substrate specificity
pocket triplet residues (Gly)
Exon 5
196 bp
(up to polyadenylation
signal)
160 to 212
Active site Ser residue, second
and third of the substrate
specificity pocket triplet residues
(Asn and Asn), 3’ untranslated
region, polyadenylation signal
188
Table 5.2: Pairwise comparison of TLGR-1 with related serine proteases.
Protein
CFGR-1
Human Granzyme A
Mus Granzyme A
Human Granzyme B
Mus Granzyme B
Rat Granzyme B
Mus Granzyme C
Mus Granzyme D
Mus Granzyme E
Mus Granzyme F
Mus Granzyme G
Human Granzyme H
Rat Granzyme J
Human Granzyme K
Mus Granzyme K
Rat Granzyme K
Human Granzyme M
Mus Granzyme M
Rat Granzyme M
Mus mast cell chymase
Human Cathepsin G
Mus Cathepsin G
Human Complement factor D
Accession #
AAQ54830
A31372
NP_034500
AAH30195
NP_038570
NP_612526
NP_034501
NP_034502
NP_034503
AAA37741
NP_034505
A32692
AAC53168
P49863
AAC17930
NP_058815
AAH25701
NP_032530
Q03238
P15119
AAA51919
CAA55290
AAH57807
189
% Identity
% Similarity
37.1
34.5
31.6
34.6
38.0
39.1
34.9
35.3
35.7
36.2
38.3
33.0
35.0
33.7
36.2
37.0
33.2
32.4
32.6
35.5
34.9
32.8
31.8
47.7
47.2
45.7
44.4
48.3
48.5
46.4
46.4
46.4
46.4
48.9
41.6
45.3
46.5
47.7
46.6
50.2
47.5
47.3
45.7
46.9
43.3
46.4
CHAPTER 6
CONSTITUTIVE EXPRESSION OF TUMOR NECROSIS FACTOR-ALPHA IN
CYTOTOXIC CELLS OF TELEOSTS AND ITS ROLE IN REGULATION OF CELLMEDIATED CYTOTOXICITY1
1
Reprinted from publication Praveen, K., D. L. Evans and L. Jaso-Friedmann. 2005. Constitutive
expression of tumor necrosis factor-alpha in cytotoxic cells of teleosts and its role in
regulation of cell-mediated cytotoxicity. Molecular Immunology, in press. ©2005,
Reprinted here with permission from Elsevier.
190
ABSTRACT
Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are the main killer cell
populations of the immune system. The mechanisms by which these cells recognize target cells
vary considerably, while the effector molecules used to facilitate target cell death are highly
conserved. The main pathways utilized by killer cells consist of granule exocytosis and those
mediated by members of the TNF superfamily. Nonspecific cytotoxic cells (NCC) are the first
identified cytotoxic cell population in teleosts. We have previously demonstrated the expression
of granzymes and Fas ligand in these cells. This is the first report of the expression of tumor
necrosis factor-alpha in these killer cells. A cDNA coding for TNF was cloned and sequenced
from NCC purified from Nile tilapia (Oreochromis niloticus). Factors regulating the
transcriptional modulation of TNF in these cells were identified by RT-PCR analysis. The
mature form of tilapia TNF was expressed as a recombinant protein and biological activities
were analyzed. Using a cross-reacting anti-TNF polyclonal antibody, analysis of TNF expression
suggested that tilapia NCC constitutively express the membrane-bound as well as secreted forms
of TNF. Recombinant tilapia TNF effectively induced cytotoxicity in the mammalian cell line
WEHI, although to a lesser extent compared to the murine TNF. Treatment with recombinant
TNF protected NCC from activation-induced cell death. Recombinant tilapia TNF was also
effective in upregulation of granzyme transcription in tilapia NCC. These data suggest that
teleost TNF may play a role in diverse effector functions of cytotoxic cells from ectotherms,
similar to the biological functions described for mammalian TNF.
Keywords: Tumor Necrosis Factor-alpha, Cytotoxicity, Nonspecific Cytotoxic Cells,
Activation-induced cell death, Stress Activated Serum Factors
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1.
INTRODUCTION
Apoptotic cellular death is initiated by two main signaling mechanisms called the
intrinsic and extrinsic pathways. The intrinsic pathway triggers apoptosis as a result of DNA
damage, cell cycle checkpoint defects, loss of survival factors and other types of severe cellular
stress conditions. In all of those circumstances death occurs as a consequence of the activation of
the pro-apoptotic arm of the Bcl-2 gene superfamily. This engages mitochondria to cause the
cytosolic release of apoptogenic factors such as cytochrome C and SMAC/DIABLO (Adams and
Cory, 1998, Green, 2000, Hunt and Evan, 2001). The extrinsic pathways of cellular death are
initiated by binding of members of the TNF superfamily ligands to their death receptors
(Ashkenazi, 2002).
The TNF ligand super-family is composed of members such as TNF, Fas ligand and
TRAIL that are grouped by their structural and functional similarities. These ligands recognize
different specific receptors, which together constitute the TNF receptor super family (Kwon et
al., 1999, Locksley et al., 2001, Wallach et al., 1999). TNF binds to two membrane receptors of
55-60 kDa (TNF-R1) and 75-80 kDa (TNF-R2). TNF-R1 is widely expressed on a variety of cell
types and mediates most of the TNF functions. TNF-R2 expression is tightly regulated and
restricted primarily to cells of lymphoid tissue (Grell et al., 1995b). Like other members of the
TNF super-family, TNF is synthesized as type II transmembrane protein that may be expressed
in either of two forms: the membrane embedded “pro” molecule or the cleaved “mature” form
(Idriss and Naismith, 2000a). Both forms are active and self assemble into non-covalent trimers,
whose individual chains fold into compact jelly roll β-sandwiches and interact at hydrophobic
interfaces (Fesik, 2000).
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The physiological significance and difference between the membrane and soluble forms
of TNF super-family members has been the focus of many investigations. Recombinant soluble
TNF-α and lymphotoxin are capable of inducing apoptotic cell death in mouse as well as human
T lymphocyte blasts in vitro (Sarin et al., 1995b). It has also been demonstrated that the
membrane-bound TNF and FasL are more potent inducers of cytotoxicity than their soluble
trimeric forms (Eissner et al., 1995, Grell et al., 1995a, Schneider et al., 1998, Tanaka et al.,
1998). Membrane-bound FasL was cytotoxic for human peripheral blood lymphocytes, while the
soluble form blocked the killing (Suda et al., 1997). Although these studies have shed light into
the possible mechanisms of actions of the different forms of TNF, the role of TNF alpha in
immune functions is still unclear. In mammalian NK cells, expression of TNF alpha alone was
not sufficient to induce tumor cell killing while simultaneous interactions of at least three TNF
family ligands (TNF, LT- 1 2 and FasL) with their corresponding receptors effectively
generated pro-apoptotic signals in tumor cells (Kashii et al., 1999).
Teleost TNFs have been sequenced and expressed from a variety of species (GarciaCastillo et al., 2002, Hirono et al., 2000, Laing et al., 2001, Saeij et al., 2003, Zou et al., 2003b).
Comparisons of the gene structure and phylogenetic analysis of the amino acid sequences appear
to indicate that the teleost TNFs are more closely related to mammalian TNF alpha than beta
(Goetz et al., 2004). It has been suggested that fish do not have a TNF beta lymphotoxin-like
gene. Although this could suggest a more prevalent role for TNF alpha in fish species, there are
many unanswered questions about its functional role. While it is clear that fish TNF is produced
by macrophages in response to bacterial stimulus, other cell sources for this cytokine have not
been determined (Goetz et al., 2004). Most of the studies on teleost TNF have utilized mixed
populations of myeloid cells, which make the functional characterization difficult to achieve
193
(Goetz et al., 2004). Using long-term hematopoietic cell-lines, expression of TNF in channel
catfish was shown to be mostly in T cells and macrophages, but not in B cells and fibroblasts
(Zou et al., 2003b). In the present study we have cloned and expressed the TNF-alpha gene from
freshly purified nonspecific cytotoxic cells (NCC) of tilapia. NCC have been well characterized
in a number of fish species and they appear to be functionally related to mammalian NK cells
(Carlson et al., 1985, Evans et al., 1984, Faisal et al., 1989, Graves et al., 1985, Greenlee et al.,
1991, Jaso-Friedmann et al., 2002, Jaso-Friedmann and Evans, 1999, McKinney and Schmale,
1994). We have previously shown that tilapia NCC expressed a molecule functionally
characterized as Fas ligand and we have molecular information about teleost granzymes. (Bishop
et al., 2002, Jaso-Friedmann et al., 2000a, Praveen et al., 2004). However, this is the first report
on the molecular identity of a death ligand in tilapia. Our results show that, much like its
mammalian counterpart, TNF alpha from tilapia NCC appears to have pleiotropic immunological
functions.
2.
MATERIALS AND METHODS
2.1.
Experimental animals and isolation of NCC.
Outbred tilapia (Oreochromis niloticus) weighing 60-100 g were obtained from
Americulture, Inc. Animas, NM. Fish were maintained in fiberglass aquaria equipped with a
constant flow through system at 23-25o C. Fish were fed a commercial diet of pelleted fish food
(Southern States Co-operative Inc, VA). Fish were acclimatized for a minimum of 3 months
prior to the study and were free from any clinical infections. NCC were purified from peripheral
blood, anterior kidney or spleen of tilapia as previously described (Bishop et al., 2000). Purity of
cell preparation was verified by flowcytometric analysis using 5C6 (a monoclonal antibody
194
detecting NCCRP-1, which is an activation marker found exclusively on NCC (Evans et al.,
1998, Jaso-Friedmann et al., 2002).
2.2.
Construction and screening of tilapia cDNA library
Construction of cDNA libraries from NCC of different fish species has been previously
described (Praveen et al., 2004). Conserved regions in various teleost TNF-alpha sequences were
identified by multiple sequence alignments using Clustal W provided with vector NTI package,
version 6 (InforMax Inc). Two degenerate primers (TNFDGN1F: 5’GNGCHAARGCHGCHATYCA-3’ and TNFDGN3R: 5’-CARRTARATDGCRTTRTACCA-3’)
were used to amplify a portion of tilapia TNF, using tilapia NCC cDNA library as template.
Amplicons were TA cloned in to pDrive cloning vector using a PCR cloning kit (Qiagen,
Valencia, CA). Inserts were sequenced in two directions and compared with the known
sequences in DDBJ/EMBL/GenBank databases using BLAST version 2.2.5 (Altschul et al.,
1997). Sequence from one of the amplicons which had closest similarity to the known TNF
sequences was used to design primers to amplify the entire 5' and 3' ends of the tilapia TNF as
described previously (Praveen et al., 2004).
2.3.
Phylogenetic analysis and protein modeling
Similar analyses as those previously done with catfish granzyme and NCCRP-1 of
zebrafish were performed (Jaso-Friedmann et al., 2002, Praveen et al., 2004). The three
dimensional structure of tilapia TNF was modeled using SWISS-MODEL in the first approach
mode accessible via the internet (http://www.expasy.org/swissmod). The co-ordinate files were
195
imported to RasWin software version 2.6 for analyzing bond lengths and other conformational
features of the molecule.
2.4.
RT-PCR analysis
Purified NCC were subjected to various treatments and total RNA was isolated using
RNeasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer’s recommendations.
Synthesis of cDNA was done using a cDNA synthesis kit (Invitrogen, Carlsbad, CA) using
olido-dT primer. Primers used for the PCR amplification were as follows: TLTNFRTF1: 5’ggttagttgagaagaaatcacctgca-3’ and TNTNFRTR1: 5’-gtcgtcgctattcccgcagatca-3’. PCR was done
with initial 2 minutes denaturation at 94oC followed by 25 cycles of following cycle:
denaturation at 94oC for 30 s, annealing at 57oC for 30 s, extension at 72oC for 30 s. The
products were resolved on a 1.5% agarose gel and ethidium bromide-stained bands were
quantified using a Kodak DC290 digital camera and digitized using UN-SCAN-IT software (Silk
Scientific, Orem, UT). Band intensities (pixel values) were normalized to that of beta-actin.
2.5.
Cloning and expression of recombinant tilapia TNF
A region at the C-terminus, which corresponds to the mature extracellular domain of
tilapia TNF was amplified by PCR using a proofreading DNA polymerase (Ex-Taq: Takara
Mirus Bio, Madison, WI). The forward PCR primer was designed to introduce a 6-His-Tag and a
Nhe I site at the 5’ end (TNFRECF1: 5’CGTAGCTAGCCATCACCATCACCATCACCTAAAACGGATCAGCAGCA-3’), while the
reverse primer was engineered to bear an Xho I site (TNFRECR1: 5’CTAGCTCGAGTCAAAGTGCAAACACGCCAAAGAAGGTC-3’). The stop codon of tilapia
196
TNF was kept intact to ensure a native C-terminus for the recombinant protein. A full-length
tilapia TNF cDNA clone, whose sequence was verified by sequencing both the strands, was used
as a template for PCR reaction. The amplicon was purified and directionally cloned in to the
pET-21b(+) expression vector (Novagen, San Diego, CA) between Nhe I and Xho I sites. The
recombinant plasmid (pET-21-TLTNF) was sequenced in both direction and was electroporated
in to expression strain of E. coli (BL21-DE3: Novagen, San Diego, CA). Bacteria transformed
with pET-21-TLTNF and empty vector were cultured in LB-ampicillin medium to an OD600 of
0.6-1.0. Production of recombinant protein was induced with 1 mM isopropyl-beta-Dthiogalactopyranoside (IPTG: Fisher Scientific, Fair Lawn, NJ). Optimization of protein
expression was verified by harvesting bacteria at various time points by centrifugation of culture
medium followed by lysis of the pellet and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). Lysates were prepared from IPTG-induced cultures by sequential
incubations in lysozyme (1 mg/ml), Triton X-100 (0.5%), DNAse I (5 µg/ml) and RNAse A (10
µg/ml) in extraction buffer containing protease inhibitors (Sigma, St. Louis, MO). Recombinant
His6-tagged tilapia TNF was purified by metal affinity chromatography using Ni-NTA agarose
(Qiagen, Valencia, CA) according to manufacturer’s instructions. The positive fractions were
pooled, desalted and concentrated using Centricon YM-3 centrifugal filters (Millipore, Billerica,
MA). Aliquots were stored at -80oC. Protein concentrations were estimated using Biorad Protein
Assay Kit (Biorad, Hercules, CA).
2.6.
Identification of antibody binding to tilapia TNF
Recombinant tilapia TNF was boiled in sample buffer and was resolved on a 12%
acrylamide gel and transferred on to nitrocellulose membrane. Membranes were probed with
197
commercially available TNF antibodies and control antibodies (anti-Fas ligand). A polyclonal
antibody raised against mouse TNF-alpha (P350: Pierce, Rockford, IL) was identified as the only
cross-reacting antibody.
2.7.
Flowcytometry
Tilapia NCC from various tissues were purified as previously described. Constitutive
expression of TNF in NCC was analyzed by one and two-color flowcytometry using an EPICS
XL-MCL analyzer (Coulter Electronics). NCC were incubated in PBS containing BSA and
0.05% Sodium Azide (PAB) before staining with polyclonal anti-TNF antibody and FITC
conjugated anti-rabbit IgG (Sigma Immunochemicals). Control rabbit IgG and appropriate
conjugate controls were used to differentiate the non-specific binding of mAbs in the
permeabilized cells. For the two color analysis, NCCRP-1 expression was detected by staining
cells with mAb 5C6 (Evans et al., 1998, Jaso-Friedmann and Evans, 1999) followed by
biotinylated anti-Mouse IgM (Sigma Immunochemicals) and phycoerythrin conjugated
streptavidin.
2.8.
Cytotoxicity
Ability of recombinant tilapia TNF to kill TNF-sensitive targets was assessed using a
murine fibrosarcoma cell line WEHI-164S (ATCC, Manassas, VA). The cells were seeded into
flat bottom 96-well microplate (5 x 104/well) and grown to confluent monolayers in RPMI-1640
supplemented with L-glutamine, sodium pyruvate, MEM vitamin solution, MEM amino acid
solution, MEM nonessential solution (Cellgro Media Tech, Washington, DC), 50 mg/ml
gentamicin and 10% fetal bovine serum (FBS). After overnight culture at 37oC under 5% CO2,
198
the medium was removed and replaced with 100 µl RPMI-1640 containing 10% FBS and 8
µg/ml of actinomycin D (Sigma, St. Louis, MO). Serially diluted recombinant tilapia TNF (100
µl volume) in RPMI was added to each well and incubated for 16 hours. The induction of
cytotoxicity was assessed by DNA hypoploidy analysis.
For chromium release assay, WEHI cells (2×106 cells/ml) were labeled with 20 µCi of
sodium chromate (Amersham, IL) for 3 h at 37°C. Cells were washed three times in medium
containing 8 µg/ml of actinomycin D and diluted to 1×105 cells/ml. Labeled targets were mixed
with effector cells (tilapia NCC) at different E:T ratios and the plates were centrifuged at 500 x
g. In order to demonstrate the specificity of killing, NCC were incubated with anti-TNF
polyclonal antibody or control rabbit IgGs for 2 hours, before mixing with the target cells. After
18 hours of incubation 100 µl of each supernatant was removed to determine radioactivity
(Beckman Biogamma II counter; Beckman Instruments, CA). The results are expressed as
percentage specific release (SR).
Percent SR = (Test Release – Spontaneous Release/ Total Release – Spontaneous release) ×100.
2.9.
DNA hypoploidy analysis
Fragmentation of DNA in WEHI cells, induced by recombinant TNF was determined as
described before for Tetrahymena (Jaso-Friedmann et al., 2000a). Briefly, treatments were
stopped by the addition of nuclear isolation media (NIM, propidium iodide [50 µg/ml], Triton X100 [0.01%] and RNase [1 mg/ml] in phosphate-buffered saline, pH 7.3). Cells were
resuspended in NIM for 10 min at 4oC in the dark. The isolated nuclei were then analyzed for
DNA content using flowcytometry.
199
3.
RESULTS
3.1.
Molecular cloning of TNF cDNA from tilapia NCC
Highly conserved regions in the known TNF sequences were used to design degenerate
primers to amplify a portion of tilapia TNF. The PCR reactions using cDNA library and fresh
cDNA from tilapia NCC as a template yielded a band of expected size. The PCR product was
purified and cloned into pDrive TA cloning vector and sequenced to verify the identity. This
product had highest similarity to known TNF-alpha sequences. Using this sequence information,
non-degenerate primers were designed to obtain full-length tilapia TNF cDNA by 5’ and 3’
RACE as described before (Praveen et al., 2004).
The complete coding sequence of tilapia TNF is shown in Fig. 1 (NCBI accession #
AY428948). The cDNA contains an open reading frame of 744 bp encoding a polypeptide of 247
residues with a predicted molecular weight of 27488.28 Da. The length of the 5’ untranslated
region (UTR) is 114 bp, while 3’ UTR has a length of 448 bp. Tilapia TNF cDNA has a
polyadenylation signal (AATAAA) and multiple mRNA instability motifs in the 3’ UTR.
The signal peptide prediction algorithms using McGeoch's (McGeoch, 1985) or von
Heijne's (von Heijne G., 1986) methods failed to detect any signal peptide in tilapia TNF.
However, using Singer's classification for membrane topology (Singer, 1990), a transmembrane
domain (residues numbers 35-51, Fig. 1) can be predicted with N-terminus inside the cytoplasm,
giving an indication for the production of tilapia TNF as a membrane-bound form.
Comparison of known TNF sequences revealed that many of the signature residues are
conserved in tilapia TNF (Fig. 2a). Most of the sequence identities are confined to the C-terminal
domain, as reported for other fish sequences. The site used by mammalian TNF-alpha converting
enzyme to release the mature (secreted) form of TNF is conserved in all the fish sequences. The
200
two cysteine residues, which are crucial in the correct folding of the mature TNF is found to be
conserved in the majority of sequences (Fig. 2a). Thus, the overall predicted structure of mature
tilapia TNF was compared with other known 3D structures of mammalian TNF (Fig. 2b). Using
this model, we could predict similar biological functions of tilapia TNF like its mammalian
counterparts.
Pair-wise comparisons of tilapia TNF with other teleost sequences indicated its close
relation with TNF identified from other spiny-rayed fish species. To determine the evolutionary
position of tilapia TNF, phylognetic analysis were done using various methods. Independent
analysis using different methods yielded similar trees indicating the clustering of tilapia TNF
with TNF from other spiny-rayed fish. Meanwhile a parallel evolution of TNF can be observed
between teleost and mammalian TNF (Fig. 3).
3.2.
Transcriptional regulation of tilapia TNF
Constitutive expression of tilapia TNF was observed in purified NCC from tilapia tissues
(Fig. 4a). Expression of TNF in various tissues was verified by RT-PCR suggesting the
ubiquitous nature of its expression as reported for other species (Fig. 4b). The TNF gene
transcription was not upregulated in NCC upon treatment with LPS or PMA/Ca2+ ionophore
(data not shown). However, exposing tilapia to acute stress treatment was found to upregulate the
transcription of TNF in NCC (Fig. 4c). We have previously shown the enhancement of NCC
activity upon exposure to stress-activated serum factors (Bishop et al., 2000, Jaso-Friedmann et
al., 2000b, Ruiz et al., 2001).
201
Similarly, cDNA from catfish NCC was used to verify the expression of TNF-alpha in
those cells using primer pairs designed based on the TNF-alpha sequence published (NCBI
accession number: AJ417565) for that species (data not shown).
3.3.
Production of recombinant tilapia TNF
Recombinant tilapia TNF was expressed in E. coli using pET-21b expression vector. The
mature form of tilapia TNF with a His-Tag at the N-terminal was expressed at high levels upon
induction with IPTG. The recombinant protein was purified using Nickel affinity
chromatography (Fig. 5a and b). The reactivity of commercially available anti-TNF antibodies
towards recombinant tilapia TNF was assessed by Western blot analysis. Among those
antibodies, a polyclonal antibody developed against murine TNF was cross-reactive with this
protein (data not shown).
3.4.
Tilapia NCC constitutively express membrane-bound form of TNF
Expression of membrane-bound form of TNF by NCC was determined by flowcytometry.
NCC were purified from peripheral blood and non-permeabilized cells were stained with the
polyclonal anti-TNF antibody and secondary antibody labeled with FITC. Expression of TNF on
the surface was verified by one and two-color analysis. Control rabbit IgGs and conjugate
controls were used to discriminate between non-specific binding of antibodies to the cells (Fig.
6a). Approximately 50% of cells were double positive for TNF antibody binding and 5C6
(monoclonal antibody binding to NCCRP-1, an activation marker on NCC). Similar analysis
using NCC purified from other tissues yielded similar results.
202
Figure 6a shows a population of NCCRP-1 positive cells that is negative for surface
expression of TNF (single positive population). We have detected the presence of lower
molecular weight forms of TNF (secreted/mature form) by Western blot analysis of tilapia NCC
lysates (data not shown). The possibility that repeated handling of the cells during the
purification steps could have resulted in the activation of NCC and release of the soluble form of
TNF-alpha resulting in the single positive cell population was investigated. Leukocytes were
purified in the presence of sodium azide (to prevent secretion) and analyzed for the surface
expression of TNF-alpha. Under these conditions, anti-TNF polyclonal antibody bound to 90%
of purified NCC.
The specificity of staining with the anti-TNF antibody was verified by adsorption of the
antibody with Ni-NTA- immobilized recombinant tilapia TNF (Fig. 6b). Staining of NCC with
the adsorbed antibody was reduced.
3.5.
Tilapia TNF can induce cytotoxicity in susceptible targets
WEHI-164S cells are commonly used to assess the TNF bioactivity. Recombinant tilapia
TNF induced dose-dependant cytotoxicity of these cells in the presence of actinomycin D. As
expected, the cytotoxicity induced by murine TNF was significantly higher than that of the
tilapia TNF, and was even apparent in the absence of actinomycin D (Fig. 7a). Actinomycin D
treatment completely abolished the cytotoxicity caused by fish TNF. Similar results were
reported for recombinant TNF from other fish species (Garcia-Castillo et al., 2004). Tilapia NCC
were also effective in killing actinomycin D-treated WEHI target cells. The specificity of the
killing was demonstrated in inhibition experiments using anti-TNF polyclonal antibody to block
NCC cytotoxicity (Fig. 7b).
203
3.6.
Recombinant tilapia TNF protects NCC from activation-induced cell death
We have previously reported the regulation of activation-induced cell death in NCC by
stress-activated serum factors (Bishop et al., 2000, Evans et al., 2001). The role of the secreted
form of TNF in this process was assessed by incubating NCC in the presence of increasing
concentrations of recombinant TNF followed by measurement of DNA hypoploidy.
Recombinant TNF appears to have a dose-dependent protective effect on NCC. Maximum
protection was achieved at 100 ng/ml of the recombinant protein, and diminished at higher
concentrations (Fig. 8). To demonstrate the specificity of this action, recombinant TNF was
treated with anti-TNF polyclonal antibody and immunoprecipitated before adding the
supernatants to the purified NCC. Depletion of recombinant TNF with the antibody diminished
the protective effect (Fig. 8).
3.7.
Recombinant TNF upregulates transcription of granzyme in tilapia NCC
The partial sequence of a granzyme expressed in tilapia NCC has already been reported
(Praveen et al., 2004). Primers were designed based on that sequence and semi-quantitative RTPCR was used to measure the granzyme gene transcription upon treatment of tilapia NCC with
recombinant TNF. A very significant increase in the transcription of granzyme gene was
observed within two hours of treatment, indicating a possible role of TNF in enhancing the cellmediated cytotoxicity induced by NCC (Fig. 9).
4.
DISCUSSION
In this study, we present the molecular cloning of TNF from nonspecific cytotoxic cells
of teleost fish and we suggest its role in the effector functions of those cells. The transcript has a
204
long 3’ untranslated region with multiple RNA instability motifs (Fig. 1), which are particularly
prevalent among mRNAs encoding many cytokines and other proteins involved in inflammation
(Caput et al., 1986). The deduced tilapia TNF protein showed highest degree of amino acid
homology with TNF alpha from other spiny-rayed fish. Various sequence analysis algorithms
suggested a type II transmembrane topology for tilapia TNF, giving rise to the possibility of
membrane bound as well as secreted forms. In mammals the conversion of membrane-bound to
the soluble form of TNF is achieved by a metalloproteinase called TNF-alpha converting
enzyme (Blobel, 1997). Multiple sequence alignment analysis of the tilapia TNF revealed the
conservation of the Thr-Leu motif which is associated with the protease processing of TNF. This
would suggest that TNF from NCC is processed by a similar enzyme as in the case of
mammalian TNF.
Although tilapia TNF has a signature domain structure that is conserved among other
members of the family, the deviations from mammalian TNF sequences are well in agreement
with other fish TNF sequences (Fig. 2a). The cysteine residues responsible for the conserved
tertiary structure among TNF alpha sequences as well as many other C-terminal residues are
conserved in tilapia TNF. The molecular modeling studies predicted a -jellyroll structure for
tilapia TNF (Fig. 2b) similar to human TNF-alpha (Gruss, 1996, Idriss and Naismith, 2000b).
The results obtained thus far indicate that fish have only TNF-alpha. This argument is
strengthened by the discovery of this molecule in tilapia which also has more similarity to TNFalpha sequences. Conservation of two cysteine residues crucial in correct folding as well as the
lack of similarity of residues surrounding the point of cleavage, suggest that the tilapia TNF is
more closely related to mammalian TNF-alpha than to TNF-beta. Phylogenetic analysis of
205
members of the TNF superfamily indicated the clustering of tilapia TNF with other TNF-alpha
sequences (Fig. 3).
In mammals, TNF is produced by macrophages, monocytes, neutrophils, NK cells and T
cells. We report expression studies using RT-PCR analysis that indicate the constitutive
expression of tilapia TNF in all tissues examined (Fig. 4b), including purified NCC. High level
of expression of TNF in NCC was verified by RT-PCR (Fig. 4a). Evidence for the expression of
TNF in channel catfish cytotoxic cell-line has been presented before (Zou et al., 2003b). Reports
on transcriptional upregulation of TNF are highly variable among various fish species. TNFalpha expression was up-regulated by stimulating isolated primary leukocytes of flounder and
trout with LPS, PMA, recombinant IL-1 or ConA (Hirono et al., 2000, Laing et al., 2001).
However, TNF is constitutively expressed in all tissues examined in seabream. Both LPS and
MAF failed to increase the TNF gene expression in cultured seabream macrophages (GarciaCastillo et al., 2002). Treatment of tilapia NCC with LPS or PMA/Ca2+ ionophore also failed to
upregulate TNF expression (data not shown). However, there was an increase in the expression
of TNF with treatment of NCC with stress-activated serum from tilapia (Fig. 4c). We have
previously demonstrated the role of stress-activated serum factors (SASF) in the regulation of
NCC functions (Bishop et al., 2000, Jaso-Friedmann et al., 2000b, Ruiz et al., 2001).
Upregulation of TNF in NCC by SASF is indirect evidence for the role of TNF in upregulation
of cytotoxicity induced by NCC purified from stressed fish.
Biological activities of recombinant TNF from rainbow trout and seabream have
previously been reported (Garcia-Castillo et al., 2004, Zou et al., 2003a). However, membrane
expression of TNF has not been reported in any population of fish cells. Tilapia TNF was
expressed as a recombinant form in bacteria using pET-21b expression vector. The protein was
206
purified using metal affinity chromatography (Fig. 5a&b) and cross-reactivity of an anti-murine
TNF polyclonal antibody with the fish protein was verified by Western blot analysis. This
antibody was used for staining of membrane TNF on NCC using one and two-color
flowcytometry analysis (Fig. 6a). The specificity of the antibody was verified by depleting the
antibody with recombinant tilapia TNF, immobilized on Ni-NTA agarose beads (Fig. 6b). These
results provided evidence for the presence of the cell surface form of TNF on fish NCC, which
demonstrates the conserved expression patterns as compared to mammalian cells. Expression of
the membrane-bound form of TNF on mammalian NK cells, macrophages and CTL and its role
in the cytotoxic functions of these cells have been previously demonstrated (Caron et al., 1999,
Monastra et al., 1996).
The murine fibrosarcoma cell line WEHI is generally used to assess mammalian TNF
cytotoxicity (Austgulen et al., 1986, Espevik and Nissen-Meyer, 1986). The species specificity
of TNF in induction of cytotoxicity has been previously reported (Lewis et al., 1991) and this is
in agreement with a recent report where recombinant seabream TNF-alpha failed to induce
cytotoxicity in L929 cells (Garcia-Castillo et al., 2004). In contrast, we now report that
recombinant tilapia TNF induced cytotoxicity of WEHI cells in the presence of actinomycin D,
though to a lesser extent than murine TNF-alpha (Fig. 7a). We propose that the discrepancies
between our results with tilapia rTNF and other fish TNF may be attributed to the use of
different apoptosis detection method. We used DNA hypoploidy, which appears to be a more
sensitive assay for WEHI cells than those previously used. The ability of NCC to kill TNF
sensitive cells was demonstrated with chromium release assays using WEHI cells as targets. The
specific role of TNF in this experiment was demonstrated by neutralizing the killing with
207
addition of anti-TNF antibodies (Fig. 7b). These results suggest a critical role of TNF in cellmediated cytotoxicity in teleosts.
The role of human recombinant TNF-alpha in negative regulation of T cell response has
been documented (Sarin et al., 1995a). In human T cells TNF-alpha as well as LT were upregulated only upon activation (Gehr et al., 1992). We have previously reported that NCC
undergo activation induced cell death, which is regulated by SASF (Bishop et al., 2000, Evans
et al., 2001). However, the actual mediators of this regulation were not identified. Incubation of
purified NCC with recombinant TNF resulted in increased survival from cell death, indicating a
protective mechanism (Fig. 8). The better protection at lower concentrations of recombinant TNF
could be explained by a concentration effect on affinity to bind to the two TNF receptors.
However, the expression of TNF receptors on tilapia NCC has not been demonstrated so far.
Using flowcytometry of non-permeabilized NCC, we have demonstrated that these cells
constitutively express membrane bound TNF-alpha (Fig. 6). Similarly, lower molecular weight
forms (mature, released form) of TNF were identified by Western blot analysis of activated NCC
lysates (data not shown). These results suggested the presence of both, membrane bound and
secreted TNF in tilapia NCC. Although there appears to be a marked difference in the mode of
action between the two forms of mammalian TNF ligand superfamily members, identification of
a more susceptible cell line for tilapia TNF would be necessary to dissect out the difference in
activities of the two forms of TNF expressed on NCC.
TNF-alpha is the most important pro-inflammatory cytokine released by many cells
during immune responses. The autocrine/paracrine growth factor effect of TNF on T cells has
been reported. Activation of T cells with TNF induces the expression of TNF receptors and
secretion of cytokines (Scheurich et al., 1987, Sung et al., 1988). Lymphokine-activated killer
208
cells and CTL are optimally induced with TNF, in combination with IL-2, resulting in an
increase in CTL granule constituents like granzyme A (Owen-Schaub et al., 1989, Robinet et al.,
1990). These studies suggested the importance of TNF in the regulation of cytotoxic effector
functions. We have previously reported the expression of a granzyme-like protease in tilapia
NCC (Praveen et al., 2004). Now we show that treatment of tilapia NCC with recombinant TNF
resulted in transcriptional upregulation of granzyme-like protease in those cells (Fig. 9). Thus, a
mechanism of regulation of cytotoxicity by TNF similar to that in mammals is suggested in
NCC.
Our results demonstrated the constitutive expression of both membrane-bound as well as
secreted forms of TNF-alpha in the cytotoxic cells of a teleost. The recombinant analog could
induce target cell death of a mammalian cell line, to a lesser extent than its murine counterpart.
Recombinant TNF was also effective in protecting NCC from cell death and up-regulating
effector molecules of cytotoxicity. By analogy to the mammalian system, TNF-alpha can be
expected to play a significant role in the regulation of cytotoxic cell functions in teleosts.
ACKNOWLEDGEMENTS
We acknowledge the assistance of Mr. John Leary, Dr. Neelesh Sharma, Dr. Jayakumar
Poovassery, Dr. Quanren He and Dr. Brian McNeal at the College of Veterinary Medicine,
University of Georgia for providing valuable suggestions, reagents and facilities. Research was
supported by BARD # 10-21-RR211-236 and Veterinary Medical Experimental Station Grants at
the College of Veterinary Medicine, University of Georgia.
209
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Figure 6.1. Nucleotide and deduced amino acid sequence of tilapia TNF. Start and stop codons
are represented in bold letters. The predicted transmembrane domain is highlighted and ATTTA
motifs are bold with underline. Polyadenylation signal and poly-A tail are italicized.
215
1
51
101
151
201
251
301
351
401
451
501
551
601
651
701
751
801
851
901
951
1001
1051
1101
1151
1201
1251
1301
ACTCTTAACACAAACAGCACACTCAAAGAGAGAAGTGTCAGCACAGAAGA
CACAGACAACTGTTCGGTTTCTGGATTTCTTTAAAGAGCAGACAGCAGAG
M V A Y T T T P V D V E
GCTTTGTGCACAGCATGGTTGCATACACAACCACACCAGTTGACGTCGAG
A G P E A K T V V L V E K K S P A
GCTGGTCCTGAGGCGAAGACTGTAGTTTTAGTTGAGAAGAAATCACCTGC
E W I W K V C A V L V V V A L C
AGAGTGGATATGGAAAGTGTGCGCCGTCCTCGTCGTCGTGGCTCTTTGTT
L A G V L L F A W Y W N T R P E R
TAGCAGGTGTCCTGCTGTTTGCCTGGTACTGGAATACAAGGCCAGAAAGG
M T Q L G Q P E A L K A K N T G D
ATGACACAATTAGGACAGCCAGAAGCACTAAAGGCGAAGAACACTGGCGA
K T E P H S T L K R I S S K A K
CAAAACAGAGCCCCACTCCACGCTAAAACGGATCAGCAGCAAAGCCAAGG
A A I H L E G S D S K G H L E W R
CAGCCATCCATCTAGAAGGCAGCGACTCAAAAGGTCATCTGGAGTGGAGG
N G Q G Q A F A Q G G F K L E A N
AATGGTCAAGGCCAGGCGTTTGCTCAGGGCGGCTTCAAGCTGGAGGCCAA
K I I I P H T G L Y F V Y S Q A
TAAAATCATCATCCCACACACTGGCCTGTACTTCGTCTACAGCCAGGCGT
S F R V I C G N T D E N E D E E K
CCTTCAGGGTGATCTGCGGGAATACTGACGAAAATGAAGATGAGGAAAAA
S L T I L S H R I W R Y S E S M G
AGCCTCACAATTCTCAGCCACAGGATCTGGCGCTACTCAGAGTCTATGGG
S S S T L M S A L R S A C Q D T
AAGCAGCTCCACTCTGATGAGCGCCCTGAGGTCGGCGTGCCAAGACACCA
I Q D S F S D H G W Y N A I Y L G
TTCAGGATAGCTTCTCAGACCACGGCTGGTACAACGCCATTTACCTGGGC
A V F Q L N E G D T L W T E T N Q
GCTGTGTTTCAGCTGAACGAAGGAGACACGCTGTGGACGGAAACCAACCA
L S E L E T D E G R T F F G V F
GCTATCAGAGCTGGAGACTGACGAAGGAAGGACCTTCTTTGGCGTGTTTG
A L *
CACTTTGAAATGACTCTCTGCTTTGAGCAGAAAAGCTGCACAGAGCCAGA
TGTTTTGGCTTAAAAACTATGTGTATATATAGTTTTTTATTTCATCCACA
TGGTGATGTTTAAATATTGAAATCTCAATGGAGATGAAATTTCTAGCTGA
AAACGCTAGGCTGATTTTTGAAAACTATATAAACTGTAACAGTTTGCAAC
ATTGTTTCTACTTTAGGCACTTTTGACATATTATTTATACTGACCTGAGA
TTGTATTAGGCTAGATTTCCTCTGCTGTATCAGATGATGACGTAGCTCTT
TGGCTCAAGAGTTAAGATACAGAGGTGCTGTCATAGCGTGTATGTATTTA
TTTGTATTTATTTGTATTTAAATCACTGGGACCGGGGCTTTAAAAGTATT
TATATAAGTTTTGAAAGGAATAAAAATGCAGAAAACTACAAAAAAAAAAA
AAAAAA
216
12
29
45
62
79
95
112
129
145
162
179
195
212
229
245
247
Figure 6.2. (a) Multiple sequence alignment of tilapia TNF with other teleosts and mammalian
TNF sequences. The analysis was performed using Clustal W provided with vector NTI package,
version 6 (InforMax Inc). Darker shading represents identical or residues with similar properties
in 100% of the sequences and lighter shading represents identical or residues with similar
properties in 80% or more of all the sequences. (b) Predicted 3D model of mature tilapia TNF
monomer in comparison to human TNF. Models were generated using SWISS-MODEL in the
first approach mode accessible via the internet (http://www.expasy.org/swissmod).
217
A
Tilapia TNF AY428948
Pagrus TNF-a AAP76392
Sparus TNF-a Q8JFG3
Flounder TNF-a BAA94969
Brook Trout TNF-a AAF8633
Trout TNF-a-1 CAB89521
Danio AAR06286
Catfish TNF-a CAD10389
Carp TNF-a-1 CAC84641
Human TNF-a AAA61198
Mouse TNF-a NP-038721
Rat TNF-a NP-036807
:
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:
-MVAYTTTPVDVEAG-----------PEAKTVVLVEKKSPAEWIWKVCAVLVVVALCLAG
-------------------------------------------MWKLSVTLLVAALCFGG
-MGAYTTAPCDLEMG-----------PEERTVVLIEKKSSTGWMWKVSVALLIAALCFAG
-------------------------------------------MCKVLGGLFIVALCLGG
-MEGYAMTTGDMERGLENSLVDSGPVYKTTVTAVAERKASRGWLWRLCGVLLVAALCAAA
-MEGYAMTPEDMER---------GPVYNTTVTAVAEGKASRGWLWRLCGVLLIAGLCAAA
-MKLESRAFLDVEEGE--------LPLPLVMVSRRKAGSSKSGVWRVFGTILAVGLCAAA
-MASDSQVVLDVD-------------GPRVTIVREKASWSSSGVWRTCGVLLAVALCAAA
MMDLESQLLEEG--GL--------LPLPQVMVSRRKSGSSKSGVWRVCGVLLAVALCAAA
--MSTESMIRDVELA--------------EEALPKKTGGPQGSRRCLFLSLFSFLIVAGA
--MSTESMIRDVELA--------------EEALPQKMGGFQNSRRCLCLSLFSFLLVAGA
--MSTESMIRDVELA--------------EEALPKKMGGLQNSRRCLCLSLFSFLLVAGA
:
:
:
:
:
:
:
:
:
:
:
:
48
17
48
17
59
50
51
46
50
44
44
44
Tilapia TNF AY428948
Pagrus TNF-a AAP76392
Sparus TNF-a Q8JFG3
Flounder TNF-a BAA94969
Brook Trout TNF-a AAF8633
Trout TNF-a-1 CAB89521
Danio AAR06286
Catfish TNF-a CAD10389
Carp TNF-a-1 CAC84641
Human TNF-a AAA61198
Mouse TNF-a NP-038721
Rat TNF-a NP-036807
:
:
:
:
:
:
:
:
:
:
:
:
VLLFAWYWNTRPERMTQLGQPEALKAKNTGDKTEPHSTLKRISSKAKAAIHLEG--SDSK
VLLFAWYWNGKPQTLIQSGQTEALTKTDTAEKTDPHSTLRRISSKAKAAIHLEGSYDEDE
VLLFAWYWNGKPEILIHSGQSEALTKKDHAEKTDPHSTLKRISSKAKAAIHLEGSYDEDE
VLAFSWYTN-KSEMMTQSGQTAALSQKDCAEKTEPHNTLRQISSRAKAAIHLEGRDEEDE
ALLFAWCQHGRLETMQDGMEP---QLEILIGAKDTHHTLKQIAGNAKAAIHLEG--EYNP
ALLFAWCQHGRPSTMQDEIEP---QLEILIGAKDTHHTLKQIAGNAKAAIHLEG--EYNP
AVCFTLHKT-------QGNQQ----DGS-VLRLTLRDRISQGNFTSKAAIHLTGGYN--S
AVCFSQNKT-------HNKPD----ETQ-EIKHSLRQ-IS---QTAKAAIHLSGHYNPQV
AVCFTLNKS-------QNNQE----GGN-ALRLTLRDHLSKANVTSKAAIHLIGAYEPKV
TTLFCLLHFGVIGPQREE-SPRDLSLISPLAQAVRS---SSRTPSDKPVAHVVAN----TTLFCLLNFGVIGPQRDEKFPNGLPLISSMAQTLTLR-SSSQNSSDKPVAHVVAN----TTLFCLLNFGVIGPNKEEKFPNGLPLISSMAQTLTLR-SSSQNSSDKPVAHVVAN-----
:
:
:
:
:
:
:
:
:
:
:
:
106
77
108
76
114
105
97
90
98
95
98
98
Tilapia TNF AY428948
Pagrus TNF-a AAP76392
Sparus TNF-a Q8JFG3
Flounder TNF-a BAA94969
Brook Trout TNF-a AAF8633
Trout TNF-a-1 CAB89521
Danio AAR06286
Catfish TNF-a CAD10389
Carp TNF-a-1 CAC84641
Human TNF-a AAA61198
Mouse TNF-a NP-038721
Rat TNF-a NP-036807
:
:
:
:
:
:
:
:
:
:
:
:
G----HLEWRNGQGQAFAQGGFKLEANKIIIPHTGLYFVYSQASFRVI-CGNTDEN--ED
GSKD-QVGWKSGQGQAFAQGGFRLVDNKIVIPQTGLYFVYSQASFRVS-CSDGEE---EG
GLKD-QVEWKNGQGQAFAQGGFRLVDNKIVIPHTGLYFVYSQASFRVS-CSDGDE---EG
ETSENKLVWKNDEGLAFTQGGFELVDNHIIIPRSGLYFVYSQASFRVSCSSDDADDGKEA
NLTADTVQWRKDDGQAFSQGGFKLQGNQILIPHTGLFFVYSQASFRVK-CNGPGE----NLSADTVQWRKDDGQAFSQGGFELQGNQILIPHTGLFFVYSQASFRVK-CNSPGE----ESK--TLDWRDDQDQAFSSGGLKLVNREIIIPDDGIYFVYSQVSLHIS-CTSELTEEQ-SSV--SMQWFDNADQSFSSG-LKLEDNEIKILRDGLYFVYSQASYRLL-CKAEGDETEGE
STE--TLDWKKNQDQAFTSGGLKLVEREIIIPTDGIYFVYSQVSFHIN-CKTNMTEDHDPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPS-------HQVEEQLEWLSQRANALLANGMDLKDNQLVVPADGLYLVYSQVLFKGQGCP--------HQAEEQLEWLSQRANALLANGMDLKDNQLVVPADGLYLIYSQVLFKGQGCP---------
:
:
:
:
:
:
:
:
:
:
:
:
159
132
163
136
168
159
152
146
154
147
149
149
Tilapia TNF AY428948
Pagrus TNF-a AAP76392
Sparus TNF-a Q8JFG3
Flounder TNF-a BAA94969
Brook Trout TNF-a AAF8633
Trout TNF-a-1 CAB89521
Danio AAR06286
Catfish TNF-a CAD10389
Carp TNF-a-1 CAC84641
Human TNF-a AAA61198
Mouse TNF-a NP-038721
Rat TNF-a NP-036807
:
:
:
:
:
:
:
:
:
:
:
:
EEKSLTILSHRIWRYSESMGSSSTLMSALRSACQDTIQDSFSDHG--WYNAIYLGAVFQL
AGGHHTPLSHRISRSSESMGSDVSLMSAVRSACQNTAQDDSYSDGRGWYNTIYLGAVFQL
AGRHLTPLSHRISRYSESMGSDVSLMSAVRSACQNTAQEDSYSDGRGWYNTIYLGAVFQL
AEKHLTSISHRVWLFTESLGTQVSLMSAVRSACQ-KSQEDAYRDGQGWYNAIYLGAVFQL
---RTTPLSHVICRYSDSIGVNANLLSGVRSVCQQNYGNAESNIGEGWYNAVYLSAVFQL
---HTTPLSHIIWRYSDSIGVNANLLSGVRSVCQQNYGDAESEIGEGWYNAVYLGAVFQL
-----VLMSHAVMRFSESYGGKKPLFSAIRSICTQEPE--S---ENLWYNTIYLGAAFHL
----VMHMSHKVSRWSDSYSSWKPLLSATRSACKKTTE--EY--QKYWYGAVYLGAAFNL
----LVHMSHTVLRYSDSYGRYMPLFSAIRTACAQASN--T---DDLWYNTIYLGAAFKL
---THVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETP--EGAEAKPWYEPIYLGGVFQL
---DYVLLTHTVSRFAISYQEKVNLLSAVKSPCPKDTP--EGAELKPWYEPIYLGGVFQL
---DYVLLTHTVSRFAISYQEKVSLLSAIKSPCPKDTP--EGAELKPWYEPMYLGGVFQL
:
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:
217
192
223
195
225
216
202
198
205
202
204
204
Tilapia TNF AY428948
Pagrus TNF-a AAP76392
Sparus TNF-a Q8JFG3
Flounder TNF-a BAA94969
Brook Trout TNF-a AAF8633
Trout TNF-a-1 CAB89521
Danio AAR06286
Catfish TNF-a CAD10389
Carp TNF-a-1 CAC84641
Human TNF-a AAA61198
Mouse TNF-a NP-038721
Rat TNF-a NP-036807
:
:
:
:
:
:
:
:
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:
:
:
NEGDTLWTETN--QLSELETDEGRTFFGVFAL
NRGDRLWTETN--QLSELETEEGKTFFGVFAL
NRGDKLETETN--QLSELETDEGKTFFGVFAL
NEGDKLWTETN--MLSELETESGKTFFGVFAL
NEGDKLWTETN--RLTDVEPEHGKNFFGVFAL
NEGDKLWTETN--RLTDVEPEQGKNFFGVFAL
REGDRLGTDTTTALLPMVENDNGKTFFGVFGL
KAGDRLRTVMDEKLLPKVESAGGKTFFGTFSL
RAGDRLRTETTEELLPSVETGDGKTFFGVFAL
EKGDRLSAEINRPDYLDFAES-GQVYFGIIAL
EKGDQLSAEVNLPKYLDFAES-GQVYFGVIAL
EKGDLLSAEVNLPKYLDITES-GQVYFGVIAL
218
:
:
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:
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:
:
247
222
253
225
255
246
234
230
237
233
235
235
B
219
Figure 6.3. Phylogenetic analysis of tilapia TNF: Phylogram showing relationships of tilapia
TNF other TNF family members. The tree was derived by parsimony analysis, with Mega
version 2. Numbers shown above the branches are bootstrap values based upon 1000 replicates
for parsimony. A separate analysis using maximum likelihood and neighbor joining methods
produced a tree with similar topology. The tree was rooted on a sub-tree containing mammalian
fas ligand and lymphotoxin.
220
85
100
Sparus TNF-a Q8JFG3
Acanthopagrus TNF-a AAP94278
Pagrus TNF-a AAP76392
99
Tilapia TNF AY428948
97
Flounder TNF-a BAA94969
Brook Trout TNF-a AAF86331
100
100
Trout TNF-a 1 CAB89521
Catfish TNF-a CAD10389
Danio AAR06286
100
Carp TNF-a 1 CAC84641
97
Carp TNF-a 2 CAC84642
99
80
Carp TNF-a 3 BAC77690
Wallaby TNF-a O77764
99
100
Rat TNF-a NP 036807
Mouse TNF-a NP 038721
Horse TNF-a P29553
99
Human TNF-a AAA61198
84
86
100
Monkey TNF-a Q8MKG8
Mouse FasL AAB33780
Human FasL P48023
Human Lymphotoxin AAA61199
74
Mouse Lymphotoxin AAA18593
100
100
221
Rat Lymphotoxin AAA16276
Figure 6.4. Expression of tilapia TNF gene (a) RT-PCR analysis of tilapia NCC. Lib: cDNA
library, cDNA: fresh cDNA from purified NCC, NTC: No Template Control. (b) RT-PCR
analysis of tilapia TNF expression in various tissues. Beta-Actin was used as a control for
amount and quality of RNA. AK: anterior kidney, LV: Liver, HT: heart, GL: gill, MSL: muscle,
PBL: peripheral blood leukocytes. (c) Transcriptional regulation of tilapia TNF gene by stress
activated serum factors (SASF). NCC were exposed to cold stress and RNA was extracted from
the cells. RT-PCR analysis using beta-actin as a normalizer was used to compare the expression
of TNF gene.
222
A
B
LV
AK
HT
GL
MSL PBL
TNF
β-Actin
C
1.6
1.4
TNF/Actin
1.2
1
0.8
0.6
0.4
0.2
0
Control
SASF
223
Figure 6.5. Purification of recombinant tilapia TNF using Ni-affinity chromatography. (a)
Purification of His-tagged proteins from bacteria with pET-21-TLTNF plasmid. M: molecular
weight markers, L: total lystates from the bacterial pellet, F: flow-through from the column, W1
and W2: washings of the column (buffer with 20 mM imidazole), E1-E4: first four elutions of
the column (buffer with 250 nM imidazole). (b) Similar purification procedure from bacteria
with empty vector. Gels were stained with Coomassie Gel Code staining reagent (Pierce, IL).
224
225
Figure 6.6. Expression of TNF on tilapia NCC membranes. (a) Two-color flowcytometric
analysis of tilapia NCC using anti-TNF polyclonal antibody (followed by FITC-labeled
secondary antibody) and 5C6, a monoclonal antibody detecting NCCRP-1 on NCC (followed by
PE-labeled secondary antibody). (b) Single color analysis of tilapia NCC for surface expression
of TNF-alpha. Tilapia NCC were purified in the presence of sodium azide and non-permeablized
cells were stained for TNF. The specificity of binding was verified by treating the antibody with
polyhistidine tagged recombinant tilapia TNF immobilized on Ni-NTA agarose beads or NiNTA agarose beads as a control.
226
A
B
100
90
80
% Positive cells
70
60
50
40
30
20
10
0
Anti-TNF pAb
Anti-TNF pAb
depleted with TNF
227
Anti-TNF pAb
depleted with NiNTAAgarose
Figure 6.7. Cytotoxicity induced by recombinant TNF on susceptible WEHI cells. (a) WEHI
cells were treated with murine or fish TNF in the presence or absence of actinomycin-D. DNA
hypoploidy analysis was done to determine the cell death. (b) Cytotoxicity of WEHI cells by
tilapia NCC were determined by chromium release assay. The specificity of killing was verified
by blocking the killing by anti-TNF antibody.
228
A
B
35
% Specific Release
30
25
Media Control
20
Control Serum
Anti-TNF pAb
15
10
5
0
160
80
40
E:T Ratio
229
Figure 6.8. Recombinant TNF protects activation-induced cell death in NCC. Purified tilapia
NCC were centrifuged at low speed to facilitate cell-to-cell contact and incubated in the presence
or absence of recombinant tilapia TNF. Cell death after 18 hours was measured by analysis of
DNA hypoploidy. Recombinant tilapia TNF (100 ng/ml) was incubated with anti-TNF
polyclonal antibody or control rabbit IgGs followed by immunoprocipitation. The TNF depleted
supernatants were added to the cells to verify the specificity of protection.
230
TNF IP Control Rb Igs
TNF IP anti-TNF pAb
TNF 6.25 ng/ml
TNF 12.5 ng/ml
TNF 25 ng/ml
TNF 50 ng/ml
TNF 100 ng/ml
TNF 200 ng/ml
TNF 400 ng/ml
Media
0
5
10
% DNA Hypoploidy
231
15
20
Figure 6.9. Recombinant TNF upregulates the transcription of granzyme in tilapia NCC.
Granzyme gene expression was analyzed by RT-PCR after treating NCC with 100 ng/ml of
recombinant tilapia TNF. Results are expressed as ratios of granzyme to beta-actin, which was
used as a normalizer.
232
1
0.9
0.8
TLGR/Actin
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0H
2H
233
4H
CHAPTER 7
MOLECULAR CLONING OF CELLULAR APOPTOSIS SUSCEPTIBILITY (CAS)
GENE IN OREOCHROMIS NILOTICUS AND ITS ROLE IN REGULATION OF
NONSPECIFIC CYTOTOXIC CELL (NCC) FUNCTIONS1
1
Praveen, K., J. H. Leary, D. L. Evans and L. Jaso-Friedmann. 2005. Submitted to Fish and
Shellfish Immunology, 02/07/2005.
234
Cellular Apoptosis Susceptibility (CAS) gene is a homologue of the Chromosome
Segregation gene (CSE) in yeast [1-4] and appears to be involved in multiple cellular
mechanisms. The roles that have been attributed to CAS include mitotic spindle checkpoints, cell
proliferation and apoptosis [5,6]. Attenuation of CAS reduces the sensitivity of cells to TNF-α
and -β mediated apoptosis, probably due to its involvement in nuclear transport of apoptosisrelated proteins [7]. CAS is highly expressed in proliferating cells but at a lower level in
quiescent cells and tissues [8-12].
CAS mutants are unable to degrade cyclin B during key check points in mitosis and
subsequently become arrested during cell cycle. Thus, CAS appears to play an important role in
cancer development [5]. Some of the functions attributed to CAS can be partly related to its role
as nuclear export factor for importin-α [13-15]. It has been demonstrated that phosphorylation of
N-terminal Tyr residues in CAS by MEK-like kinases regulates the nuclear localization of CAS
[8]. In normal cells, CAS is considered to function as a "switch", determining whether a cell will
proliferate or undergo apoptosis [7].
Nonspecific cytotoxic cells (NCC) are the first identified cytotoxic cell population in
teleosts. Since their initial description in the channel catfish [16-19], these cells have been well
characterized in a number of lower vertebrates [20-25]. NCC are functionally similar to
mammalian NK cells. It has been reported that human NK cells are recycled 3-4 times between
different target cells in 3-h in vitro killing assays [26].
Binding of NCC to target cells (or cross linking of the activation marker nonspecific
cytotoxic cell receptor protein-1, NCCRP-1 with a monoclonal antibody) has been shown to
induce activation induced programmed cell death (AIPCD) in NCC, thus preventing the
possibility of recycling [27,28]. NCC are active participants of the innate immune response so as
235
such, NCC must activate regulatory mechanisms of protection from AIPCD. Cells undergoing
apoptosis are immunologically incompetent because they are unable to effect cytotoxic functions
and they cannot secrete growth factors or cytokines necessary for regulation of immune activities
[29]. One of the mechanisms of regulation of AIPCD in NCC has been identified as the soluble
cytokine-like serum factors released into the serum of tilapia following acute stress (stressactivated serum factors: SASF) [30,31].
CAS was previously identified in tilapia NCC with a cross-reacting monoclonal antibody.
Its expression was up-regulated in apoptosis-protected NCC treated with stress-activated serum
[31]. Here we report the molecular cloning and expression of CAS in NCC, and we propose CAS
expression as a mechanism of regulation of AIPCD in these cytotoxic cells.
Full-length cDNA sequence of tilapia CAS (Accession number AF547173) has 3572 bp
with an open reading frame (ORF) of 2916 bp. The putative polypeptide sequence encoded by
this ORF consists of 971 amino acids (Fig. 1). Homology analysis of this protein using different
algorithms suggested it has closest similarity to cellular apoptosis susceptibility (CAS) genes
identified from various organisms. Multiple motifs with L(X)nP sequence, which is generally
found in other CAS sequences are highly conserved in tilapia CAS. By searching the NCBI’s
conserved domain database [32], a C-terminal CAS-CSE-1 signature motif was identified.
There are two putative N-glycosylation sites (456-459 and 537-540) as well as multiple
phosphorylation sites that were identified in the tilapia CAS sequence. Two of the tyrosine
kinase phosphorylation sites (425-432 and 281-289) have been predicted to play an important
role in regulating the movement of CAS in and out of the nucleus [8]. A basic region (positions
372-385), which is believed to be a nuclear localization or DNA binding site [2], was also found
to be well conserved between fish and other CAS genes (Fig. 2). Similar to other CAS genes,
236
there is a putative Zn finger domain (His-(X)2-His-(X)6-Cys-(X)6-Cys) present in tilapia CAS at
positions 613-630. Phylogenetic analysis of similar proteins identified from various species
revealed these close similarities between tilapia CAS and other teleost CAS sequences (Fig. 3).
Expression of CAS transcripts in various tissues was investigated by RT-PCR (Fig. 4).
Higher level of expression of CAS was observed in tissues which are rich in NCC, indicating
NCC as a major site of CAS expression in tilapia. A possible role of CAS in the regulation of
NCC functions was identified by RT-PCR of NCC following exposure to stress-activated serum
factors and recombinant tilapia TNF-alpha. We have previously reported the role of stressactivated serum components in protection of NCC from activation-induced cell death [30,31].
Transcriptional up-regulation of CAS upon exposure to stress-activated serum is an indication of
the involvement of CAS as one of the mechanisms of protection against apoptotic cell death. The
specific serum factors that are responsible for up-regulation of CAS transcription were not
identified.
In order to better understand the factors involved in AIPCD protection of NCC,
experiments were next done with recombinant tilapia TNF-alpha. We have reported that this
cytokine has a protective effect on NCC (Praveen et al. 2005. Molecular Immunology, In press).
RT-PCR analysis of NCC exposed to recombinant tilapia TNF-alpha revealed a transcriptional
up-regulation of CAS, indicating that CAS may be one of the mediators of protective effects of
TNF on NCC.
Although further studies are necessary to show the exact nature of the events involved in
the protective mechanisms in response to higher levels of CAS, it is relevant to note that teleost
cytotoxic cells appear to have complex pathways of regulation of activation-induced cell death.
Previous studies have demonstrated the inability of NCC to recycle in in vitro assays, similar to
237
what has been described for mammalian NK cells [27]. We propose that a mechanism of tight
regulation of cell death in response to adverse conditions, as in the case of SASF or TNF alpha
secretion, could be an effective substitute for recycling of effector cells to maintain a heightened
state of innate immunity. It is important to identify other key players involved in these complex
pathways in order to design effective prophylactic therapies.
ACKNOWLEDGEMENTS
This research was funded in part by grants from BARD US-3159-99C.
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receptor (NCCRP-1) on catfish and zebrafish nonspecific cytotoxic cells belongs to a new
gene family characterized by an F-box-associated domain. Journal of Molecular
Evolution 54, 386-395.
[26] Ullberg, M. & Jondal, M. (1981) Recycling and target binding capacity of human natural
killer cells. Journal of Experimental Medicine 153, 615-628.
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cells in fish (Ictalurus punctatus). IV. Target cell binding and recycling capacity.
Developmental & Comparative Immunology 8, 823-833. doi:10.1016/0145305X(84)90065-X
[28] Bishop, G.R., Jaso-Friedmann, L. & Evans, D.L. (2000). Activation-induced
programmed cell death of nonspecific cytotoxic cells and inhibition by apoptosis
regulatory factors. Cellular Immunology 199, 126-137. doi:10.1006/cimm.1999.1609
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immunity in tilapia: activation of nonspecific cytotoxic cells by cytokine-like factors.
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(2000). In vivo activation of tilapia nonspecific cytotoxic cells by Streptococcus iniae and
amplification with apoptosis regulatory factor(s). Fish & Shellfish Immunology 10, 419434. doi:10.1006/fsim.1999.0250
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Gwadz, M., He, S., Hurwitz, D.I., Jackson, J.D., Ke, Z., Lanczycki, C.J., Liebert, C.A.,
Liu, C., Lu, F., Marchler, G.H., Mullokandov, M., Shoemaker, B.A., Simonyan, V.,
Song, J.S., Thiessen, P.A., Yamashita, R.A., Yin, J.J., Zhang, D. & Bryant, S.H. (2005).
CDD: a Conserved Domain Database for protein classification. Nucleic Acids Research
33, D192-D196.
[33] Praveen, K., Evans, D.L. & Jaso-Friedmann, L. (2004). Evidence for the existence of
granzyme-like serine proteases in teleost cytotoxic cells. Journal of Molecular Evolution
58, 449-459.
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Directed cosmid isolation of bovine markers for physical assignment by fish. Animal
Biotechnology 8, 167-177.
[35] Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman,
D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database
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evolutionary genetics analysis software. Bioinformatics 17, 1244-1245.
241
Figure 7.1. Compiled full-length cDNA sequence of tilapia CAS. The start and stop codons are
represented in bold. Polyadenylation signal is highlighted and poly-A tail is underlined. The
highlighted portion in the amino acid chain represents the highly conserved Zn finger domain.
Construction of cDNA libraries from NCC of different fish species has been described
previously [33]. A directed PCR-based iterative screening protocol was used to identify positive
clones for CAS [34]. Specific primers were designed based on conserved regions in various CAS
sequences were identified by multiple sequence alignments using Clustal W provided with vector
NTI package, version 6 (InforMax Inc, Carlsbad, CA). Sense (5’-cagtctgtgaaaggccacactataag-3’)
and antisense (5’-tcattggcttgtgttattccatgcttctg-3’) PCR primers were used to amplify a portion of
tilapia CAS gene using tilapia NCC cDNA and bacterial pellet of tilapia NCC cDNA library as a
template. The amplicons were TA cloned in to pDrive cloning vector using a PCR cloning kit
(Qiagen, Valencia, CA) and sequenced to verify the identity. After several rounds of screening,
the culture-enriched for target DNA was spread on solid medium and colonies were randomly
selected for assay by PCR. Several CAS positive clones were isolated, expanded and frozen in
glycerol stocks for further analysis. These clones were sequenced in a 373 A DNA sequencer
(Applied Biosystems, CA). The full length tilapia CAS cDNA sequence was obtained by doing
5’ and 3’ RACE on fresh mRNA isolated from tilapia NCC. The entire sequence was compared
with the known sequences in DDBJ/EMBL/GenBank databases using BLAST version 2.2.5 [35].
242
1
51
101
151
201
251
301
351
401
451
501
551
601
651
701
751
801
851
901
951
1001
1051
1101
1151
1201
1251
1301
GCATTTGAAAGCTGCCCTGGCTCGGGAAAGCGGCACGTTCTGTACGAACT
TGTGGTGGCTCTCCATCCCATCCCTAAATTAAACTCACTATCACGTTTTC
M E L N D A N L Q T L T E F L R
ATCATGGAGCTGAATGATGCTAACCTACAGACCCTGACTGAGTTCCTTAG
K T L D P D P T V R R P A E K F
AAAAACCCTCGACCCAGATCCAACAGTCAGACGCCCTGCTGAAAAGTTTC
L E S V E G N Q N Y P L L L L T L
TTGAATCTGTAGAAGGAAACCAGAACTATCCACTGTTACTTCTCACGTTG
L E K S Q D N V I R V C A A V T F
CTGGAGAAGTCCCAGGACAACGTGATCCGTGTCTGTGCTGCTGTGACATT
K N Y I K R N W R I V E D E P N
CAAAAACTATATAAAAAGAAACTGGAGAATTGTTGAAGATGAACCCAACA
K I S D P D R T A V K A N I V N L
AAATCTCTGATCCTGACCGAACGGCAGTAAAAGCAAACATTGTAAATTTG
M L S S P E Q I Q K Q L S D A I S
ATGCTGAGCAGCCCAGAACAGATTCAGAAACAGTTAAGTGATGCCATCAG
I I G R E D F P Q K W P D L L T
CATCATAGGACGTGAAGACTTTCCACAAAAATGGCCAGACCTTCTAACAG
E M V A R F R S G D F H I I N G V
AGATGGTGGCTCGCTTCAGAAGTGGAGATTTCCACATCATCAATGGTGTG
L R T A H S L F K R Y R H E F K S
CTGCGCACTGCACATTCGCTCTTCAAGAGGTATCGCCATGAGTTCAAGTC
N E L W S E I K L V L D T F A L
AAATGAGCTTTGGTCTGAGATAAAACTGGTTCTGGACACGTTTGCTCTAC
P L T E L F K A T I E L C Q T H A
CTCTGACTGAACTATTCAAGGCCACAATCGAGCTGTGTCAAACTCATGCT
T D V N A L K V L F S S L T L I A
ACAGATGTCAATGCCTTGAAGGTCCTCTTTTCATCACTCACACTCATCGC
K L F Y S L N F Q D L P E F F E
CAAGCTTTTCTACAGTCTTAACTTTCAGGACCTTCCAGAGTTTTTTGAAG
D N M E T W M T N F H G L L T L D
ACAATATGGAAACCTGGATGACCAATTTCCACGGCCTACTGACTTTGGAT
N K L L Q T D D E E E A G L L E L
AATAAACTTTTACAAACAGATGATGAGGAGGAGGCAGGTCTCCTGGAGCT
L K S Q I C D N A A L Y A Q K Y
GCTGAAGTCTCAGATCTGTGACAATGCGGCTCTTTATGCTCAGAAGTACG
D E E F Q P Y L P R F V T A I W N
ATGAGGAATTTCAGCCATACCTGCCGCGCTTTGTTACTGCAATCTGGAAC
L L V S T G Q E V K Y D L L V S N
CTTCTAGTTTCTACTGGCCAGGAAGTCAAATATGACTTGCTCGTAAGCAA
A I Q F L A S V C E R P H Y K H
TGCTATCCAGTTCTTGGCATCAGTCTGTGAAAGGCCACACTATAAGCATT
L F E D Q N T L T S I C E K V I V
TGTTTGAGGACCAGAATACACTCACAAGCATTTGTGAGAAGGTCATTGTG
P N M E F R S A D E E A F E D N S
CCCAACATGGAGTTCAGAAGTGCGGATGAGGAGGCCTTTGAAGATAACTC
E E Y I R R D L E G S D I D T R
GGAGGAGTACATCCGAAGAGACCTGGAGGGATCTGACATTGACACTCGCC
R R A A C D L V R G L C K F F E G
GTAGGGCTGCATGTGATTTGGTGAGAGGGCTTTGTAAATTTTTTGAGGGA
P V T A I F S G Y V N S M L S E Y
CCAGTCACAGCAATCTTCTCCGGCTACGTGAACTCGATGCTGAGCGAGTA
243
16
32
49
66
82
99
116
132
149
166
182
199
216
232
249
266
282
299
316
332
349
366
382
399
416
1351
1401
1451
1501
1551
1601
1651
1701
1751
1801
1851
1901
1951
2001
2051
2101
2151
2201
2251
2301
2351
2401
2451
2501
2551
2601
A K N P R E N W K H K D A A I Y
TGCCAAGAACCCTCGGGAGAACTGGAAACACAAGGATGCTGCCATCTATT
L V T S L A S K A Q T Q K H G I T
TGGTCACATCGCTGGCATCGAAAGCCCAGACACAAAAGCATGGAATAACA
Q A N E L V N L T E F F V N H I L
CAAGCCAACGAGTTGGTGAATCTGACAGAGTTCTTTGTGAACCACATTCT
P D L K S P N V N E F P V L K A
CCCTGATTTAAAATCCCCCAATGTTAATGAGTTCCCAGTGCTGAAGGCTG
D A I K Y V M I F R S Q L P K E Q
ATGCCATCAAGTATGTAATGATCTTCAGAAGTCAGCTTCCTAAAGAGCAG
L L Q A V P L L I T H L Q A E S T
CTGCTGCAGGCAGTCCCTCTACTAATAACTCACCTGCAGGCAGAGAGCAC
V E H T Y A A H A L E R L F T M
AGTGGAGCACACTTATGCTGCACATGCTTTGGAGAGACTGTTCACTATGA
R G P N N A T L I T A A E M A P F
GGGGACCCAACAACGCAACACTTATCACTGCTGCAGAGATGGCACCGTTT
T E Q L L N N L F K A L A F P G S
ACTGAACAGCTGCTCAACAACTTGTTCAAGGCACTGGCTTTTCCTGGTTC
A E N E Y I M K A I M R S F S L
TGCAGAAAATGAATACATCATGAAAGCCATCATGCGCAGCTTCTCCCTGC
L Q E S I V P Y I P T L I G Q L T
TGCAGGAGTCAATTGTTCCCTACATCCCAACACTGATTGGTCAGCTCACT
H K L L Q V S K N P S K P H F N H
CATAAGCTCCTCCAAGTCAGCAAGAATCCCAGCAAACCTCACTTTAACCA
Y L F E S L C L S V R I T C K A
CTACCTGTTTGAGTCCCTGTGCCTGTCTGTCCGCATCACCTGCAAGGCAA
N P T T V S S F E E A L F P V F T
ACCCCACTACTGTCAGCAGTTTCGAGGAAGCACTCTTCCCTGTCTTCACA
E I L Q N D V Q E F L P Y V F Q V
GAGATCCTTCAGAACGATGTTCAGGAGTTTCTTCCATATGTGTTCCAGGT
M S L L L E I H S N S I P A S Y
GATGTCTCTCCTCCTAGAGATCCACTCCAACTCTATTCCCGCTTCCTATA
M A L F P H L L Q P V L W E R T G
TGGCTTTATTCCCTCACCTGCTGCAGCCTGTACTTTGGGAACGAACAGGG
N I P P L V R L L Q A Y L E K G G
AACATCCCCCCTCTGGTGCGCCTCCTTCAGGCTTACCTGGAGAAGGGAGG
E T I A R S A A D K I P G L L G
TGAAACTATTGCTAGGTCTGCTGCTGATAAAATACCTGGCTTGCTTGGAG
V F Q K L I A S K A N D H Q G F Y
TTTTCCAAAAGCTTATAGCATCCAAGGCCAATGATCATCAAGGATTTTAC
L L N S I I E H M P P E S L T Q Y
CTTCTCAACAGCATCATAGAGCACATGCCCCCAGAATCACTCACTCAGTA
R K Q I F I L L F Q R L Q S S K
CAGGAAACAGATCTTCATTTTGCTCTTCCAGAGGCTACAAAGTTCCAAAA
T T K F I K S F L V F V N L Y C V
CCACCAAGTTCATCAAGAGTTTCCTGGTGTTTGTCAATTTGTATTGTGTC
K Y G A I A L Q E I F D S I Q P K
AAATATGGAGCTATAGCACTTCAGGAGATTTTTGACAGCATCCAGCCAAA
M F G M V L E K I V I P E V Q K
AATGTTTGGTATGGTGCTGGAGAAAATAGTTATTCCAGAGGTTCAGAAGG
V S G P V E K K I C A V G I T K V
TGTCTGGACCAGTCGAGAAGAAGATCTGTGCTGTCGGCATTACAAAGGTC
244
432
449
466
482
499
516
532
549
566
582
599
616
632
649
666
682
699
716
732
749
766
782
799
816
832
849
2651
2701
2751
2801
2851
2901
2951
3001
3051
3101
3151
3201
3251
3301
3351
3401
3451
3501
3551
L T E C P A M M D T E Y T K L W T
CTCACTGAGTGTCCTGCAATGATGGACACGGAGTACACGAAACTCTGGAC
P L L Q A L I G L F E L P E D D
CCCACTGCTCCAGGCCCTCATTGGTTTATTTGAGCTACCTGAAGATGACA
S I P D D E H F I D I E D T P G Y
GCATCCCAGACGACGAGCACTTCATTGACATCGAAGACACACCGGGCTAT
Q T A F S Q L A F A G K K E H D P
CAGACCGCCTTCTCACAGCTGGCCTTTGCTGGCAAGAAGGAGCACGACCC
I G D A V G N P K I L L A Q S L
AATTGGAGATGCTGTTGGCAATCCAAAAATTCTGCTGGCCCAGTCACTGC
H K L S T A C P G R V P S M L S T
ACAAGCTTTCTACTGCCTGTCCTGGAAGGGTTCCTTCAATGCTGAGCACG
S L N A E A L Q F L Q G Y L Q A A
AGTCTGAATGCAGAAGCTCTCCAGTTCCTGCAGGGGTACTTGCAGGCGGC
T V Q L V *
TACTGTGCAGCTGGTTTAAAACCAAAGCGAGGGTGTTCACACAAAGCAAA
ATCTCAGTACACTGCAGGAATAGCCCCCAATGGCCCCCCAGTCACATGAC
AGTCCTGCTTCAGTTGAACTCGAGACCTGAAAGTTAAATGATGGATTTAC
CAGAGAAGTAAATTTTGCCTGAATTCAGGGTGTGTACTCCAGTGGAAGTC
ATTTTAAGGTTTTTCTTTTAAAGAAAAAAGGAACACAACCAAAATGTTAT
TACTTTAAGATATCTGGGCCTTTTTATTTCCTGTGTTAAAGGCTAACAAG
GATTTGGATGTCAGGTTGTGAAGCACTTGCCGGTTTATTTCAAAAACATC
AGCAATAGAAGCAGATGTAAAAAACTGAACATGTGGGACACTGAACATCT
TTCAAGCACCGCATCCGCAAAGAGGAGTCCATGCGCTGGTTCCAGCAGAA
ATACGACGGCATCATCCTCCCCGGCAAGTAAAGGACCACCTTTTGCTCTT
GTTTGTAACAAAGTAATAAAATGGAAAAAATAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAA
245
866
882
899
916
932
949
966
971
Figure 7.2. Multiple sequence alignment of tilapia CAS with similar proteins from other species.
Multiple sequence alignments were done with Clustal W using Vector NTi suite version 6
(InforMax Inc).
246
Tilapia CAS AAN52370
Pagrus CAS BAA89430
Danio CAS AAH52479
Xenopus CAS AAH73735
Rat XP_342582
Mouse CAS AAG24636
Human CAS AAC50367
:
:
:
:
:
:
:
MELNDANLQTLTEFLRKTLDPDPTVRRPAEKFLESVEGNQNYPLLLLTLLEKSQDNVIRVCAAVTFKNYIK
MELNDANLQTLTEFLRKALDPDPTVRRPAEKFLESVEGNQNYPLLLLTLLEKSQDNVIRVCAAVTFKNYIK
MELNDGNLQTLTEYLQKTLSADPAVRRPAEKFLESVEGNQNYPILLLTVLEKSQNEVIRVCSAVTFKNYIK
MELSEGNLQGLTEYLKKTLDPDPAVRRPAEKYLESVEGNQNYPLLLLTLVERSQDNVIKVCSAVTFKNYIK
MELSDANLQTLTEYLKKTLDPDPAIRRPAEKFLESVEGNQNYPLLLLTLLEKSQDNVIKVCASVTFKNYIK
MELSDANLQTLTEYLKKTLDPDPAIRRPAEKFLESVEGNQNYPLLLLTLLEKSQDNVIKVCASVTFKNYIK
MELSDANLQTLTEYLKKTLDPDPAIRRPAEKFLESVEGNQNYPLLLLTLLEKSQDNVIKVCASVTFKNYIK
:
:
:
:
:
:
:
71
71
71
71
71
71
71
Tilapia CAS AAN52370
Pagrus CAS BAA89430
Danio CAS AAH52479
Xenopus CAS AAH73735
Rat XP_342582
Mouse CAS AAG24636
Human CAS AAC50367
:
:
:
:
:
:
:
RNWRIVEDEPNKISDPDRTAVKANIVNLMLSSPEQIQKQLSDAISIIGREDFPQKWPDLLTEMVARFRSGD
RNWRVIEDEPNKVSDPDRTAIKANIVNLMLSSPEQIQKQLSDAISIIGREDFPQKWPDLLTEMVTRFRSGD
RNWRIVEDEPNKISDPDRTAIKANIVNLMLTSPEQIQKQLSDAISIIGREDFPLKWPDLLTEMVNRFQSGD
RNWRIVEDESNKICEADRIAVKSSIINLMLRSPEQIQKQLSDAISIIGREDFPQKWPNLLTEMVNRFQSGD
RNWRIVEDEPNKICEADRVAIKANIVHLMLSSPEQIQKQLSDAISIVGREDFPQKWPDLLTEMVNRFQSGD
RNWRIVEDEPNKICEADRVAIKANIVHLMLSSPEQIQKQLSDAISIIGREDFPQKWPDLLTEMVNRFQSGD
RNWRIVEDEPNKICEADRVAIKANIVHLMLSSPEQIQKQLSDAISIIGREDFPQKWPDLLTEMVNRFQSGD
:
:
:
:
:
:
:
142
142
142
142
142
142
142
Tilapia CAS AAN52370
Pagrus CAS BAA89430
Danio CAS AAH52479
Xenopus CAS AAH73735
Rat XP_342582
Mouse CAS AAG24636
Human CAS AAC50367
:
:
:
:
:
:
:
FHIINGVLRTAHSLFKRYRHEFKSNELWSEIKLVLDTFALPLTELFKATIELCQTHATDVNALKVLFSSLT
FHIINGVLRTAHSLFKRYRHEFKSNELWSEIKLVLDTFALPLTELFKATIELCQTHATDVNALKVLFSSLT
FHIINGVLRTAHSLFKRYRHEFKSNELWSEIKLVLDTFAQPLTELFKATIELCQTHATDINALKVLFSSLT
FHVINGVLHTAHSLFKRYRHEFKSSELWTEIKLVLDTFAGPLTDLFKATIELCNTHANDVGALKVLFSSLN
FHVINGVLRTAHSLFKRYRHEFKSNELWTEIKLVLDAFALPLTNLFKATIELCSTHANDASALRILFSSLI
FHVINGVLRTAHSLFKRYRHEFKSNELWTEIKLVLDAFALPLTNLFKATIELCSTHANDASALRILFSSLI
FHVINGVLRTAHSLFKRYRHEFKSNELWTEIKLVLDAFALPLTNLFKATIELCSTHANDASALRILFSSLI
:
:
:
:
:
:
:
213
213
213
213
213
213
213
Tilapia CAS AAN52370
Pagrus CAS BAA89430
Danio CAS AAH52479
Xenopus CAS AAH73735
Rat XP_342582
Mouse CAS AAG24636
Human CAS AAC50367
:
:
:
:
:
:
:
LIAKLFYSLNFQDLPEFFEDNMETWMTNFHGLLTLDNKLLQTDDEEEAGLLELLKSQICDNAALYAQKYDE
LISKLFYSLNFQDLPEFFEDNMETWMTNFHGLLTLDNKLLQTDDEEEAGLLELLKSQICDNAALYAQKYDE
LISKLFYSLNFQDLPEFFEDNMETWMTNFHNLLTLDNKLLQTDDEEEAGLLELLKSQICDNAALYAQKYDE
LIAKLFHSLNFQDLPEFFEDNMETWMTNFHNLLTLDNKLLQTDDEEEAGLLELLKSQICDNAALYAQKYDE
LISKLFYSLNFQDLPEFFEDNMETWMNNFHTLLTLDNKLLQTDDEEEAGLLELLKSQICDNAALYAQKYDE
LISKLFYSLNFQDLPEFFEDNMETWMNNFHTLLTLDNKLLQTDDEEEAGLLELLKSQICDNAALYAQKYDE
LISKLFYSLNFQDLPEFWEGNMETWMNNFHTLLTLDNKLLQTDDEEEAGLLELLKSQICDNAALYAQKYDE
:
:
:
:
:
:
:
284
284
284
284
284
284
284
Tilapia CAS AAN52370
Pagrus CAS BAA89430
Danio CAS AAH52479
Xenopus CAS AAH73735
Rat XP_342582
Mouse CAS AAG24636
Human CAS AAC50367
:
:
:
:
:
:
:
EFQPYLPRFVTAIWNLLVSTGQEVKYDLLVSNAIQFLASVCERPHYKHLFEDQNTLTSICEKVIVPNMEFR
EFQPYLPRFVTAIWNLLVSTGQEVKYDLLVSNAIQFLASVCERPHYKHLFEDQNTLTSICEKVIVPNMEFR
EFQPYLPRFVTAIWNLLVTTGQEVKYDLLVSNAIQFLASVCERPHYKHLFEDQNVLTSICEKVIVPNMEFR
EFQPYLPRFVTAIWNLLVTTGQEVKYDLLVSNAIQFLASVCERPHYKNLFEDPSTLTSICEKVIVPNMEFR
EFQRYLPRFVTAIWNLLVTTGQEVKYDLLVSNAIQFLASVCERPHYKNLFEDQNTLTSICEKVIVPNMEFR
EFQRYLPRFVTAIWNLLVTTGREVKYDLLVSNAIQFLASVCERPHYKNLFEDQNTLTSICEKVIVPNMEFR
EFQRYLPRFVTAIWNLLVTTGQEVKYDLLVSNAIQFLASVCERPHYKNLFEDQNTLTSICEKVIVPNMEFR
:
:
:
:
:
:
:
355
355
355
355
355
355
355
Tilapia CAS AAN52370
Pagrus CAS BAA89430
Danio CAS AAH52479
Xenopus CAS AAH73735
Rat XP_342582
Mouse CAS AAG24636
Human CAS AAC50367
:
:
:
:
:
:
:
SADEEAFEDNSEEYIRRDLEGSDIDTRRRAACDLVRGLCKFFEGPVTAIFSGYVNSMLSEYAKNPRENWKH
SADEEAFEDNSEEYIRRDLEGSDIDTRRRAACDLVRGLCKFFEGPVTAIFSGYVNSMLAEYAKNPGENWKH
SADEEAFEDNSEEYIIRDLEGSDIDTRRRAACDLVRGLCKFFEGPVTGIFSGYVNSMLAEYAKNPGVNWKH
AADEEAFEDNSEEYIRRDLEGSDIDTRRRAACDLVRGLCKFFEGPVTNIFSGYVNSMLQEYAKNPSVNWKH
AADEEAFEDNSEEYIRRDLEGSDIDTRRRAACDLVRGLCKFFEGPVTGIFSGYVNSMLQEYAKNPSVNWKH
AADEEAFEDNSEEYIRRDLEGSDIDTRRRAACDLVRGLCKFFEGPVTGIFSGYVNSMLQEYAKNPSVNWKH
AADEEAFEDNSEEYIRRDLEGSDIDTRRRAACDLVRGLCKFFEGPVTGIFSGYVNSMLQEYAKNPSVNWKH
:
:
:
:
:
:
:
426
426
426
426
426
426
426
Tilapia CAS AAN52370
Pagrus CAS BAA89430
Danio CAS AAH52479
Xenopus CAS AAH73735
Rat XP_342582
Mouse CAS AAG24636
Human CAS AAC50367
:
:
:
:
:
:
:
KDAAIYLVTSLASKAQTQKHGITQANELVNLTEFFVNHILPDLKSPNVNEFPVLKADAIKYVMIFRSQLPK
KDAAIYLVTSLASKAQTQKHGITQANELVNLNEFFVNHILSDLKSHNVNEFPVLKADAIKYVMIFRSQLPK
KDAAIYLVTSLASKAQTQKHGITQANELVNLSEFFLNHILIDLKSPNVNEFPVLKSDAIKYVMTFRSQLPK
KDAAIYLVTSLASKAQTQKHGITQANELVNLTEFFVNHILPDLKSANINQYPVLKADGIKYIMFFRSQIPR
KDAAIYLVTSLASKAQTQKHGITQANELVNLTEFFVNHILPDLKSNNVNEFPVLKADGIKYIMIFRNQVPK
KDAAIYLVTSLASKAQTQKHGITQANELVNLTEFFVNHILPDLKSNNVNEFPVLKADGIKYIMIFRNQVPK
KDAAIYLVTSLASKAQTQKHGITQANELVNLTEFFVNHILPDLKSANVNEFPVLKADGIKYIMIFRNQVPK
:
:
:
:
:
:
:
497
497
497
497
497
497
497
Tilapia CAS AAN52370
Pagrus CAS BAA89430
Danio CAS AAH52479
Xenopus CAS AAH73735
Rat XP_342582
Mouse CAS AAG24636
Human CAS AAC50367
:
:
:
:
:
:
:
EQLLQAVPLLITHLQAESTVEHTYAAHALERLFTMRGPNNATLITAAEMAPFTEQLLNNLFKALAFPGSAE
EQLLQAVPLLISHLQAESTVEHTYAAHALERLFTMRGPNNTTLITPVEMAPFTEQLLNNLFKSLALPGSAE
EQLLQAVPLLVSHLQAESIVQHTYAAHALERLFTMRGGNNTTLITPTEMAPFTEQLLNHLFKALAIPGSSE
EQLLVTIPLLIAYLQAESIVVHTYAAHALERFFTMKGAATTTLIVAADMMPYVELLLANLFKALSLPGSTE
EHLLVSIPLLISHLGAESIVVHTYAAHALERLFTMRGPNNATLFTAAEIAPFVGILLTNLFKALTLPGSSE
EHLLVSIPLLISHLEAESIVVHTYAAHALERLFTMRGSNNTTLFTAAEIAPFVEILLTNLFKALTLPGSSE
EHLLVSIPLLINHLQAGSIVVHTYAAHALERLFTMRGPNNATLFTAAEIAPFVEILLTNLFKALTLPGSSE
:
:
:
:
:
:
:
568
568
568
568
568
568
568
Tilapia CAS AAN52370
Pagrus CAS BAA89430
Danio CAS AAH52479
Xenopus CAS AAH73735
Rat XP_342582
Mouse CAS AAG24636
Human CAS AAC50367
:
:
:
:
:
:
:
NEYIMKAIMRSFSLLQESIVPYIPTLIGQLTHKLLQVSKNPSKPHFNHYLFESLCLSVRITCKANPTTVSS
NEYIMKAIMRTFSLLQEAIVPYIPTLIGQLTHKLLLVSKNPSKPHFNHYLFESLCLSVRITCKANPATVSS
NEYIMKAIMRSFSLLQEAIVPYIPTLIGQLTHKLLLVSKNPSKPHFNHYLFESLCLSIRITCKANPDTVSS
NEYIMKAIMRSFSLLQEAIIPYIPSVISQLTQKLLAVSKNPSKPHFNHYMFEAICLSIRITCRANPAAVAS
NEYIMKAIMRSFSLLQEAIIPYIPTLITQLTQKLLAVSKNPSKPHFNHYMFEAICLSIRITCKANPAAVVN
NEYIMKAIMRSFSLLQEAIIPYIPTLITQLTQKLLAVSKNPSKPHFNHYMFEAICLSIRITCKANPAAVVN
NEYIMKAIMRSFSLLQEAIIPYIPTLITQLTQKLLAVSKNPSKPHFNHYMFEAICLSIRITCKANPAAVVN
:
:
:
:
:
:
:
639
639
639
639
639
639
639
247
Tilapia CAS AAN52370
Pagrus CAS BAA89430
Danio CAS AAH52479
Xenopus CAS AAH73735
Rat XP_342582
Mouse CAS AAG24636
Human CAS AAC50367
:
:
:
:
:
:
:
FEEALFPVFTEILQNDVQEFLPYVFQVMSLLLEIHSNSIPASYMALFPHLLQPVLWERTGNIPPLVRLLQA
FEEALFPVFTEILQNDVQEFLPYVFQVMSLLLEIHSSSIPSSYMALFPHLLQPALWERTGNIPPLVRLLQA
FEEALFPVFTEILQNDVQEFVPYVFQVMSLLLEIHSNSIPSSYMALFPHLLQPVLWERTGNIPPLVRLLQA
FEDALFLVFTEILQSDVQEFIPYVFQVMSLLLEIHTTDIPPSYMALFPHLLQPVLWERTGNIPPLVRLLQA
FEEALFLVFTEILQNDVQEFIPYVFQVMSLLLETHKNDIPSSYMALFPHLLQPVLWERAGNIPALVRLLQA
FEEALFLVFTEILQNDVQEFIPYVFQVMSLLLETHKNDIPSSYMALFPHLLQPVLWERTGNIPALVRLLQA
FEEALFLVFTEILQNDVQEFIPYVFQVMSLLLETHKNDIPSSYMALFPHLLQPVLWERTGNIPALVRLLQA
:
:
:
:
:
:
:
710
710
710
710
710
710
710
Tilapia CAS AAN52370
Pagrus CAS BAA89430
Danio CAS AAH52479
Xenopus CAS AAH73735
Rat XP_342582
Mouse CAS AAG24636
Human CAS AAC50367
:
:
:
:
:
:
:
YLEKGGETIARSAADKIPGLLGVFQKLIASKANDHQGFYLLNSIIEHMPPESLTQYRKQIFILLFQRLQSS
YLEKGGATIAASAADKIPGLLGVFQKLIASKANDHQGFYLLNSIIEHMPPESITQYRKQIFILLFQRLQSS
YLEKGAAAIANTASDKIPGLLGVFQKLIASKANDHQGFYLLNSIVEHMPAEAITQYRKQIFILLFQRLQSS
YLERGATTIAASASDKIPGLLGVFQKLIASKANDHQGFYLLNSIIEHLPAECIEQYKRQIFIVLFQRLQSS
FLERGSSTIATAAADKIPGLLGVFQKLIASKANDHQGFYLLNSIIEHMPPESVDQYRKQIFILLFQRLQNS
FLERGSSTIATAAADKIPGLLGVFQKLIASKANDHQGFYLLNSIIEHMPPESVDQYRKQIFILLFQRLQNS
FLERGSNTIASAAADKIPGLLGVFQKLIASKANDHQGFYLLNSIIEHMPPESVDQYRKQIFILLFQRLQNS
:
:
:
:
:
:
:
781
781
781
781
781
781
781
Tilapia CAS AAN52370
Pagrus CAS BAA89430
Danio CAS AAH52479
Xenopus CAS AAH73735
Rat XP_342582
Mouse CAS AAG24636
Human CAS AAC50367
:
:
:
:
:
:
:
KTTKFIKSFLVFVNLYCVKYGAIALQEIFDSIQPKMFGMVLEKIVIPEVQKVSGPVEKKICAVGITKVLTE
KTTKFIKSFLVFVNLYSVKYGAIALQEIFDSIQPKMFGMVLEKIIIPEVQKVSGAVEKKICAVGITKVLTE
KTTKFVKSFLVFINLYSVKYGAIALQEIFDDIQPKMFGMVVEKIVIPEVQKVSGQVEKKICAVGIIKILTE
KTTKFVKSFLVFLNLFCIKFGAIALQEMFDSIQPKMFGMVVEKIIIPEIQKVSGPIEKKICAVGLTKVLTE
KTTKFIKSFLVFINLYCIKYGALALQEIFDGIQPKMFGMVLEKIIIPEIQKVSGNVEKKICAVGITKLLTE
KTTKFIKSFLVFINLYCIKYGALALQEIFDGIQPKMFGMVLEKIIIPEIQKVSGNVEKKICAVGITKLLTE
KTTKFIKSFLVFINLYCIKYGALALQEIFDGIQPKMFGMVLEKIIIPEIQKVSGNVEKKICAVGITNLLTE
:
:
:
:
:
:
:
852
852
852
852
852
852
852
Tilapia CAS AAN52370
Pagrus CAS BAA89430
Danio CAS AAH52479
Xenopus CAS AAH73735
Rat XP_342582
Mouse CAS AAG24636
Human CAS AAC50367
:
:
:
:
:
:
:
CPAMMDTEYTKLWTPLLQALIGLFELPEDDSIPDDEHFIDIEDTPGYQTAFSQLAFAGKKEHDPIGDAVGN
CPAMMDTEYTKLWTPLLQALIGLFELPEDDSIPDDEHFIDIEDTPGYQTAFSQLAFAGKKEHDPIGDAVGN
CPAMMDTEYTKLWAPLLQALIGLFELPEDDSIPDDEHFIDIEDTPGYQTAFSQLAFAGKKEHDPIGDAVSN
CPVMMDTEYTKLWTPLLQALIGLFELPEDDTIPDDEHFIDIEDTPGYQAAFSQLAFAGKKEHDPIGEMVNN
CPAMMDTEYTKLWTPLLQSLIGLFELPEDDSIPDEEHFIDIEDTPGYQTAFSQLAFAGKKEYDPVGQTVNN
CPPMMDTEYTKLWTPLLQSLIGLFELPEDDSIPDEEHFIDIEDTPGYQTAFSQLAFAGKKEHDPVGQMVNN
CPPMMDTEYTKLWTPLLQSLIGLFELPEDDTIPDEEHFIDIEDTPGYQTAFSQLAFAGKKEHDPVGQMVNN
:
:
:
:
:
:
:
923
923
923
923
923
923
923
Tilapia CAS AAN52370
Pagrus CAS BAA89430
Danio CAS AAH52479
Xenopus CAS AAH73735
Rat XP_342582
Mouse CAS AAG24636
Human CAS AAC50367
:
:
:
:
:
:
:
PKILLAQSLHKLSTACPGRVPSMLSTSLNAEALQFLQGYLQAATVQLV
PKILLAQSLHKLSTACPGRVPSMLSTSLNAEALQFLQGYLQAATVQLV
PKILLAQSLHKLSTACPGRVPSMLSTSLPTEALQFLQGYLQAATVQLV
PKILLAQSLHKLSTACPGRVPSMISTSLNAEALQFLQGYLQAGSVSLV
PRVHLAQALHRLSTACPGRVPSMVSTSLNAEALQYLQGYLQAASVTLL
PKIHLAQSLHKLSTACPGRVPSMVSTSLNAEALQYLQGYLQAASVTLL
PKIHLAQSLHMLSTACPGRVPSMVSTSLNAEALQYLQGYLQAASVTLL
248
:
:
:
:
:
:
:
971
971
971
971
971
971
971
Figure 7.3. Phylogenetic analysis of tilapia CAS. Similar analyses as those previously done with
catfish granzyme and NCCRP-1 of zebrafish were performed [25,33]. Briefly, multiple sequence
alignments were done using Clustal W, followed by phylogenetic analysis using Mega version
2.1 [36]. The aligned data set was analyzed by the criteria of maximum parsimony using the
branch-and-bound algorithm. The reliability of the trees was tested using 1000 bootstrap
replicates. The alignment was also analyzed by the neighbor joining method as implemented by
Mega with 1000 bootstrap replications. For neighbor joining method, Poisson correction was
used with the complete deletion option.
249
Rat XP 342582
100
Mouse CAS AAG24636
Human CAS AAC50367
100
Xenopus CAS AAH73735
Tetraodon CAS-like CAG06818
Danio CAS AAH52479
99
Pagrus CAS BAA89430
99
Tilapia CAS AAN52370
Drosophila CAS NP 523588
100
Anopheles CAS XP 311424
Arabidopsis CAS AAD20163
100
250
Yeast CSE O13671
C-elegans CAS NP 490716
Figure 7.4. Expression of tilapia CAS in different tissues. Total RNA was isolated from various
tissues using RNeasy Mini Kit (Qiagen, CA) according to manufacturer’s recommendations.
Synthesis of cDNA was done using a cDNA synthesis kit (Invitrogen, CA) using oligo-dT
primer. RT-PCR analysis was performed with tilapia CAS specific primers using cDNA from
various tissues. Lane 1, purified NCC; lane 2, total white blood cells; lane 3, kidney; lane 4,
spleen; lane 5, red blood cells; lane 6, heart; lane 7, liver; lane 8, gill; lane 9, muscle.
251
252
Figure 7.6. Transcriptional regulation of tilapia CAS in NCC. Purified NCC were subjected to
various treatments, total RNA was isolated and synthesis of cDNA was done using a cDNA
synthesis kit (Invitrogen, CA) using olido-dT primer. PCR was done with initial 2 minutes
denaturation at 94oC followed by 25 cycles of following cycle: denaturation at 94oC for 30 s,
annealing at 57oC for 30 s, extension at 72oC for 30 s. The products were resolved on a 1.5%
agarose gel and ethidium bromide-stained bands were quantified using a Kodak DC290 digital
camera and digitized using UN-SCAN-IT software (Silk Scientific, UT). Band intensities (pixel
values) were normalized to that of beta-actin.
253
A
0.5
0.45
0.4
CAS/Actin
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Non-Stressed
Stressed
B
0.35
0.3
CAS/Actin
0.25
0.2
0.15
0.1
0.05
0
0H
2H
254
4H
CHAPTER 8
SUMMARY AND CONCLUSIONS
255
Cell-mediated cytotoxicity is an important component of the immune system to destroy
virally infected cells and transformed cells of a tumor. Cytotoxic lymphocytes are the main
mediators of this function. These cells vary considerably with regard to the mechanisms they use
to recognize the target cells. However, once the target is identified, effector cells use similar
pathways to induce cellular death. The main pathways employed by these cells include the use of
their cytotoxic granules and/or ligation of TNF superfamily members to corresponding death
receptors on the target cells. The activation of these mechanisms leads to cell death though
different cell death pathways.
Even though the importance of these effector pathways in target cell death has been
demonstrated, much remains to be understood regarding the molecular identity of effector
molecules and their relative contribution to the cell death pathways. Novel components of
cytotoxic granules are being described, while functions of many known components are still
unknown. For example, multiple granzymes with different substrate specificities and unknown
physiological functions are described from these killer cells. Furthermore, the roles of various
TNF-superfamily members in different cytotoxic functions are still poorly understood. One of
the most significant outcomes of understanding the effector molecules of cell-mediated
cytotoxicity is that it will lead to the discovery of novel cell death and proliferation pathways. In
addition, it is significant to understand the role of these molecules in the context of immune
reactions, so that efficient anti-tumor and anti-viral strategies can be adopted to enhance the
immune system during the course of an infection.
Granzymes are members of the serine protease family expressed in the cytoplasmic
granules of professional killer cells. Multiple granzymes with variable substrate specificities
have been identified in cytotoxic lymphocytes. Most of the studies on granzymes were
256
concentrated on granzymes A and B, and evidence suggests very complicated pathways of cell
death initiated by these proteases. Results from recent studies suggest that, in addition to A and
B, other granzymes also play a significant role in inducing cell death pathways.
Nonspecific cytotoxic cells are the first identified cytotoxic cell population in teleost and
they are believed to be the evolutionary precursors of NK cells and CTLs. NCC can induce cell
death in a variety of targets including virally infected cells, protozoan parasites and even
mammalian tumor cells. This last characteristic is important because it appears to indicate that
similar killing mechanisms may be used by cytotoxic cells of ectothermic and endothermic
vertebrates. NCC do not require any prior activation and the killing is not MHC-restricted. Ease
of purification of these cells and their cytotoxic effects on a variety of mammalian targets make
them an ideal model system to study the cell-mediated cytotoxicity. Moreover, NCC play a
significant role in the immune functions and in the health of teleosts.
Previous studies have demonstrated various aspects of cytotoxicity mediated by NCC,
including the interaction of NCC with the target cells. However, there was no molecular
evidence that could be used to identify the effector molecules involved in the cytotoxic activity
of NCC. In the present study, the identity of cytotoxic molecules involved in cell death pathways
from two different species of fish is presented for the first time. The two cell death pathways
initiated by NCC induce target cell death through granule exocytosis as well as TNF-superfamily
ligands.
Teleost cytotoxic cells express multiple granzymes and other components of the granule
exocytosis pathway. Substrate specificities of individual teleost granzymes were assessed by
expressing them as recombinant proteins and measuring the hydrolysis of thiobenzyl ester
substrates. Teleost granzymes identified so far have tryptase and chymase activity, but no Asp-
257
ase or Met-ase activities. Tryptase, Asp-ase, Met-ase and chymase activities are the major
substrate specificities associated with mammalian granzymes. Teleost cytotoxic cells exocytose
the contents of their granules upon conjugate formation with the targets and cytotoxicity
correlates with the residual granzyme activity in the supernatants. Catfish NCC constitutively
express high level of granzymes, while tilapia NCC can be induced to upregulate the granzyme
expression. Teleost granzymes do have a novel S1 specificity pocket triplet, which is not seen in
any mammalian granzymes. Phylogenetic analysis of all known granzyme sequences suggests
clustering of teleost chymases into a separate group.
Teleost cytotoxic cells also express two forms (membrane-bound as well as secreted) of
TNF-alpha. Both forms of TNF can induce cell death in susceptible targets. We have previously
demonstrated the role of the secreted form of Fas ligand in inducing cell death by NCC. Here we
show that TNF also functions as a cytokine by protecting the cells from activation-induced cell
death and activating the NCC to express higher levels of granzymes. These results suggest a
major role played by TNF superfamily members in NCC functions.
Unlike mammalian NK cells, NCC lack the ability to recycle and kill multiple target cells
during in vitro cytotoxicity assays. The importance of regulators of apoptosis in protecting NCC
from activation-induced cell death during immune reactions has been proposed. This study
demonstrates the upregulation of cellular apoptosis susceptibility (CAS) protein upon treatment
with stress-activated serum factors and recombinant TNF-alpha, leading to protection of cells
from AICD. It is proposed that CAS may be an important modulator of NCC functions.
In conclusion, in this study, molecular evidence is presented for the first time for a
parallel evolution of effector molecules of cell-mediated cytotoxicity in teleosts. These findings
are significant because they provide new insights into the evolution of cell-mediated
258
cytotoxicity. Understanding the molecular pathways initiated by teleost granzymes and death
ligands that lead to target cell death, is critical in discovering novel cell death and proliferation
pathways.
259