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Blood First Edition
prepublished
online
March
20, 2003;
DOI 10.1182/blood-2002-10-3055
Sorting of soluble TNF-receptor for granule storage in hematopoietic
cells as a principle for targeting of selected proteins to inflamed sites
Short title: Sorting for granule storage in hematopoietic cells
1
Ying Gao, 1Hanna Rosén, 1Ellinor Johnsson, 2Jero Calafat, 3Hans Tapper,
and 1Inge Olsson4
1
Department of Hematology, C14, BMC, SE-221 84 Lund, Sweden.
2
The Netherlands Cancer Institute, Amsterdam, The Netherlands.
3
Department of Cell and Molecular Biology, B14, BMC, SE-22184 Lund,
Sweden
Financial support: This work was supported by the Swedish Cancer
Foundation, the Swedish Childhood Cancer Foundation, the Alfred
Österlund Foundation, and funds from Lund University Hospital.
4
Corresponding author:
Inge Olsson MD
Department of Hematology,
C14, BMC,
SE-221 84 Lund, Sweden
telephone: +46-46
- 2220737
telefax:
+46-46
- 184493
e-mail:
[email protected]
Total word count:4611 Abstract word count:154
Scientific heading: Phagocytes
1
Copyright (c) 2003 American Society of Hematology
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Abstract
Hematopoietic cells have secretory lysosomes that degranulate at the
inflammatory site upon stimulation. We asked whether one could target
exogenous proteins with a therapeutic potential to secretory lysosomes in
hematopoietic cells. For this purpose, we expressed a soluble TNF receptor
form (sTNFR1) in hematopoietic cell lines. In order to accomplish targeting
to secretory lysosomes both ER-retention and constitutive secretion has to be
prevented. ER-retention was facilitated by addition of a transmembrane (tm)
sequence and constitutive secretion was overcome by incorporating a
cytosolic sorting signal (Y) from CD63. This signal directed the resulting
sTNFR1-tm-Y to secretory lysosomes. Confirmation of these results was
provided
from
biosynthetic
radiolabeling,
subcellular
fractionation,
immunofluorescence microscopy and immunoelectron microscopy. The tmY fragment was cleaved by proteolysis resulting in generation of the
membrane-free sTNFR1 in secretory lysosomes. Our results suggest a
potential for using the storage organelles of hematopoietic cells as vehicles
for targeting sites of inflammation with therapeutically active agents.
[email protected]
2
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Introduction
Hematopoietic cells such as neutrophil granulocytes, natural killer (NK)
cells and cytotoxic T-lymphocytes (CTLs) have a critical role in host
defense. As a consequence, individuals that lack neutrophils can be stricken
by serious bacterial infections since elimination of microorganisms relies on
phagocytosis by these cells followed by the release of cytolytic proteins into
the phagosome for killing.1 In contrast, NK cells and CTLs destroy virusinfected and malignant cells through regulated secretion of cytolytic proteins
to the exterior.2,3 Although they have different biological functions in host
defense, the various hematopoietic cells are characteristically furnished with
many bioactive agents. These are specific for each cell type, and are stored
in subsets of granules manufactured during hematopoietic differentiation.
Bioactive agents play a critical role after they are released from the
granules.4,5 Specific hematopoietic cells, such as neutrophils have many
subsets of storage granules among which the lysosome-like azurophil
granules are the first to be manufactured. Such granules are equipped with
antimicrobial proteins and cationic serine proteases as well as lysosomal
hydrolases.4,5 At a later maturation stage, specific (secondary) granules are
manufactured in these cells that contain a unique composition of
antimicrobial proteins, matrix metalloproteases, and other constituents.4
Clearly, hematopoietic cells have lysosome-related organelles that package
and
store
specialized
cytolytic
proteins
together
with
lysosomal
hydrolases.2,3 These organelles are designated secretory lysosomes as they
display a dual function and are distinguished from conventional lysosomes
by the additional ability to undergo secretion upon stimulation.3 Compelling
evidence indicating a common critical secretory mechanism for cells with
secretory lysosomes comes from observations in the Chediak-Higashi
3
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syndrome, a syndrome characterized by mutation of the LYST gene.2,3 In
this syndrome the immune function of cells with secretory lysosomes is
impaired while the non-secretory function of conventional lysosomes is
retained and is normal.
The granule targeting process requires retrieval of newly synthesized
proteins from the constitutive secretory pathway. While the delivery of the
membrane proteins into secretory lysosomes relies on cytoplasmic sorting
peptide sequences,3 the mechanism for delivery of matrix proteins into these
organelles is less well defined.5 Secretory lysosomes have the morphology
of multivesicular bodies that commonly contain internal vesicles as well as
dense cores. These bodies may serve different functions, e.g. a lytic function
in the case of internal vesicles and a storage function in the case of dense
cores.3 A role of the endosomal pathway for organelle biogenesis is obvious
and consistent with the involvement of the mannose-6-phosphate receptor
(MPR) system normally used for targeting lysosomal enzymes.3 The
secretory granzymes of NK cells and activated CTLs are sorted by means of
the mannose-6-phosphate pathway.3 Neutrophil precursors, on the other
hand, do not depend on this pathway for targeting into lysosome-related
azurophil granules.5 Granule formation for the regulated secretory pathway
in endocrine, neuroendocrine and exocrine cells does not utilize the
endosomal pathway. In these cells, proteins are routed to dense secretory
vesicles within the trans Golgi network (TGN) followed by a granule
maturation process involving removal and re-routing of miss-sorted, soluble
proteins and membrane proteins to lysosomes.6-8 A similar pathway may, in
addition to endosomal sorting, be utilized also in hematopoietic cells.
4
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The above results suggested to us that we could take advantage of the
granule targeting in hematopoietic cells and devise a therapeutic intervention.
If a ”foreign” protein were targeted for storage it could be delivered at an
inflamed site during degranulation. Targeting should be possible as we have
observed that certain exogenous non-hematopoietic proteins can be retrieved
and sorted in hematopoietic cells.9,10 The targeting mechanism(s) in
hematopoietic cells may in fact not be unique for endogenous granule
proteins. The coexistence of lysosomal enzymes and hematopoietic serine
proteases with several antibiotic proteins in secretory lysosomes indicates
that co-storage is possible without autodigestion. To explore this possibility,
we have expressed a soluble form of a TNF receptor (sTNFR1)11 for storage
in hematopoietic granules. sTNFR1 was chosen for the experiments because
anti-TNF therapy is beneficial to patients with certain inflammatory
disorders. For instance, if a local release of sTNFR1 from hematopoietic
cells could be achieved at an inflamed site where TNF needs to be
inactivated, this approach may have a potential therapeutic effect in such
disorders.
As a first step to accomplish targeting of sTNFR1, characterisation of
sorting and processing was performed after expression in the murine
leukemia 32D12 and the rat basophilic leukemia (RBL)13 hematopoietic cell
lines. These cells have been extensively used in studies of targeting and
processing,5 and the RBL cells are known to exhibit regulated secretion
upon stimulation. For successful targeting of sTNFR1 to secretory
lysosomes ER-retention and constitutive secretion has to be prevented. ERexport was facilitated by utilizing a transmembrane form of sTNFR1, and
constitutive secretion was overcome by the incorporation of a cytosolic
sorting signal from CD63, a normal membrane protein of secretory
lysosomes.14,15
5
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Materials and methods
Materials
Constructs were created for eukaryotic expression using the vector pcDNA3
(Invitrogen, Groningen, The Netherlands) for transfection of RBL and 32D
cells. [35S] Methionine/[35S] cysteine (cell radiolabeling grade) were from
ICN Pharmaceuticals, Inc. (Costa Mesa, CA). Heat-inactivated fetal bovine
serum (FBS) was from BioWhittaker (Verviers, Belgium). L-glutamine was
from GIBCO BRL (Life Technologies, Grand Island, NY). Percoll and
protein A-Sepharose CL-4B were from Pharmacia (Uppsala, Sweden). 2mercaptoethanol were from Sigma-Aldrich Co. (St. Louis, MO). Geneticin,
endoglycosidase
Complete
H
(Endo-H),
N-glycosidase
F
(N-glycanase)
and
(protease inhibitor cocktail tablets) were from Boehringer
Mannheim (Mannheim, Federal Republic of Germany). Novex Pre-Cast
Gels (10-20% Tris-Glycine Gel) were from Novex (San Diego, CA).
Polyclonal antiserum against sTNFR1 was obtained by immunization of
rabbits and used for immunoprecipitation.11 A monoclonal antibody, antisTNFR1 (SC8436) (Santa Cruz Biotechnology, Inc. Santa Cruz, CAL) was
used in detection of sTNFR1 by Western blotting. Rabbit anti-lgp120
antiserum was from Dr I Mellman.
The following buffer systems were used: Lysis buffer for cells (1 M NaCl,
50 mM Tris-HCl, pH 8.0, 0.5% Triton X-100 (v/v)), radioimmune
precipitation buffer (RIPA) (0.75 M NaCl, 0.15 M HEPES, pH 7.3, 0.5%
SDS (v/v), 5% Triton X-100 (v/v), 5% sodium deoxycholate (w/v)), lysis
buffer for immunoblotting (86 mM Tris, pH 6.8, 11% (v/v) glycerol, 2.3%
(w/v) SDS, 1.2% (v/v) ß-mercaptoethanol, 0.005% (v/v) bromophenol blue).
Protease inhibitors were added to all buffers before use.
6
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Construction of Expression Vectors
The cDNA fusion constructs sTNFR1-tm and sTNFR1-tm-Y were generated
(Fig. 1). The tm corresponds to the transmembrane domain of human
TNFR1
with
flanking
sequences
(VKGTEDSGTTVLLPLVIFFGLCLLSLLFIGLMYRYORWKSKLYSIV).
Y represents the cytosolic tyrosine-sorting signal (SIRSGYEVM) for
secretory lysosomes
of CD63.2 All PCRs were performed in 20-cycle
reactions in a Perkin-Elmer 480 Thermal Cycler using Pfu polymerase
(Stratagene) according to the manufacturers instructions. By design of the
primers, the Kozak consensus leader sequence for maximal translational
efficiency16 was introduced 5´ to the ATG initiation codon. All constructs
were cloned into the pcDNA3 vector.
The sTNFR1-tm cDNA construct was created by adding flanking restriction
enzyme sites for BamH1 and EcoR1 using pADTNF-R as a template. The
upstream
primer
was
5´-
TTCGGAGGATCCGCCACCATGGGCCTCTCCACCGTG (A1), and the
downstream
primer
was
3´-
TTCGGAGAATTCTCAAACAATGGAGTAGAGCTTGGAC (A4).
The sTNFR1-tm-Y cDNA construct was generated by using ADTNF-R as
template in a PCR reaction with the following primers: A1 plus 3´TTCGGAGAATTCTCACATCACCTCGTAGCCACTTCTGATACTAAC
AATGGAGTAGAGCTTGGAC (A6 long) leading to the creation of
sTNFR1-tm-GY with BamH1 and EcoR1 restriction enzyme sites.
7
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Transfections
32D cl3 cells12 or RBL cells13 were transfected with the pcDNA3 constructs
described above using the Bio-Rad Gene Pulser
(Bio-Rad, Hercules, CA),
with electrical settings of 960 µF and 260-300 V as described
previously.17,18 After electroporation, 1 mg/ml geneticin was added and
recombinant clones of pcDNA3 containing the geneticin-resistance were
selected. Antibiotic-resistant cell clones were expanded in suspension and
screened for the expression of the transfected protein by biosynthetic
radiolabeling. Clones with appropriate expression were chosen. Cells were
incubated in 5% CO2 at 37 °C in a fully humidified atmosphere.
Exponentially growing cells were utilized in all experiments.
Biosynthetic radiolabeling
Biosynthetic radiolabeling of newly synthesized protein was carried out with
[35S]methionine/[35S]cysteine as described previously.19
Immunoprecipitation
All steps were performed at 4°C. Cells (2 × 106/ml) were solubilized in lysis
buffer for cells followed by freezing and thawing. Percoll-containing
subcellular fractions were diluted with one volume of H2O and half a
volume of 5-fold concentrated RIPA. All samples were cleared by
centrifugation at 32000 g for 60 min. Antiserum, protein A-Sepharose, and
protein G-agarose were added, the mixture was rotated overnight, and the
pellet was dissolved in electrophoresis sample buffer followed by SDSPAGE in Pre-Cast 10-20% Tris-glycine gels. The gels were exposed to x-ray
film at -80°C. Densitometry was performed in a Molecular Imager
FX
(BIORAD). Endoglycosidase H (0.24 U) and N-glycosidase F (2U)
8
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digestions were performed at 37˚C for 24 h on immunoprecipitates
corresponding to 20 x 106 cells.
Subcellular fractionation
Subcellular fractionation was carried out on continuous gradients of Percoll,
as described previously.18 The cell homogenate was centrifuged in a Percoll
density gradient after which nine fractions were collected. All the cytosol
was contained in fraction 9. The distribution of secretory lysosomes and
Golgi elements in the gradient was determined by assaying for ßhexosaminidase and galactosyl transferase respectively.20,21 The peak
activities of ß-hexosaminidase and galactosyl transferase in Percoll fractions
of RBL cells were localized in fractions 2 and 6, respectively.18
Western blotting
The ECL Western blotting kit was used according to the manufacturers
instructions. Cells (5 x 106) were washed in PBS, frozen and sonicated in
100µl lysis buffer for immunoblotting. Samples were boiled for 5 min and
cleared by centrifugation at 14,000 x g at 4°C for 5 min. Lysate from 0.5 x
106 cells was loaded in each lane of a precast 10-20% Tris-glycine gel. After
electrophoresis, proteins were transferred to Hybond-P nitrocellulose
membrane in blotting buffer. Detection was performed by exposing the
membranes to Hyperfilm
ECL
for 10-30s.
Immunoelectron microscopy
RBL cells were fixed for 24 hours in 4% paraformaldehyde in 0.1M PHEM
buffer (pH 6.9) and then processed for ultrathin cryosectioning as described
before.22 Cryosections (45 nm ) were cut at –125oC using diamond knives
(Drukker Cuijk, The Netherlands) in an ultracryomicrotome (Leica
Aktiengesellschaft, Vienna, Austria) and transferred with a mixture of
9
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sucrose and methylcellulose onto formvar-coated copper grids. The grids
were placed on 35-mm petri dishes containing 2% gelatin. Double
immunolabeling was performed using the procedure described by Slot23
with 10- and 15-nm protein-A conjugated colloidal gold probes (EM Lab.,
Utrecht
University,
The
Netherlands).
After
immunolabeling,
the
cryosections were embedded in a mixture of methylcellulose and uranyl
acetate and examined with a Philips CM 10 electron microscope (Eindhoven,
The Netherlands). For the controls, the primary antibody was replaced by a
nonrelevant rabbit antiserum.
Immunofluorescence microscopy
Cells were put on ice, washed in 1.5 ml cold PBS and fixed with 1 ml 2%
(v/v) paraformaldehyde solution (Becton Dickinson, Franklin Lakes, NJ) for
15 min and subsequently incubated for an additional 45 min at room
temperature. After fixation, the cells were permeabilized in 1 ml cytoskeletal
buffer containing 100 mM KOH, 2 mM MgCl2, 5 mM EGTA, 0.05% (v/v)
Triton X-100 and 100 mM PIPES (pH 6.8) for 10 min on ice and thereafter
incubated in blocking solution (PBS containing 0.1% BSA (w/v), 0.2%
Tween 20 (v/v), and 5% (v/v) goat serum (Sigma, St. Louis, MO) for 30 min
at room temperature. Next, cells were incubated at room temperature with
the primary antibody (dilution 1:400 - 1:500) for 3 hours in blocking
solution. Following washing, cells were incubated with secondary antibody
(goat anti-rabbit Alexa FluorR 488 F(ab)2 fragment, dilution 1:400, or goat
anti-mouse Alexa FluorR 594 F(ab)2 fragment, dilution 1:400; Molecular
Probes, Eugene, OR) for 1 hour in blocking solution. After washing twice,
cells were adhered to poly-L-lysine-coated coverslips. The samples were
then overlaid with ProLong Antifade reagent (Molecular Probes, Eugene,
OR) before mounting. Images were recorded on a Nicon Eclipse TE300
inverted fluorescence microscope equipped with a Hamamatsu C4742-95
10
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cooled CCD camera, using a Plan Apochromat 100X objective and a high
N.A. oil-condenser. Adobe Photoshop was used to pseudo-color and overlay
the recorded images.
Results
Expression of sTNFR1-tm
Pulse-chase-radiolabeling experiments were performed in RBL cells
expressing sTNFR1-tm shown in Fig. 2. Immunoprecipitation of cell lysates
showed a major band corresponding to full-length 37-kDa sTNFR1-tm that
still predominated after 12 hours chase (Fig. 2A). A 34-kDa processed
component was also observed. Secretion into the culture medium of the 34kDa component was visible after 2-4 hours and increased during a 4-12
hour chase (Fig. 2A). Densitometry performed at 12 hours (data not shown)
indicated 59 % of the immunoreactive species to be intracellular and 41 %
to be extracellular, the latter representing constitutive secretion.
Results
from
biosynthetic
radiolabeling
followed
by
subcellular
fractionation (Fig. 2B) indicated accumulation of the full-length 37-kDa
sTNFR1-tm and a component of lower molecular weight (34-kDa) in
fractions corresponding to the ER, Golgi and plasma membrane. Only a
minor accumulation in the densest secretory lysosome-containing fractions
was observed after chase of the radiolabel as is obvious also from the
densitometry data shown in Fig. 2B. In addition, results from Ip-Western of
lysates from subcellular fractions support this interpretation (data not shown).
In addition, no association of expressed sTNFR1 with dense organelles has
been observed previously in subcellular fractionation experiments.10 We can
therefore conclude that significant sorting of sTNFR1-tm into secretory
lysosomes is unlikely to occur. sTNFR1-tm may instead be transferred to the
11
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cell membrane from which the 34-kDa sTNFR1 could be released after
cleavage as suggested by the results in Fig. 2A. In conclusion, these data
show sTNFR1-tm to be synthesized followed by secretion of generated
sTNFR1.
Expression of sTNFR1-tm-Y
Pulse-chase radiolabeling of RBL cells expressing sTNFR1-tm-Y is shown
in Fig. 3. Immunoprecipitation visualized a 37-kDa sTNFR1-tm-Y after
pulse radiolabeling. A series of processing steps were observed during the
radiolabel chase period. Initially, a slight increase in molecular weight was
observed most likely corresponding to carbohydrate processing. In addition
34-kDa and 31-kDa forms were generated with time. A minor fraction of the
31/34-kDa processed forms was not retained but rapidly released into the
culture medium. The 31/34-kDa processed forms are likely to consist of
sTNFR1 generated by proteolysis of sTNFR1-tm-Y. After prolonged
radiolabel chase a 24-kDa processsed form was also visible. A similar
processing pattern was observed in 32D cells without detectable secretion
(data not shown).
To confirm export from the ER of sTNFR1-tm and sTNFR1-tm-Y,
carbohydrate processing was also investigated. A major part of sTNFR1-tm
observed after 60 min radiolabeling was sensitive to digestion with Endo-H
while some was resistant indicating rapid ER-export to Golgi of the latter
part. Radiolabel chase for 3 hours resulted in total resistance to Endo-H (Fig.
4A). This indicates a change from high mannose to complex glycoforms
consistent with ER export and transfer through the Golgi of sTNFR1-tm.
The secreted species of sTNFR1-tm was also Endo-H resistant indicating a
similar transport through the secretory pathway. A major part of sTNFR1tm-Y observed after 45 min radiolabeling was sensitive to digestion with
12
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Endo-H as shown by a decrease in the molecular size from 37- to 29-kDa,
indicating the presence of high mannose glycoforms that are normally
produced in the ER (Fig. 4B). A similar decrease in molecular size was
achieved by digestion with N-glycanase that removes all N-linked
carbohydrates, indicating that all N-linked glycans were of the high mannose
type. After chase of the radiolabel for 3 hours complete Endo-H resistance
was observed of all processing forms. This suggested the presence of
complex glycoforms that are normally manufactured in the Golgi by
trimming of mannose and addition of new terminal sugars. In conclusion,
these results show that both newly synthesized sTNFR1-tm and sTNFR1tm-Y are rapidly exported to the Golgi for further processing.
Targeting of sTNFR1-tm-Y for secretory lysosomes
The subcellular fate of sTNFR1-tm-Y was investigated by biosynthetic
pulse-chase radiolabeling followed by subcellular fractionation. Also, IPWestern of the subcellular fractions was performed and the protein construct
was visualized by immunofluorescence microscopy and immunogold
electron microscopy.
Possible targeting of sTNFR1-tm-Y for storage in the dense secretory
lysosomes was investigated by pulse-chase radiolabeling experiments
combined with subcellular fractionation. After 30 min of radiolabeling, most
of the radiolabeled 37
- kDa sTNFR1-tm-Y was in the light density fractions
6-7 corresponding to the ER and Golgi elements, and no radiolabeling was
visible in the densest organelles (Fig. 5A). After chase of the radiolabel for 2
hours there was a considerable decrease of the sTNFR1-tm-Y in the light
fractions concomitant with an accumulation of the 31-kDa product in the
most dense organelles (Fig. 5A). This was confirmed by densitometry
analyses that showed an increase from 3 to 28 % of radiolabeled sTNFR113
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tm-Y and processing form in the densest fractions 1-3 (Fig. 5A). At the
same time, radiolabeled sTNFR1-tm-Y of fractions 6 and 7 decreased from
71 to 29 %. The accumaulation observed in the cytosol fraction of sTNFR1tm-Y and processing form may represent leakage during the homogenization.
Pulse-chase radiolabeling experiments were also performed with RMCP-II,
an endogenous constituent of secretory lysosomes. After 30 min of
radiolabeling, the RMCP-II was concentrated in light fractions (Fig. 5B)
similar to sTNFR1-tm-Y. Transfer to the densest fractions occurred upon
radiolabel chase (Fig. 5B). This was confirmed by densitometry analyses
that showed an increase from 2 % to 31 % of radiolabeled RMCP- II in the
densest fractions 1-3 (Fig. 5B). At the same time, the radiolabeled RMCP-II
of fractions 6 and 7 decreased from 66 % to 35 %. In conclusion, the results
on RBL cells indicated export to the Golgi of newly synthesized sTNFR1tm-Y, targeting for the densest organelles corresponding to secretory
lysosomes, and concomitant proteolytic processing into lower molecular
weight forms. Similar results were noticed for 32D cells (data not shown).
Results from IP-Western of subcellular fractions indicated a slight
accumulation of the 31-kDa processed form of sTNFR1-tm-Y in the densest
fractions of both RBL and 32D cells under steady state conditions (data not
shown). These observations are consistent with the results from the
biosynthetic radiolabeling experiments.
To confirm the targeting for storage in secretory lysosomes, we performed
immunoelectron-microscopy with double immunogold labeling in order to
verify co-localization of sTNFR1-tm-Y and constituents of these organelles
in RBL cells. sTNFR1-tm-Y was visible in multivesicular bodies as well as
in the trans-Golgi network (TGN) (Fig. 6). Multivesicular bodies mature as
prelysosomal compartments and correspond to the abnormal granules of
14
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RBL cells. sTNFR1-tm-Y co-localized with RMCP-II, the major constituent
of the granules of RBL cells13 (Fig. 6B). sTNFR1-tm-Y also colocalized
with rat lysosomal glycoprotein 120 (lgp120) in the multivesicular bodies
(Fig. 6C). Lgp120 corresponds to hLAMP-1 and mLAMP-1 in humans and
mice, respectively, indicating involvement of the endosomal/lysosomal
pathway.24 sTNFR1-tm-Y was located mostly in the interior of the
multivesicular bodies while lgp 120 was along its outer membrane (Fig. 6C).
In conclusion, the data are consistent with targeting of sTNFR1-tm-Y to
secretory lysosomes of RBL cells.
We then used immunofluorescence microscopy to confirm the colocalization
of sTNFR1-tm-Y and the marker of secretory lysosomes, lgp120. Cells that
expressed sTNFR1-tm-Y consistently showed a very similar staining pattern
for the TNFR and for lgp120 (Fig. 7A-D, where two of the cells express
high levels of sTNFR1-tm-Y). This staining was frequently highly localized
to one pole of the cells. On the other hand, cells expressing sTNFR1-tm did
not exhibit colocalization with lgp120 (Fig. 7E-H). Instead, the staining of
the TNFR in these cells often appeared in smaller punctate structures,
possibly representing the ER / endosomal apparatus. These structures were
often accumulated in a different region of the cell than the staining for
lpg120 (Fig. 7E-H). In non-transfected cells, a low intensity and diffuse
punctate staining was observed that could represent nonspecific staining or a
low endogenous expression of TNFR (Fig. 7I-L). The immunofluorescence
analysis thus confirms the different targeting of sTNFR1-tm-Y and
sTNFR1-tm in transfected RBL cells, and shows that sTNFR1-tm-Y
accumulates in secretory lysosomes.
15
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Discussion
We expressed a soluble TNF receptor form in hematopoietic cell lines with
the aim of showing targeting of an exogenous protein to secretory lysosomes
for storage and subsequent regulated secretion during degranulation. The
major finding is that this was accomplished by utilizing a transmembrane
form of the protein to facilitate ER-export, and by incorporating a cytosolic
sorting signal for secretory lysosomes to overcome constitutive secretion and
achieve the targeting. This result suggests a potential for using the storage
organelles of hematopoietic cells as vehicles for targeting sites of
inflammation with therapeutically active agents.
In the first compartment of the secretory pathway, the ER, secretory and
membrane proteins acquire native conformations before export to the Golgi
through the assistance of ER-resident and cytosolic chaperones.25 As a
consequence, conformation-based biosynthetic quality control identifies
improperly folded or unassembled oligomeric proteins enabling them to be
diverted for proteolysis by the proteasomes instead of being exported to the
Golgi. An initial problem with our goal was to prevent the retention and
degradation of sTNFR1. What was ER-exported was secreted through
constitutive-like carriers and not retained.10 Other experience with truncated
proteins has been similar, e.g. deletion of the propeptide of myeloperoxidase
(MPO) rendered the enzyme highly susceptible to degradation in the ER.19
In addition, we have observed that chimeric proteins e.g. MPO propeptide
fused to other proteins, were retained in the ER to a greater extent than the
native proteins.10 However, anchoring sTNFR1 through a transmembrane
domain facilitated ER-export.
16
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How is protein cargo selected in the Golgi and packaged into vesicles for
dispatch to its final destination in hematopoietic cells? Timing of gene
expression4 and selective post-transcriptional regulation26 determine the
composition of the mixture of newly synthesized proteins that is offered at
any given time. Consequently, the filling of forming granules depends, in
the first place, on what is offered. The targeting for granule storage in the
transformed cells utilized in this study may differ from that in normal
hematopoietic cells whose granules are the final vehicles for delivering
agents at an inflamed site. The secretory lysosomes of RBL cells, used in
this work, link the endocytic and exocytic pathways to become the regulated
secretory organelle.27,28 Further evidence indicates that, in these cells, the
targeting may occur through either an MPR-dependent endosomal pathway
or an MPR–independent non-endosomal pathway or both.27 The endosomal
pathway, essential for MPR recycling, has however not been proven to be
involved in filling of the azurophil granules of neutrophils. In addition, these
granules lack the endosome-derived LAMP-1 and LAMP-2 membrane
proteins.29,30 On the other hand, azurophil granules, as well as typical
secretory lysosomes of hematopoietic cells, contain the membrane protein
CD63 (LAMP-3). Consequently, the strategy employed here, that takes
advantage of the sorting signal in CD63 for targeting of sTNFR1 to granules,
should be generally applicable for targeting secretory lysosomes of
hematopoietic precursors.
We have observed previously that retrieval and sorting are not exclusive for
endogenous proteins in hematopoietic cells in as much as constitutive
expression
of
exogenous
proteins
such
as
1-microglobulin
and
lipopolysaccharide binding protein (LBP) resulted in equal sorting for
storage.9,10 On the other hand, we have evidence to suggest that
17
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hematopoietic cells can not route all proteins for storage. For instance,
sTNFR1 that escaped ER-retention and was exported to the Golgi was not
sorted for storage but rather secreted.10 Moreover, when cleavage of
transmembrane sTNFR1 was induced in the Golgi through a furin cleavage
site, constitutive secretion of released sTNFR1 was again observed and not
targeting (data not shown). For this reason it is suggested that not all that is
offered is targeted to granules; what is not accepted for sorting may, in this
case, be secreted. Granule targeting was, however, specifically accomplished
by generating a transmembrane protein with a cytosolic signal for secretory
lysosomes. This signal from CD63 was important, as transmembrane
sTNFR1 without the signal was lost by degradation or constitutive secretion.
Non-native protein, if generated in post-ER compartments, may be
eliminated by accelerated degradation. This seems to be the case for certain
aggregated membrane proteins such as the integral membrane protein furin.
The aggregated form of furin is directed to lysosomal degradation from the
Golgi without reaching the plasma membrane.31 This pathway is consistent
with a quality control for the disposal of aggregated proteins in
compartments of the secretory pathway in addition to those of the ER.
A prerequisite for reliable storage of an exogenous protein prior to its release
through degranulation is stability. Secretory lysosomes have a proteolytic
environment, but proteins that are part of their characteristic dense cores
may resist to proteolysis. In addition, a polyanionic matrix of proteoglycan
is likely to play a protective role for stored proteins.32 In fact, almost equal
protection was observed in RBL cells for the endogenous protein RMCP-II,
the major constituent of the granules of RBL cells, and sTNFR1-tm-Y (data
not shown). Thus, sTNFR1-tm-Y seems to be rather stable in this
environment, except for when the tm-Y fragment is cleaved by proteolysis
18
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resulting in generation of the membrane-free-sTNFR1 in secretory
lysosomes.
Although our results are based on studies of secretory lysosomes in
hematopoietic cell lines, platelets, neutrophils, mast cells, macrophages,
CTLs and NK cells all use compartments with lysosomal properties for
storage and for secretion.33 Thus, these cells, and the hematopoietic cell
lines used in our experiments, have secretory lysosomes. The majority of the
secretory granules of RBL cells biochemically and functionally overlap with
endosomes and lysosomes.27 Even though the environment is similar
between the granules of the cellular models used and the secretory
lysosomes of hematopoietic cells, the biogenesis of the respective granules
may still be different. Therefore, the results reported need to be extended to
neutrophil precursor cells, and other non-transformed hematopoietic
precursor cells. Many cytoplasmic sequences that are sufficient for targeting
membrane proteins to conventional lysosomes also target P-selectin to the
secretory lysosomes of RBL cells.28 While the secretory lysosome content
of NK cells and CTLs is destined for extracellular discharge upon
stimulation, the content of azurophil granules of activated neutrophils is
released both into the phagosome and the extracellular space. Consequently,
the azurophil granule may be particularly suitable for delivering proteins
into phagosomes as a means of promoting antibacterial defense. NK cells
and CTLs on the other hand may be particularly suitable for extracellular
delivery as a means of promoting defense against tumor cells.
In summary, we asked whether one can target exogenous proteins with a
therapeutic potential for granules in hematopoietic cells, that might act as
vehicles for sites of inflammation.The successful targeting of sTNFR1-tm-Y
into secretory lysosomes of hematopoietic cells answers both questions.
19
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Another aspect of the results is that exposure to a proteolytic milieu, such as
that of secretory lysosomes, is not necessarily destructive to an exogenous
protein that is not normally adapted to this environment. If our results from
hematopoietic cell lines can be adopted for cells such as neutrophils, the
latter could be used as vehicle cells for site-specific delivery into inflamed
tissue enabling release both into the phagocytic vacuole and the exterior
milieu. What is more, the principle may be adopted for other cells with
secretory lysosomes e.g. NK cells and CTLs.
Acknowledgements
We would like to thank Hans Janssen and Nico Ong for their expert
technical assistance.
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Legends to figures
Fig. 1. Schematic view of the cDNA constructs used. sTNFR1-tm
corresponds to the sequence for sTNFR1 with a transmembrane domain (tm).
sTNFR1-tm-Y contains the cytosolic sorting signal from CD63 (Y).
24
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25
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Fig.2. Processing and subcellular distribution of sTNFR1-tm. (A) RBL
cells expressing sTNFR1-tm were radiolabeled (pulse) for 1 hour and the
radiolabel chased for up to 12 hours. At the indicated time points, 20 x 106
cells and incubation medium were withdrawn, immunoprecipitated and
analyzed as described in Materials and Methods. The position of sTNFR1tm is indicated with an arrow and a processed form (that is the secreted form)
with a dotted line. Molecular weight markers are shown to the left in kDa.
(B) Cells were biosynthetically radiolabeled for 1 hour (pulse), followed by
a radiolabel chase for 2 hours. At the pulse and chase intervals, 100 x106
cells were removed and homogenized and the postnuclear supernatant was
subcellular
fractionated
by
centrifugation
in
Percoll.
Extraction,
immunoprecipitation and analysis were performed as described in the
Materials and Methods. The densitometry data are shown for pulse (
and chase (
)
) experiments as % of total sTNFR1-tm and processed forms.
26
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Fig.3. Processing of sTNFR1-tm-Y. RBL cells were radiolabeled (pulse)
for 1 hour followed by chase of the label for up to 16 hours. At the indicated
time points, 20 x 106 cells and incubation medium were withdrawn,
extracted, immunoprecipitated and analyzed as described in Materials and
Methods. The position of sTNFR1-tm-Y is indicated with arrow and the
various processed forms are indicated with dotted lines. Molecular weight
markers are shown to the left in kDa.
27
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28
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Fig. 4. Oligosaccharide side chains of processing forms for sTNFR1-tm
and sTNFR1-tm-Y. (A) RBL cells transfected with sTNFR1-tm were
biosynthetically radiolabeled for 60 min (pulse) and chased for 3 hours
before analysis of cell extracts.
Due to the slow secretion, the cell
supernatant was obtained after a 15-hour chase to have enough secreted
sTNFR1-tm for analysis. After pulse and chase, 20 x 106 cells were
withdrawn, and after lysis, subjected to immunoprecipitation with antisTNFR1. Aliquoted immunoprecipitates were incubated either with
Endoglycosidase-H (Endo-H) or N-glycosidase F (N-Glyc), or remained
untreated as control, and analyzed by SDS-PAGE. The pulse cell lysate
showed partial Endo-H sensitivity while the chase cell lysate showed total
Endo-H resistance. The cell supernatant showed Endo-H resistance. The
position of sTNFR1-tm is indicated with an arrow. Molecular weight
markers are shown to the left in kDa. (B) Similar experiments were
performed with RBL cells transfected with sTNFR1-tm-Y. Cells were
biosynthetically radiolabeled for 45 min (pulse) and chased for 3 hours
before analysis of cell extracts. The pulse sample showed partial Endo-H
sensitivity while the chase sample showed total Endo-H resistance. Secretion
was too low to yield enough material for analysis.
29
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30
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Fig.5. Subcellular distribution of radiolabeled sTNFR1-tm-Y (A), and
endogenous RMCP-II (B). RBL cells were biosynthetically radiolabeled
for 0.5 hour (pulse), followed by chase of the label for 2 hours (chase). At
the pulse and chase intervals, 100x106 cells were removed and homogenized
and
the
postnuclear
supernatant
was
subcellular
fractionated
by
centrifugation in Percoll. The fractions were immunoprecipitated and
analyzed as described in the Materials and Methods. The densitometry data
are shown for pulse (
) and chase (
and processed forms of
) experiments as % of total mature
sTNFR1-tm-Y or RMCP-II. The position of
sTNFR1-tm-Y, and RMCP-II are indicated with arrow and the various
processed forms are indicated with dotted lines. Molecular weight markers
are shown to the left in kDa.
31
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Fig. 6. Colocalisation of sTNFR1 with a matrix protein (RMCP) and a
membrane-associated protein (lgp120) of secretory lysosomes. Ultrathin
cryosections from RBL cells transfected with sTNFR1-tm-Y were labeled
accordingly. A. Labeled with rabbit anti-sTNFR1. B. Double labeled with
rabbit anti-sTNFR1 (10 nm) and mouse anti-RMCP (15 nm). C. Double
labeled with rabbit anti-sTNFR1 (10 nm) and rabbit anti-lgp 120 (15 nm).
A. Area of a cell showing granules highly labeled with sTNFR1. The Golgi
(G) and the trans Golgi network (TGN) are also labeled. B. Granules are
shown double labeled for sTNFR1 and RMCP on the matrix. C. A granule is
seen with labeling for lgp 120 on the membrane and for sTNFR1 on the
matrix. Nucleus (n). Bar, 200 nm.
32
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Fig. 7. Subcellular localization of sTNFR1-tm and sTNFR1-tm-Y by
immunofluorescence microscopy. Indirect immunolocalization of the TNFR and a marker for secretory lysosomes, lgp120. The cells used were RBL
cells transfected with sTNFR1-tm-Y (A-D), sTNFR1-tm (E-H), and wild
type RBL cells (I-L). The cells were fixed, permeabilized and stained, as
described in the Methods section, before being attached to poly-lysine
coated cover-slips and the images recorded. Briefly, the cells were incubated
with a rabbit anti-lgp120 antibody and a mouse monoclonal antibody against
the sTNFR1. Thereafter, the cells were stained with Alexa Fluor
-labeled
anti-rabbit and anti-mouse secondary antibodies. The localization of TNF-R
and lgp120 is shown in red and green, respectively, whereas the rightmost
images show an overlay of the red and green staining patterns. The
corresponding Nomarski images are also shown. Results are representative
of 4 separate experiments. (Bar = 10 µM).
33
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Prepublished online March 20, 2003;
doi:10.1182/blood-2002-10-3055
Sorting of soluble TNF-receptor for granule storage in hematopoietic
cells as a principle for targeting of selected proteins to inflamed sites
Ying Gao, Hanna Rosen, Ellinor Johnsson, Jero Calafat, Hans Tapper and Inge Olsson
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