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REVIEW ARTICLE
Hematopoietic Differentiation Antigens That Are Membrane-Associated
Enzymes: Cutting Is the Key!
By Margaret A. Shipp and A. Thomas Look
M
ANY CELL SURFACE glycoproteins expressed during specific stages of lymphoid and myeloid cell development were initially characterized on the basis of their
reactivity with monoclonal antibodies (MoAbs). Subsequent analysis of cloned cDNAs identified four of these hematopoietic differentiation antigens as membrane-associated enzymes with common structural and regulatory
features: CDlO/neutral endopeptidase 24.11 (CDlO/NEP),
CD 13/aminopeptidase N (CD 13/APN), BP- 1/6C3/aminopeptidase A (BP-1/6C3/APA), and CD26/dipeptidyl peptidase IV (CD26/DPPIV).’-9All four of these enzymes are
type I1 integral membrane proteins with a single hydrophobic sequence that functions as both a signal peptide and a
transmembrane region.’-’ Three of these enzymes (CD10/
NEP, CD 13/APN, and BP- 1/6C3/APA) require zinc for biologic activity and share a pentapeptide consensus sequence
that has been implicated in both zinc binding and catalys ~ s . ~Although
,~,’
CDlO/NEP, CD13/APN, BP-l/6C3/APA,
and CD26/DPPIV are expressed by different hematopoietic
cell types at unique stages of lymphoid or myeloid differentiation, they are coexpressed on the luminal surface of epithelial brush borders. All four enzymes participate in the
final stages of peptide hydrolysis in the renal proximal tubules and the small intestine. In certain instances, these enzymes also work in concert to digest common substrates.*-l2
CD73/ecto 5’-nucleotidaseis another hematopoietic differentiation antigen and cell surface enzyme that is also expressed on epithelialbrush borders. A glycosyl phosphotidylinositol-linked protein, CD73/ESN, differs from the
above-mentioned enzymes in its transmembrane orientation and its role in nucleoside dephosphorylation and trans~0rt.I~
The well-characterizedactivities of these enzymes in non-
From the Department of Medicine, Dana-Farber Cancer Institute, Haward Medical School, Boston, MA; the Department of Experimental Oncology, St Jude Children ‘s Research Hospital and the
Department of Pediatrics, University of Tennessee College ofMedicine, Memphis, TN.
Submitted March 10, 1993; accepted May 26, 1993.
Supported in part by National Institutes of Health Grants No.
CA-55095 (M.A.S.), CA-42804 (A.T.L.), and CA-21765 (A.T.L.),
and by the American Lebanese Syrian Associated Charities (ALSAC) of St Jude Children’s Research Hospital (A.T.L.). M.A.S. is
the recipient of an American Cancer Society Junior Faculty Award.
Address reprint requests to Margaret A. Shipp, MD, Dana-Farber
Cancer Institute, 44 Binney St, Boston, M A 021 15.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solelyto
indicate this fact.
0 1993 by The American Society of Hematology.
0006-49 71/93/8204-003 7$3.00/0
1052
hematopoietic tissues have stimulated research into their
potential regulatory roles in hematopoiesis. Recent studies
implicate these proteins in a variety of processes including
stromal cell-dependent B 1ymphopoiesis,”l6 peptide-mediated inflammatory response^,'^^'^ and T-cell activat i ~ n . ’ ~Although
*~’
peptide or nucleotide cleavage remains
the best-known activity of each enzyme, CDl3/APN,
CD26/DPPIV, and CD73/ESN have additional nonenzymatic roles, as illustrated by CD 13/APN, which serves as a
coronavirus receptor.”
We review here recent progressin understanding the structural features, patterns of expression, and biologic functions
of these hematopoietic differentiation antigens and ectoenzymes, emphasizing their roles in the regulation of hematopoiesis.
CDlO/NEP (EC 3.4.24.1 1)
Pattern of Expression
The common acute lymphoblastic leukemia antigen
(CALLA; CD10) was originally identified with heterosera
and subsequently with MoAbs as a 100-Kd tumor-associated cell surface antigen expressed by the majority ofacute
lymphoblastic leukemia^.^'*'^ Additional lymphoid malignancies, including lymphoblastic, Burkitt’s, and nodular
poorly differentiated lymphocytic lymphomas, as well as
chronic myelogenous leukemias in lymphoid blast crisis,
were CDIOf, whereas acute myelogenous leukemias and
lymphomas with a mature B- or T-cell phenotype lacked
CD 10 expre~sion.’~
For this reason, CD 10was a useful diagnostic marker long before its function was known.24 Although it was originally considered to be a tumor-specific
antigen, CDlO was subsequently identified on normal early
human lymphoid progenitorswithin the bone marrow, fetal
liver, and thymus (Table 1). The phenotypic characteristics
of these CDlO+cells indicated that they were either uncommitted to the B- or T-cell lineage or committed to only the
earliest stages of B-cell differentiati~n.~”~~
In fact, the expression of CDlO on certain lymphoid malignancies was
thought to reflect the restricted expression of the antigen
during the earliest stages of normal lymphoid differentiat i ~ nHowever,
.~~
CDlO was also identified on multiple myelomas with an immature histologic appearance, suggesting
that there may be discordant expression of the antigen in
certain B-cell malignan~ies.~~”~
CD 10 was also expressed on terminally differentiated
granulocytes and a variety of nonhematopoietic cell types,
including bronchial epithelial cells, cultured fibroblasts,
bone marrow stromal cells, renal proximal tubular epithelial cells, breast myoepithelium, biliary canaliculi, fetal intestine, and certain solid tumor cell lines (Table 1),34,4”6
indicating that the biologic function of the antigen was not
restricted to lymphoid development.
Blood, VOI 82, NO 4 (August 15). 1993: pp 1052-1070
B-cell progenitors
Abelson-transformed
lymphoblastoid lines
Immature thymocytes
Subpopulations of
peripheral blood T cells
Splenic B cells
EBV-transformed
lymphoblastoid lines
B-cell lymphomas and
surface lg+ acute
lymphoblastic leukemias
Subpopulations of
peripheral B and T cells
EBV-transformed
lymphoblastoid lines
Many pre-B and progenitor
B-cell leukemias
Reduces cellular responses
to peptide hormones
Reduces cellular responses
to peptide hormones
Adhesion molecule
Regulates uptake of
extracellular purines
Completes processing of
extracellular ATP by
dephosphorylating AMP
to adenosine
BP-l/GCB/APA
CDZB/DPPIV
CD73/ESN
Abbreviations: CML, chronic myeloid leukemia; CNS, central nervous system.
Myeloid progenitors (CFUGM)
Monocytes
Granulocytes
Myeloid leukemias
Reduces cellular responses
to peptide hormones
Serves as coronavirus
receptor
CD 13/APN
Tissue Distribution
Lymphoid progenitors
Granulocytes
Acute lymphoblastic
leukemias
Certain nodular,
lymphoblastic, and
Burkitt’s lymphomas
CML in lymphoid blast
crisis
Certain myelomas
General Functions
Reduces cellular responses
to peptide hormones
Enzvme
CDl O/NEP
Tissue Distribution
Reaulatorv
. Function
Limits analgesic effects of
opioid peptides
Mediates coronavirus-induced
upper respiratory tract
infections and gastroenteritis
Processes peptides at the
intestinal and renal proximal
tubular brush borders
Regulates cardiovascular
homeostasis
Processes peptides at the
intestinal and renal proximal
tubular brush borders
Processes peptides at the
intestinal and renal proximal
tubular brush borders
Completes the hydrolysis of
extracellular 5’ AMP to
adenosine in intestinal crypt
abscesses
Renal proximal tubular
epithelial cells
Small intestinal epithelium
Bone marrow stromal cells
and subpopulation of
thymic cortical epithelial
cells
Placenta
Renal proximal tubular
epithelial cells
Small intestinal epithelium
Biliary canaliculae
Alveolar pneumocytes
Small intestinal epithelium
Mesynchymal cells
Follicular dendritic reticular
cells
Hepatocytes
?
Mediates antigen-induced Tcell proliferation
Maturation marker of B and T
cells
Mediates generation of
cytotoxic T cells
Regulates neutrophil
inflammatory responses
Limits the analgesic effects of
opioid peptides
Regulates peptide-mediated
inflammatory responses
including secretory diarrhea
and neurogenic inflammation
of the lungs
Inhibits the autocrine growth of
small cell carcinomas of the
lung
Regulates the proliferation and
maturation of normal fetal
bronchial epithelium
Regulates cardiovascular
homeostasis
Processes peptides at the
intestinal and renal proximal
tubular brush borders
Renal proximal tubular
epithelial cells
Small intestinal epithelium
Biliary canaliculae
Bone marrow stromal cells
Fibroblasts
CNS synaptic membranes
Placenta
Osteoclasts
Renal proximal tubular
epithelial cells
Small intestinal epithelium
Biliary canaliculae
Bone-marrow stromal cells
Fibroblasts
CNS synaptic membranes
Placenta
Bronchial epithelial cells
Breast myoepithelium
Certain solid tumor cell
lines
Placenta
Nonhematopoietic Cells
Limits neutrophil inflammatory
responses
Promotes monocyte antigen
processing
Reciulatorv
. Function
Regulates stromal celldependent B lymphopoiesis
Limits neutrophil inflammatory
responses
Augments IL-2 production by
certain T cells
Hematopoietic Cells
Table 1. Tissue-Specific Expression and Functions of Hematopoietic Differentiation Antigens Identified as Ectoenzymes
w
cn
0
A
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SHIPP AND LOOK
1054
Biochemical and Moleciilar Characterization
Biochemical analysis of CDIO/NEP suggested that the
glycoprotein contained -20% to 25% carbohydrate and
that there were tissue-specific differences in its posttranslational modification?’ In fibroblasts and kidney, the CDI O/
NEP molecular mass was -90 Kd,”.42 whereas CD 1 O/NEP
in lymphoid progenitors and neutrophils was 100 Kd and
1 10 Kd,
The CDlO cDNA sequence predicts a 750 amino acid
type I1 integral membrane protein with a single 24 amino
acid hydrophobic segment that functions as both a transmembrane region and a signal peptide (Fig
The
COOH-terminal700 amino acids compose the extracellular
region, whereas the 25 NH,-terminal amino acids remaining after cleavage of the initiation methionine form the cytoplasmic tail (Fig l).’v3 There are six potential N-linked
glycosylation sites in the extracellular domain, suggesting
that the tissue-specificdifferences in CDIO molecular mass
may be related to glycosylation patterns. CD I O transcripts
range in size from 2.7 to 5.7 kb, with majortranscriptsof 3.7
and 5.7 kb’; the multiple mRNAs result, at least in part,
from the use of multiple polyadenylation signals in the 3’
untranslated region.’
The CDlO cDNA sequence is virtually identical to the
reported cDNA sequence for a cell surface zinc-dependent
metalloprotease, neutral endopeptidase 24.1 1 (NEP, “enThe CDlO cDNA was
kephalinase,” EC 3.4.24. I 1).’-3*48*50
formally shown to encode this enzyme by generating
CDIO’ stable transfectants that had cell surface NEP enzymatic activity., In all cell types examined to date, the levels
of the cell surface enzyme correspond to the levels ofdetectable CD 1 O/NEP transcript^.'^*^'-^^
The enzyme was first described in 1974 as a neutral metalloendopeptidase from rabbit kidney brush b ~ r d e r , ~ ~ . ~ ~
and subsequently in 1978 as an “enkephalinase” from
brain.56Thereafter, the separatelycharacterized neutral metalloendopeptidase and “enkephalinase” were shown to be
the same enzyme in a variety of t i s s ~ e s . ~Although
~ * ~ * cell
types including fibroblasts, reticular cells, bronchial epithelial cells, and peripheral blood granulocytes and tissues including placenta and intestine were known to express NEP,
the presence of the enzyme on lymphoid cells was not confirmed until CDlO and NEP were found to be identicaL2s3
CD I O/NEP hydrolyzes a variety of physiologically active
peptides, including chemotactic peptide (fMLP), substance
P, atrial natriuretic factor, endothelin, neurotensin, oxy-
-
-
COOH
co
KEY
69 aa
waa
Dmltide Bond
c
COOH
-.
--
8
c
8
*
.
*
’t
Active Site
(H-E-I-T-H)
Extracellular
Domain
c
c
8
c
8
Site
NH
523 88
I Activesite
(H-E-L-V-H)
(H-E-L-A-H)
-
GIyc&ylation
..
e
c
3 Activesite
-8
COOH
c
‘-? TS
-.
j
c
c
c
SUB-!
c
aa
.
.
,.::.:.
.......,.
..;....;:...
... ,.;>;.:;., .
.:..:.,:..;..
.; ’.,.
.:.,:;.,:.......
2
CD101
NEP
CD131
APN
BP-116C3 /
APA
CD26 I
DPPIV
CD731
E5N
Fig 1. Molecular structures of CD1O/NEP, CD13/APN, BP-l/GCB/APA, CD26/DPPIV, and CD73/E5N. The size, monomeric or dimeric
structure, and orientation of each protein with respect to the cytoplasm. transmembrane (TM) region, and extracellular domain are shown.
Proven or potential active sites are indicated, as are zinc-binding (ZB), substrate-binding (SUB), and potential tyrosine sulfation (TS) sites.
Glycosylation sites, cysteines, and known disulfide bonds are included.
Opioid peptides, fMLP, substance P,
bombesin-like peptides, atrial
natriuretic factor, endothelin,
oxytocin, bradykinin, angiotensins
I and II
Opioid peptides, enkephalins,
tuftsin, fMLP
Angiotensins, ?IL-7
Substance P, casomorphin, kenzin,
a chain of fibrin, growth hormone
releasing hormone
Purine and pyrimidine ribonucleoside
and deoxyribonucleoside
monophosphates
CD13/APN
BP-I/GCB/APA
CDPB/DPPIV
CD73IE5N
Known Substrates
CDlO/NEP
Enzyme
Catalyzes the dephosphorylation
of purine and pyrimidine
ribonucleoside and
deoxyribonucleoside
monophosphates
Cleaves Xaa-Pro and Xaa-Ala
dipeptides from the NH,
terminus of polypeptides
and proteins
Catalyzes the removal of NH,
terminal acidic amino acids
from peptides
Catalyzes the removal of NH,
terminal neutral amino
acids from peptides
Cleaves small peptides on the
NH, terminal side of
hydrophobic amino acids
Enzymatic Activity
NH,
NH,
1
Phe
Leu
Glu
Pro
-
IHI
-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa
IHI
Xaa-Xaa-Xaa
TYr -Xaa-Xaa-Xaa-Xaa-Xaa-Xaa
Ala
Phe
-
...Xaa-Xaa-Xaa-
1
R
Cleavage Site t i )
...
-
COOH
COOH
COOH
COOH
Table 2. Hematopoietic Differentiation AntisenslCeIl Surface Enzvmes: Known Substrates, Enzvmatic Activities, and Inhibitors
5-diphosphate
a,p methylene adenosine-
Di-isopropyl fluorophosphate,
diprotin A
Bestatin, amastatin
Bestatin, amastatin, actinonin
Phosphoramidon, thiorphan,
SCH32615
Inhibitors
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SHIPP AND LOOK
1056
tocin, bradykinin, angiotensins 1 and 2, the bombesin-like
peptides, and the opioid peptides met- and leu-enkephalin
(Table 2).59-66
Comparison of the CDlO/NEP cDNA sequences from
several human, murine, rat, and rabbit sources, including
leukemic and bone marrow stromal cell lines, kidney, brain,
and placenta, indicates that the gene has been tightly conserved throughout evolution. Additional studies also predict the existence of a CDlO/NEP invertebrate homo10gue.l~ There is approximately 90% identity of the
predicted CD 10/NEP amino acid sequences and 100%conservation of critical functional motifs.'-3~48,49~5'
The CD10/
NEP sequence includes 12 conserved cysteine residues in
the extracellular domain that are known to participate in
the formation of disulfide bridges required for CD 1O/NEP
structure and enzymatic activity (Fig l).67
The CD 1O/NEP extracellular segment also contains the
characteristic pentapeptide sequence (His-Glu-[Ile, Leu,
Met]-Xaa-His) associated with zinc binding and catalytic
activity in a variety of cell surface and secreted zinc-dependent metalloproteases (Fig 1).68This pentapeptide consensus sequence resides within a region of homology between
CD 1O/NEP and thermolysin, a bacterial metalloprotease
with similar substrate specificity and known crystallographic structure.69 Specific CD lO/NEP amino acids were
predicted to facilitate substrate binding, function as zinccoordinating residues, and promote catalysis because they
were homologous to specific residues in the active site of
thermolysin. The contribution of these amino acids to
CD 1O/NEP enzymatic activity was further characterized by
site-directed mutagenesis (Fig 1).70.72 Additional amino
acids required for zinc binding, catalytic activity, and substrate binding have also been identified (Fig 1).73-75
The similarities between amino acids comprising the active sites of
thermolysin and C D 1O/NEP68*69,73,76
allowed the construction of a three-dimensional model of the CD lO/NEP active
site" that provides preliminary insights into the nature of
the zinc-dependent substrate binding and catalysis.
The CDlO/NEP gene spans more than 80 kb and is composed of 24 exons.77The first two exons encode 5' untranslated sequences, whereas the third exon encodes the initiation codon, cytoplasmic domain, and transmembrane
region.77Twenty short exons (exons 4 through 23), ranging
in size from 36 to 162 bp, encode most of the extracellular
portion of the enzyme, and a single large exon (exon 24)
encodes the COOH-terminal portion of the protein and the
entire 3' untranslated region.77The structures of three previously characterized C D 1O/NEP cDNAs suggested that they
result from alternative splicing of unique 5' untranslated
region sequences into a common exon.' The CDlO/NEP
genomic organization confirmed this alternative splicing
pattern and raised the possibility that different regulatory
elements control the tissue specificity and developmentally
regulated expression of the enzyme.77
The human CDlO/NEP gene maps to chromosome
3q2 1-27, a region that contains the genes for multiple metal
binding proteins and additional type I1 integral membrane
proteins.78379
Although rearrangements of the 3q2 1-26 region have been associated with certain dysmyelopoietic syn-
dromes," to date no rearrangements of the CDlO/NEP
locus have been identified in hematopoietic malignancies
(M. Shipp et al, unpublished data)." The murine CDlO/
NEP gene is localized to the proximal half of chromosome
3.5l
Biologic Activity of CD1O/NEP
General principles. To evaluate C D I O/NEP function in
various organ systems, investigators have used a panel of
compounds that specifically inhibit C D 1O/NEP enzymatic
activity (phosphoramidon, thiorphan, SCH326 15; Table
2). l4,15,17,61,82-84 When these CD lO/NEP inhibitors are added
to in vitro or in vivo assays, the effect is that of increasing
the local concentrations of the relevant CD IO/NEP peptide
substrate(s). In all of the cell types in which CDlO/NEP
function has been studied, the enzyme appears to reduce
cellular responses to peptide hormones (Table 1). Target
cells express both the cell surface enzyme and the receptor
for the relevant CDlO/NEP peptide substrate (Fig 2). By
hydrolyzing the peptide substrate, CD lO/NEP reduces the
local concentrations of peptide available for receptor binding and signal transduction (Fig 2A).'4J5,6'*83-87
For example, CD 1O/NEP reduces atrial natriuretic factor-mediated
hypotension and diuresis,6' enkephalin-mediated analge~ i aand
, ~peptide-mediated
~
inflammatory responses (Table
I ). 17,59,83,85 The concentrations of enkephalin needed to generate a neutrophil inflammatory response differ by several
orders of magnitude in the presence or absence of the cell
surface enzyme.I7 CDlO/NEP similarly affects the required
concentrations of other inflammatory peptides such as substance P and fMLP.59,8338s,86
The cough, bronchial constriction, vasodilation, and neutrophil migration associated with
substance P-mediated neurogenic inflammation of the
lungs are significantly increased when cell surface CDlO/
NEP is inhibited (Table l).83,86In certain instances, inflammatory peptides such as fMLP are initially cleaved by
CD 1O/NEP and further hydrolyzed by CD 13/APN (Fig
2B). This pattern of cooperative cleavage of peptide substrates by CDlO/NEP and CDl3/APN occurs in a variety of
tissues.
Regulation of cellular proliferation. Recent studies suggest that CD 1O/NEP also regulates peptide-mediated cellular p r ~ l i f e r a t i o nThe
. ~ ~ enzyme hydrolyzes bombesin-like
peptides (BLP), which are potent mitogens for fibroblasts
and normal bronchial epithelial cells and essential autocrine
growth factors for many small-cell carcinomas of the lung.87
The growth and proliferation of BLP-dependent small cell
carcinomas is inhibited by CD 10/NEP and potentiated by
CD 1O/NEP inhibiti~n.'~
These results are ofparticular interest because ( 1 ) small-cell carcinomas of the lung occur almost exclusively in cigarette smokerss8; (2) smokers have
increased levels of BLP in their bronchoalveolar lavage
fluid"; (3) cigarette smoke inactivates CD10/NEP9O; and (4)
small-cell carcinomas have reduced levels of the cell surface
enzyme.87 Thus, reduced CD 1O/NEP enzymatic activity
may promote BLP-mediated proliferation of pulmonary
neuroendocrine cells and facilitate the development of
small-cell carcinomas ofthe lung.87For this reason, the characterization of a lung cancer-derived CDlO/NEP cDNA
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ANTIGENS THAT ARE MEMBRANE-ASSOCIATED ENZYMES
1057
P
Fig 2. Biologic functions of CD1O/NEP, CD13/
APN, and BP-l/6C3/APA. (A) CDlO/NEP, CD13/
APN, and BP-l/6C3/APA downregulate cellular
responses to peptide hormones by hydrolyzing
available peptide substrates. (B) CD1O/NEP and
CD13/APN cooperate to hydrolyze fMLP and regulate associated neutrophil inflammatory responses.
that lacks an exon and encodes a cell surface protein with
virtually no enzymatic activity is of particular interest?'
The temporal and cellular patterns of C D IO/NEP expression in developing fetal lung also implicate the enzyme in
the regulation of BLP-mediated normal lung
The
enzyme is expressed at the highest levels in early fetal lung
development and is primarily found on cells that are most
sensitive to the effects of BLP, the undifferentiated airway
epithelium in the distal developing airways.84These observations led to in vitro and in vivo studies that directly linked
the enzyme with peptide-mediated bronchial epithelial development (Table l).84.92 In human fetal lung organ cultures, inhibition of cell surface CDIO/NEP resulted in a
significant increase in DNA synthesiss4and, in related murine studies, the in utero administration of a specific longacting CD IO/NEP inhibitor also increased the proliferation
and maturation of developing murine fetal lungs.92
CDlO/NEP Junction in 1-vmphoid development. Although CDIO/NEP has a defined function in many organ
systems, until recently little was known about the enzyme's
role in lymphoid ontogeny. The absence of suitable human
models for the study of lymphoid CDIO/NEP prompted
investigators to turn to murine systems in which discrete
stages of B-cell development and models of in vitro and in
vivo lymphoid differentiation were well-characterized. The
majority of murine pre-B cells lacked CDIO/NEP expression; however, a subpopulation of Thy- 1'OWB220+murine
lymphoid progenitors from bone marrow and modified
Whitlock-Witte cultures had low levels ofCDlO/NEP enzymatic activity and C D IO/NEP transcripts that were detectable using RNA-based polymerase chain rea~ti0n.I~
These
Thy-1'OWB220+lymphoid progenitors were of particular interest because they were large cycling cells that could effectively reconstitute the B-lymphoid compartment of lethally
irradiated animals.93 Murine bone marrow stromal cells
that supported the B-cell differentiation of hematopoietic
progenitors including Thy- I'OWB220+cells also expressed
high levels of C D 1 O/NEP.'4,5' For these reasons, CDIO/
NEP function was evaluated in modified Whitlock-Witte
cultures in which uncommitted hematopoietic progenitors
were plated on a stromal cell line that supports the earliest
stages of B-cell differentiati~n.'~
In such assays, in which
uncommitted progenitors differentiate into Thy- llowB220+
pro-B cells and Thy-I-B220+ pre-B cells, inhibition of
C D 1O/NEP significantly increased early lymphoid colony
f ~ r m a t i o n .The
' ~ effects of CDlO/NEP inhibition on early
lymphoid development were further assessed using an in
vivo congenic mouse model in which uncommitted hematopoietic progenitors from donor animals were used to rescue
sublethally irradiated recipients treated with a specific longacting CDIO/NEP inhibitor (SCH326 1 5).15 The lymphoid
reconstitution of animals treated with the CDIO/NEP inhibitor or vehicle alone was compared by analyzing the percentages of donorderived hematopoietic cells, B-lymphocytes,
and mature B cells in the lymphoid organs in both sets of
animals." SCH326 I 5-treated animals had significantly
more donor-derived splenocytes, splenic B cells, and mature (sIg+) splenic B cells than animals treated with vehicle
alone, suggesting that inhibition of C D IO/NEP increased
the proliferation and maturation of splenic B cells.'5 Taken
together, the in vitro and in vivo studies suggest that CDIO/
NEP functions to regulate B-cell development by inactivat-
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1058
SHlPP AND LOOK
ing a peptide that stimulates B-cell proliferation and differentiation (Table l).'4,'5Alternatively, the enzyme may activate a propeptide that inhibits B-cell proliferation and
differentiation. These new insights into the role of the enzyme in regulatingearly lymphoid proliferation are of particular interest because patients with CALLA' acute lymphoblastic leukemias were originally thought to have more
favorable prognoses." These studies also underscore the
similarities between the growth-regulatoryeffects of the enzyme on lymphoid progenitors and developing epithelial
cells. l4,15,84.92
Although the majority of T cells have undetectable
CD 10/NEP transcripts and cell surface protein, a subclone
of the T-cell leukemia line Jurkat was recently reported to
express low levels of the enzyme.95This CD lO/NEP+ T-cell
subclone produced substantially lower levels of interleukin2 (IL-2) when it was activated in the presence of specific
CD 1O/NEP enzymatic inhibitors or an anti-CD 1O/NEP
MoAb, prompting speculation that CD IO/NEP may also
function to regulate IL-2 production in certain T cells (Table
Regulation of the enzyme in response to peptide stimulation. In early studies, the treatment of CDlO/NEP' cells
with an anti-CD10 antibody resulted in the rapid cell surface redistribution, internalization, and degradation of the
Subsequently, a variety
CD 1O/NEP-antibody
of inflammatory mediators were found to modulate the levels of the cell surface enzyme. Treatment of CDIO/NEP+
lymphoid, myeloid, fibroblastoid, or epithelial cells with
phorbol esters decreased cell surface CD 1O/NEP expression98-100.
, in several cell types, the cell surface protein was
rapidly internalized and the CD 1O/NEP transcripts were
also
Epstein-Barr virus (EBV)-negativeB-cell
lymphoma cell lines transfected with the EBV-associated
latent membrane protein also lost cell surface CD IO/NEP
expression.'" In contrast, inflammatory mediators such as
tumor necrosis factor, MLP, granulocyte-macrophagecolony-stimulating factor (GM-CSF), C5a, and calcium ionophore increased cell surface CD 10/NEP enzymatic activity
in human neutrophil^.^^.'^^ In additional studies, glucocorticoid treatment of transformed human tracheal epithelial
cells increased CD I O/NEP enzymatic activity and transcript~."~
Investigators postulated that steroids might exert
their anti-inflammatory effects in part by upregulating cell
surface CD10/NEP,96because the enzyme was known to
reduce substance P-mediated neurogenic inflammation of
the l ~ n g s . ~ ~ , ~ ~
Recent studies also associate the levels of CD lO/NEP enzymatic activity with cell growth. Human tracheal bronchial epithelial cells and non-B, non-T lymphoblastic leukemias express increased levels of CD I O/NEP at increased cell
density,'02,'04suggesting that one mechanism for limiting
peptide-mediated cell growth may be upregulation of the
cell surface enzyme.
'
CDlS/APN (EC 3.4.11.2)
Pattern of Expression
CD13 was originally identified as a 150-Kd cell surface
glycoprotein (gp I 50) expressed by committed granulo-
cyte-monocyte progenitors (CFU-GM) and by cells of the
granulocytic and monocytic lineages at all morphologically
distinct stages of differentiation (Table l).'05-'09The glycoprotein is also expressed by leukemic blasts in a high percentage of acute myeloid leukemia^'^^.''^''^ and by a smaller
subset of acute lymphoid leukemias (Table 1).""''6 The
molecule appears to be highly immunogenic; 17 MoAbs
individually derived from mice immunized with human myeloid cells bound CD13 epitopes in studies reported at the
Third International Workshop.'17 Antibodies specific for
CD 13 do not bind to normal B or T lymphocytes, but do
react with nonhematopoietic cells including fibroblasts,
bone marrow stromal cells, osteoclasts, and cells that line
renal proximal tubules, small intestine, and bile duct canaliculae (Table l).'" The significance ofthis pattern of CD13
expression became clear when the CDl3 cDNA sequence
was found to be identical to that of aminopeptidase N
(APN; EC 3.4. I 1.2), a prominent membrane-anchored metallopeptidase expressed by the brush borders of the small
intestine and renal t ~ b u l e s . ~ ~ "Although
* ~ ' ' ~ CD 13/APN
and CDIO/NEP are expressed by many of the same nonhematopoietic cell types,'20x'2'the former is not found on early
lymphoid pre~"sors,'~~~'''
whereas the latter does not appear on monocytes or committed myeloid precursors (Table l).'05,'17
Biochemical and Molecular Characterization
Biochemical analysis. CD 13-specific antibodies immunoprecipitate 130-Kd and 150-Kd glycoproteins (gp130
and gp 150) from human myeloid leukemia cell lines."8J22
Metabolic labeling studies established that gp 130 is an intracellular precursor of gpl50 that differs only in the composition of its carbohydrate chains. When cells were labeled in
the presence of tunicamycin, which blocks the addition of
asparagine-linked oligosaccharide chains, a single unglycosylated polypeptide of 1 I O Kd was immunoprecipitated.'18,'22Comparison of labeled tryptic cleavage products
of gp 130 and gpl50 molecules confirmed that these glycoproteins have an identical primary structure and are thus
posttranslationally modified products of a single gene.4
The biosynthesis of APN was independently characterized in epithelial cells from the small intestine and renal
proximal t u b ~ l e s . ' ~ Pulse-chase
~,'~~
experiments showed
that newly synthesized molecules of approximately 140 Kd
contained cotranslationally added asparagine-linked oligosaccharide chains rich in mannose. Within 30 to 60 minutes
of synthesis, the side chains were remodeled to form complex oligosaccharides characteristic of the mature 160-Kd
molecule on the cell surface. Endoglycosidase H treatment
of the immature form of the molecule removed the highmannose oligosaccharide chains, producing a polypeptide
backbone of 1I5 Kd.
APN is expressed as a homodimer on the surface of pig,
rat, rabbit, and human intestinal epithelial cell^.^^'^'^'^^
More recent studies by Danielsen'Z5,'26show that APN
forms non-disulfide-linked homodimers intracellularly before Golgi-associated processing and suggest that dimerization may be required for transport of the high-mannose intracellular form of APN out of the endoplasmic reticulum.
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ANTIGENS THAT ARE MEMBRANE-ASSOCIATED ENZYMES
Although myeloid cells display APN uniformly over the cell
surface, epithelial cells and hepatocytes primarily express
the enzyme on the apical surface. Cell type-specific pathways are responsible for this asymmetric expression, because APN is directly sorted to the apical membrane in
canine kidney cells, but indirectly sorted via the basolateral
membrane in hepatocyte^."^ The targeting signal in the canine kidney cells is located in the extracellular catalytic domain of the glycoprotein, rather than the intracellular domain, transmembrane domain, or the serine- and
threonine-rich talk.'^','^^
Predicted amino acid structure. Human APN and
CD 13cDNA clones from intestinal epithelium and myeloid
cells predict an identical type I1 integral membrane protein
of 967 amino acids with 1 I potential sites of asparaginelinked oligosaccharide addition (Fig l).4,'19These findings
account for the 1 IO-Kd molecular size of the unglycosylated
polypeptide and the additional mass attributable to the carbohydrate moiety of the cotranslationally modified glycoprotein."8s122
Analysis of amino-terminal protein sequences
indicated that CD 13/APN molecules are synthesized with
an uncleaved signal sequence and that proteolytic processing at the amino terminus is limited to removal of the initiator methionine. The retained signal sequence also functions
as the membrane-spanning segment, orienting the CDI 3/
APN amino terminus inside and the carboxyl terminus outside the cell in a configuration common to a subset of receptors and membrane-bound enzymes, including CD 10/NEP
(Fig 1).',L3GL35
The large extracellular carboxy-terminal domain contains a pentapeptide signature sequence (His-GluLeu-Ala-His) characteristic of members of the zinc-binding
metalloprotease family (Fig 1).2,3,48,68,'36.'37
Regulation of expression. Little is known about the
mechanisms that regulate the lineage-restricted expression
of cell surface metalloproteases. Nevertheless, it is of interest that CDI3/APN and CDlO/NEP are coexpressed by
many of the same nonhematopoietic cells, although the two
enzymes are expressed by different subsets of hematopoietic
cells. Epithelial cells of the renal proximal tubule and small
intestine, granulocytes, bone marrow stromal cells, and synaptic membranes in the central nervous system expressboth
enzymes, whereas monocytes and committed myeloid progenitors express only CD 13/APN, and early lymphoid progenitors express only CD I O/NEP (Table I ). Because CD 13/
APN and CD 1O/NEP are both type I1 metalloproteaseswith
multiple alternativelyspliced 5' untranslated regions,',4,77~138
it is possible that distinctive regulatory elements control the
expression of these enzymes in different tissues.
In the case of CD13/APN, Shapiro et all3' identified separate promoters that control transcription in myeloid and
intestinal epithelial cells and regulate independent transcripts that differ only in their 5' untranslated sequences. In
small intestinal epithelial cells, transcription is controlled by
a classical promoter containing a TATA box immediately
upstream from the translation initiation codon. This promoter, which has been characterized by Olsen et al,139appears to direct the expression of very high levels of APN on
intestinal brush border membra ne^.^*^*'^' By contrast, the
longer transcripts found in myeloid cells and fibroblastsorig-
1059
inate from several sites clustered in an upstream exon located 8 kb from the exon containing the initiator codon;
these longer RNAs include sequences from a unique untranslated exon.I3' Regulatory elements of the promoter
used by epithelial cells, including the TATA box and the
transcription origin, are included within the 5' untranslated
region of the longer myeloid cell/fibroblast t r a n s ~ r i p t . ' ~ ~
Both aminopeptidase N transcripts encode the same polypeptide, indicating that the physically distinct promoters
must have evolved to regulate expression of this cell surface
peptidase by cells of different tissues.'38
Enzymatic Activity and Functional Roles
CD13/APN catalyzes the removal of NH,-terminal
amino acids from peptides; although the enzyme has a preference for neutral amino acids, it will also cleave basic and
acidic residues' (Table 2). Natural CDI 3/APN substrates
appear to be small peptides rather than larger proteins, although the enzyme is more effective in removing residues
from oligopeptidesthan dipeptides (Table 2).9 Inhibitors of
CDl3/APN activity that have been useful in characterizing
the enzyme's function include bestatin, amastatin, and actinonin (Table 2).14' In the intestinal brush border, the
CD 13/APN carboxy-terminal enzymatic domain faces the
lumen and most likely plays an important role in the final
stages of the digestion of small peptides.','' In other tissues,
the enzyme has been postulated to interact with CD 1O/NEP
to modulate signal transduction by bioactive peptides (Fig
2).'-12 The two membrane-bound enzymes collaborate in
the hydrolysis of oligopeptides in the small intestine8,' and
appear to act in concert to inactivate opioid peptides and
enkephalins in the
as well as t u f t ~ i n ' ~and
. ' ~ the
~
chemotactic peptide Met-Leu-Phe during neutrophil-mediated inflammatory responses (Fig 2B).'433'"
CD13/APN also serves as a receptor for coronaviruses,
which are RNA viruses that cause respiratory disease in humans and several species of animals (Table 1).'45J46 In humans, coronaviruses are responsible for 15%to 20% of common upper respiratory tract infections, and probably also
cause gastroenteritis. Recent studies show that two coronavirus strains use CD l3/APN to enter respiratory and intestinal epithelial cells. One strain, 229E (HCV-229E),which is
an important cause of upper respiratory tract infections in
humans, uses CD 13/APN as its receptor." Similarly,transmissible gastroenteritis virus (TGEV), a major source of fatal gastroenteritis in newborn pigs, uses porcine APN as its
portal of entry.'47 In both instances, the receptor role of
CDI 3/APN was suggested by the finding that MoAbs able
to block infection of susceptible cells specificallyrecognized
extracellular epitopes of the enzyme. Because the host range
of these viruses was restricted, it was also possible to render
resistant cells susceptibleto infection by introducing recombinant cDNAs that encoded either human or porcine APN.
These studies provide the means to define key CDI3/APN
residues that are required for virus binding and infectivity.
For example, CD13/APN encoded by a mutant human
cDNA clone and lacking key residues near the active site did
not bind HCV-229E virus or have enzymatic activity; these
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SHIPP AND LOOK
1060
results suggest that the enzyme active site and the virus attachment site are closely linked.2'~'4*
BP-I/GCB/APA (EC 3.4.1 1.7)
Pattern of Expression
BP- 1/6C3 is expressed in a developmentally restricted
pattern by immature murine B lymphocytes(Table 1). This
finding can be traced to studies, begun nearly a decade ago,
on lineage-restricted molecules recognized by the MoAbs
BP-1 and 6C3. These antibodies were independently derived in the laboratories of Max Cooper and Irving Weissman 149.1 50 and subsequently found to recognize epitopes on
the same murine cell surface molecule (BP-1/6C3).151
The
BP-l/6C3 antigen is expressed by cytoplasmic Ig+ pre-Blymphoid cells in bone marrow and fetal liver, but not by
mature B-lineage cells in blood, spleen, Peyer's patches, or
lymph nodes (Table l).'49 The antigen is also expressed at
high levels by early B-lineage cells grown in Whitlock-Witte
cultures and by Abelson virus-transformed pre-B cells (Table l). BP-l/6C3 expression can be abolished by fusing preB cells with plasmacytoma cell^.'^^^'^^ The developmentally
restricted binding pattern of the BP-1 and 6C3 antibodies
suggested that their cell surface target was a murine early
B-lineage differentiation antigen, a finding supported by
marked upregulation of the antigen in pre-B cells stimulated
to proliferate with IL-7.'53,''4The pattern of BP-l/6C3 expression in human lymphocytes remains to be determined.
It soon became apparent that the murine BP- 1/6C3 antigen was also expressed by nonhematopoietic cell types, including bone marrow-derived stromal cell lines and epithelial cells along the brush borders of the renal proximal
tubule and small intestine, and a subset of epithelial cells in
the thymic cortex (Table l).163'55These results suggested a
role for BP- 1/6C3 beyond B-lymphocyte development and
drew attention to the similarities between the patterns of
expression of this antigen and the two human metalloproteases, CDlO/NEP and CDl3/APN.
Biochemical and Molecular Characterization
Biochemical analysis. Both the BP-1 and 6C3 MoAbs
precipitate a disulfide-linked homodimer from the surface
The protein
of B cells and bone marrow stromal ~e1ls.l~'
backbone observed in cells treated with tunicamycin is consistently 110 Kd in size, and the intracellular high-mannose
form of the glycoprotein is 125 Kd, although various glycosylated forms of the molecule are expressed at the cell surface by different cell types. For instance, the cell surface
form of the molecule expressed by pre-B cells has the apparent electrophoretic mobility of a 140-Kd homodimer,
whereas a more rapidly migrating 135-Kd homodimer is
observed on bone marrow-derived stromal cell lines.'"
Predicted amino acid structure. As was the case for
CD 1O/NEP and CD 13/APN, BP- I /6C3 was identified as a
cell surface metalloproteaseby virtue of its predicted amino
acid sequence.' cDNA clones for BP-l/6C3, isolated by Wu
et
predicted a 945 amino acid integral membrane protein with a type I1 configuration and a large extracellular
carboxyl terminal domain (Fig
Within this extracellular
region, the BP- 1/6C3 protein contains the pentapeptide
consensus sequence (His-Glu-Leu-Val-His) that is characteristic ofzinc-binding metalloproteases(Fig 1). Among previously characterized members of the metalloprotease family, the BP-l/6C3 protein is most similar, but not identical,
to aminopeptidase N, with murine BP-l/6C3 and human
APN showing 36% overall sequence id en tit^.^
Enzymatic Activity and Functional Roles
Although the predicted amino acid sequence of the BP-I/
6C3 protein suggested that it might be an aminopeptidase,
the sequence was not identical to that of a previously characterized enzyme. In recent studies, this murine enzyme
was found to have the substrate specificityand sensitivity to
inhibitors characteristic of aminopeptidase A (APA; EC
3.4.11 .7)'55;BP-l/6C3 specifically catalyzed the hydrolysis
of N-terminal glutamic or aspartic acid residues and was
inhibited by chelating agents and competitive inhibitors
In addition,
such as amastatin and bestatin (Table 2).'56-'59
BP-l/6C3 enzymatic activity was augmented by Ca2+,'58a
property characteristic of APA.'60,'6'Taken together, these
data indicated that the recently cloned BP-l/6C3 antigen
was APA, which had not previously been cloned or sequenced.
In contrast to other members of the zinc metalloprotease
family, the enzymatic activity of BP- 1/6C3/APA is inhibited rather than potentiated by increasing concentrations of
zinc.'55 This observation was initially confusing, because
the amino acid sequence BP- 1/6C3/APA3contains the pentapeptide motif required for zinc coordination and enzymatic activity by other metalloproteases (Fig 1). This discrepancy was resolved when both wild-type and mutant
BP- 1/6C3/APA cDNA clones were examined in heterologous cells, and the enzyme was found to require low levels of
zinc for activity and to be inhibited by higher concentrations of the meta1.I6' Moreover, a missense mutation that
converted one of the histidine residues of the signature pentapeptide to a phenylalanine residue completely abolished
enzymatic activity, indicating that the integrity of the zincbinding motif is required for aminopeptidase activity.l6'
The wide tissue distribution of BP-l/6C3/APA suggests
that it may function differently in different cell types. As
with other members of the cell surface metalloprotease family, BP-l/6C3/APA is thought to participate in the final
stages of the hydrolysis of peptides in the small intestine
(Table 1). It may also be involved in the termination of
peptide signals, particularly those mediated by angiotensin
APA catalyzes the removal of the N-terI1 (Table 2).'59,'63
minal amino acid aspartic acid from angiotensin 11, markedly reducing its biologic activity and rendering it susceptible to further degradation by APN.
The function of BP-l/6C3/APA in murine early B-cell
development is still not completely understood; however,
several provocative associations suggest a regulatory role.
The IL-7 growth factor induces proliferation of early B cells
and simultaneouslyupregulates cell surface BP- 1/6C3/APA
e x p r e ~ s i o n . ' ~Intriguingly,
~*'~~
both IL-7 and the IL-7 receptor contain acidic N-terminal peptides, although neither has
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ANTIGENS THAT ARE MEMBRANE-ASSOCIATED ENZYMES
yet been directly shown to be an APA substrate. Preliminary
studies suggest that the BP- 1 antibody may inhibit the proliferative effect of IL-7 on early B cells.'53In addition, the
ability of cloned bone marrow stromal cell lines to support
B lymphopoiesis is correlated with the levels of stromal cell
surface expression of BP-1/6C3.I6The identification of BP1/6C3 as APA and the availability of defined cDNA clones,
MoAbs, and enzymatic inhibitors should allow the role of
the enzyme in early B-cell development to be defined at a
mechanistic level. The human BP-l/6C3/APA gene has recently been cloned (L. Li, J. Wang, and M. Cooper, personal
communication, March 1993), so that studies of the enzyme in normal and malignant human B lymphopoiesis
will also be possible.
CD26/DPPIV (EC 3.4.14.5)
Pattern of Expression
CD26 is a cell surface glycoprotein that was originally
characterized as a T-cell differentiation antigen. The murine CD26 homolog, known as the thymocyte-activating
molecule (THAM), is expressed at high levels by immature
CD4-CD8- thymocytes and at progressively lower levels by
double-positivethymocytes and mature single-positivecells
(Table 1).7,164,165
In humans, CD26 is primarily expressed by
CD3+ medullary thymocytes166;variable numbers and
functional subsets of resting human peripheral T cells have
also been reported to be CD26' (Table l).'9,167,168
Activation
of peripheral blood T cells with mitogen, antigen, or IL-2
results in increased numbers of CD26' cells and increased
levels of the cell surface antigen.169%'70
Like CD 1O/NEP, CD 13/APN, and BP- 1/6C3/APA,
CD26/DPPIV participates in the final stages of hydrolysis of
certain peptides in both the small intestine and the renal
proximal tubules.
CD26 is also expressed by certain splenic B cells, EBVtransformed lymphoblastoid cell lines, B-cell lymphomas,
and surface Ig' B-cell ALLs. In many respects, the nonhematopoietic tissue distribution of CD26 resembles that of
CD 1O/NEP, CD 13/APN, and BP- 1/6C3/APA; CD26 is found
on renal proximal tubules, intestinal epithelium, biliary caniliculae, and alveolar pneumocytes (Table 1),'9,'643L7'-L74
with
the highest levels of protein on epithelial cell brush
borders.164
Biochemical and Molecular Characterization
The human CD26 protein is identified by multiple
MoAbs'9,L68,175-177that precipitate a 1 10- to 120-Kd protein
under reducing and nonreducing conditions. However, the
murine and rat CD26 proteins have been reported to form a
200- to 220-Kd dimer on the cell surface, raising the possibility that the protein may be a nondisulfide-linkeddimer. The
size of the CD26 protein and its wide tissue distribution on
nonhematopoietic cells prompted further studies that
showed that CD26 is the cell surface ectoenzyme dipeptidyl
peptidase IV (DPPIV).19,'64,167-177
After an incorrect identification of murine CD26 as an aminopeptida~e,'~'
its identity
with DPPIV was ~onfirmed.~
The recently characterized murine and human CD26/
1061
DPPIV cDNAs predict a highly conserved approximately
766 amino acid polypeptide with type I1 membrane topology and a cytoplasmic domain of 6 amino acids (Fig
1).6,179,'80In contrast to CDlO/NEP, CD13/APN, and BP-1/
6C3/APA, the predicted CD26/DPPIV protein sequence
contains no zinc-binding motif.180However, it does possess
structural homology with a previously described 1 10-Kd
bile caniliculus domain-specific membrane protein, differThe preing in only the 25 carboxy-terminal residues.6*'81
dicted CD26/DPPIV protein also contains regions of localized homology with a type I1 membrane dipeptidyl
aminopeptidase from yeast (DPAPB).1803'82
Enzymatic Activity and Functional Roles
CD26/DPPIV is a serine-type protease that preferentially
cleaves Xaa-Pro and Xaa-Ala dipeptides from the NH2 terminus of peptides and proteins (Table 2).'64,173,183,'84
The
optimal size and additional features of substrates for this
enzyme have not been fully characterized; however, CD26/
DPPIV hydrolyzes natural peptides including substance P,
casomorphin, growth hormone releasing hormone (GRH),
kenzin, and a chain of fibrin (Table 2).1643'85,'86
The enzyme
also cleaves a family of antibacterial peptides from insect~,"~
peptides from frog skin that resembled mammalian
hormones and neurotransmitters,'" and a potent neurotoxin from bee venom.IE9Many other human brain and gut
peptides contain NH,-terminal sequences that predict susceptibility to CD26/DPPIV hydrolysis.lE6
CD26/DPPIV in nonhematopoietic cells. Like CD 1O/
NEP, CD 13/APN, and BP- 1/6C3/APA, CD26/DPPIV participates in the final stages of hydrolysis of certain peptides
in both the small intestine and the renal proximal tubules.
This was shown by investigators who took advantage of the
fact that a specific strain of Fischer rats lack brush border
CD26/DPPIV enzymatic a ~ t i v i t y . ' ~In
~ ~these
' ~ ' animals,
the hydrolysis and absorption of Xaa-Pro-peptides in both
the kidney and small intestine was markedly reduced.19os191
Additional studies suggest that CD26/DPPIV is also the
major enzyme responsible for the inactivation of circulating
GRH in plasma.ls6
CD26/DPPIV in T cells. CD26/DPPIV was initially associated with T-cell activation and proliferation because
DPPIV inhibitors suppressed mitogen- and antigen-induced T-cell proliferation and impaired T-cell-directed Bcell differentiation and Ig p r o d ~ c t i o n . ' ~ ~Recent
~ ~ ~ "stud'~~
ies using more potent and specific DPPIV inhibitors
suggested that these compounds primarily inhibit antigeninduced T-cell proliferation (Table l).I9' In contrast,
MoAbs directed against the cell surface enzyme stimulated
T-cell proliferation; immature and mature thymocytes proliferated in response to anti-CD26/DPPIV and PMA, IG 1,
or IL-2 and mature peripheral blood T cells proliferated
after treatment with anti-CD26/DPPIV and submitogenic
doses of either anti-CD3 or anti-CD2.'65,'96s'97
Furthermore,
transfection of a CD26- T-cell hybridoma with either of the
murine or human CD26 cDNAs results in the expression of
a 10- to 128-Kd protein that functions as both an ectopeptidase and an activation signal-transducing
CD26/DPPIV may also function as an auxiliary adhe-
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1062
SHIPP AND LOOK
sion factor as well as an ectoenzyme. Purified CD26/DPPIV
protein was found to bind fibronectin,'" which was of particular interest because CD26/DPPIV and fibronectin colocalized in many tissues."' In additional studies, CD26/
DPPIV+ cell types including hepatocytes and fibroblasts
also bound collagen; this adhesive interaction was inhibited
by either a tripeptide enzyme substrate or anti-CD26/
DPPIV a n t i b ~ d i e s . ' ~The
~ . ' ~interaction
~
between CD26/
DPPIV and fibronectin or collagen in nonhematopoietic
cells prompted analyses of CD26/DPPIV-mediated adhesion in T lymphocytes.200Human CD4+ T cells proliferated
in response to a combination of anti-CD3 MoAbs and type
I, 111, or IV collagen; an anti-CD26/DPPIV antibody inhibited this effect.200
Recent studies also suggest an association between CD26/
DPPIV and the T200 tyrosine phosphatase, CD45. CD26/
DPPIV comodulates with CD45, and a CD26/DPPIV
MoAb also precipitates CD45 from the T-cell surface.20'
Because anti-CD26/DPPIV treatment also leads to enhanced phosphorylation of CD3 { on tyrosine residues and
increased CD4-associated P56lck tyrosine kinase activity, it
is possible that the antibody stimulates T-cell proliferation
in part by decreasing CD45-mediated dephosphorylation of
key substrates.201
CD73/ECTO-Sf-NUCLEOTIDASE
Pattern of Expression: Enzymatic, Immunologic, and
Biochemical Characterization
Enzymatic activity. Ecto-5'-nucleotidase (E5N) is a glycosyl phosphatidylinositol-linked cell surface enzyme that
catalyzes the dephosphorylation of purine and pyrimidine
ribonucleoside and deoxyribonucleoside monophosphates
to the corresponding ribonucleosides and deoxyribonucleosides (Table 2).202,203
The 70- to 74-Kd dimer converts nontransportable 5' nucleotides into a transportable form and
contributes to the extracellular hydrolysis of ATP by dephosphorylatingAMP to adenosine (Table 1).202,203The previously shown association between inherited enzyme deficiencies in purine nucleotide degradation and human
immunodeficiency syndrome^^^,^^^ prompted further evaluation of E5N activity in various immunodeficiency states.
Patients with adult-onset "variable" primary hypogammaglobulinemia, congenital X-linked agammaglobulinemia,
and IgA deficiency were found to have reduced levels of
E5N enzymatic activity in their peripheral blood lymphocytes,206208
raising the possibility that decreased E5N levels
in patients with immunodeficienciesresulted from another
inborn error of purine metabolism.
Cell surface E5N expression was initially evaluated by
enzymatic assays oflymphoid subpopulation^.^^^ Early studies suggested that E5N activity was a maturation marker on
both B and T cells because peripheral T cells had 10-fold
higher levels of enzymatic activity than thymocytes210and
adult peripheral B cells had fivefold to sixfold higher levels
of enzymatic activity than fetal spleen and cord blood B
cells (Table 1).2'1,2'2 Further analyses of lymphoid subpopulations from patients with congenitalagammaglobulinemia,
common variable immunodeficiency,or acquired immuno-
deficiency syndrome (AIDS)showed that these patients had
decreased numbers of E5N+ T ~ e l l s . ~ ' ~In~ ~AIDS
' ~ patients,
circulating CD8+ T cells with reduced E5N activity had additional phenotypic characteristics suggesting that the reduced enzymatic activity resulted from a maturation block
in these
These data supported the hypothesis that
E5N is a differentiation antigen on peripheral blood lymphocytes and that enzyme levels are reduced in patients
with certain immunodeficiency syndromes because these
patients have more immature circulating cells rather than a
genetic abnormality in E5N
Immunologicand biochemicalcharacterization. In studies to assess the role of E5N in lymphoid maturation, the
enzyme was purified from human placenta and used to generate a highly specific goat antihuman E5N antiserum and
murine MoAbs; an additional MoAb was made by immunization with a human pre-B-cell line.202,2'6-2'9
Thereafter, the
enzyme was given a CD designation, CD73.202Immunophenotypic analysis showed that CD73/E5N was expressed
on subpopulations of peripheral blood T cells (eg, 20% to
30% of CD3+cells, 10%to 20% of CD4+cells, 50%of CD8'
cells, and 25% of CD28' cells) and B cells (eg, 70%to 80%of
CD19+cells) (Table l).2'822'9
In frozen sections of lymphoid
tissues, CD73/E5N antibodies stained mantle zone B cells
and follicular dendritic reticulum cells in the basal part of
the light zone and the dark zone of the germinal enter.^^^,^'^
Immunophenotyping studies on peripheral blood lymphocytes from patients with immunodeficiency syndromes
confirmed the data derived from enzymatic assays. Specifically, patients with congenital agammaglobulinemia had reduced numbers of CD73/E5N+peripheral T cell^.^'^,"^ Furthermore, most hypogammaglobulinemicpatients also had
reduced levels of CD73/E5N+ B cells, suggestingthat their B
cells were blocked in maturation before expression of the
cell surface enzyme.218However, some hypogammaglobulinemia patients had normal levels of B-cell CD73,220suggesting that there may be multiple sitesat which B-cell development can be blocked.
CD73/E5N was also expressed on EBV-transformedlymphoblastoid cell lines, certain myeloma cell lines, and many
pre-B- and progenitor B-cell leukemias (Table 1).202The
B-cell leukemias that had the highest levels of CD73/E5N
expression were CD lO/NEP+/cp-/sIg- or CD 1O/NEP+-/
cp+/sIg-.zz'High levels of CD73 expression correlated with
a poor prognosis in patients with CDlO/NEP+ leukemia.222,223
Nonhematopoietic cell types, including mesenchymal cells, follicular dendritic cells, hepatocytes, and
small intestinal epithelium, also expressed the enzyme (Table 1).'02
CD73/E5N is an -70- to 74-Kd dimer with interchain
disulfide bridges (Fig 1).202MoAbs to CD73/E5N precipitate a single -70- to 74-Kd band from myeloma and pre-B
cells and placenta under reducing conditions. Treatment of
CD73/E5N+ cells with the phosphatidylinositol-specific
phospholipase C releases most of the enzyme activity, indicating that the cell surface protein is anchored in the membrane by a glycosylphosphatidylinositollinkage. This mode
of membrane attachment was confirmed by detection of
inositol in highly purified preparations of the
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ANTIGENS THAT ARE MEMBRANE-ASSOCIATED ENZYMES
1063
cDNAs show 89.4% id en tit^.'^'^^, The human CD73/E5N
cDNA encodes a 574 residue polypeptide with a calculated
CD73/E5N and lymphocyte function. The functional
size of 63.4 Kd (Fig l).I3 The NH,-terminal 26 residues
significance of CD73/E5N expression in human lymphocomprise
a signal peptide that is followed by the NH,-termicyte subpopulations was further evaluated by separating ennal sequence of the purified protein (Fig l).I3 The mature
zyme-positive and -negative cells and performing standard
CD73/E5N protein lacks the predicted COOH-terminal eximmunologic assays in the presence or absence of a specific
tension (aa524 through aa548), which has been replaced by
biochemical inhibitor of CD73/E5N activity (a,0 methglycophospholipid
functioning as the membrane anchor
ylene adenosine 5'-diphosphate [AOPCP]; Table 2)20*227,228
(Fig l).13The cDNA contains four potential N-linked glycoor an inhibitory antiserum or MoAbs.
sylation sites, suggesting that glycosylation may account for
B lymphocytes. CD73/ESN-positive and -negative huthe
larger size of the cell surface protein (7 1 Kd).I3 CD73/
man B-cell subpopulations synthesized equivalent quantiE5N
has been reported to bind zinc and to contain three
ties of IgM in response to a T-cell-dependent or T-cell-incysteine and histidine residues that may function as potendependent stimulus; however, CD73/E5N+ lymphocytes
tial Zn+'-binding sites (Fig 1).233,234In contrast to CD10/
synthesized 8- to 26-fold more IgG per cell than B cells
NEP, CD 13/APN, and BP-l/6C3/APA, CD73/E5N does
lacking this activity.227These data provided further evinot contain a characteristic zinc-binding pentapeptide sedence that the enzyme is a marker for the functional maturaquence.
tion of human B cells (Table I ) and supported the hypothe-
Biologic Activity of CD73/E5N
sis that CD73/E5N deficiency in hypogammaglobulinemic
patients results from a block in B-cell development.227
T lymphocytes. In peripheral T cells, the inhibition of
CD73/E5N with a specific inhibitor (AOPCP) selectively
suppressed the cytotoxic responses of alloreactive T cells.20
In additional studies, CD73/E5N+ peripheral T cells proliferated when they were cocultured with submitogenic doses
of PMA and either goat antiserum or murine MoAbs to
CD73/E5N.229Pretreatment of the T cells with phospholipase C removed the majority of CD73/E5N from the cell
surface and inhibited the ability of the cells to proliferate in
response to the CD73/E5N antibodies and PMA.229Immobilized CD73/E5N MoAbs also increased T-cell activation
via the CD3 or CD2 pathway,23oalthough it is not clear
whether the CD73/E5N antibodies enhanced T-cell proliferation by modulating enzymatic activity or by affecting the
enzyme's glycophospholipid anchor. In recent studies, T
cells from transgenic animals expressing a glycophospholipid-anchored form of a nonmitogenic protein proliferated
in response to antibodies directed against that protein,
prompting speculation that glycophosolipid membrane anchors of phosphoinositol-linked cell surface proteins like
CD73/E5N may function directly in transmembrane signaling.231
CD73/E5N in nonhematopoieticcells. Like CD 1O/NEP
and CD13/APN, E5N has also been implicated in the regulation of neutrophil inflammatory responses (Table l)." In
the intestine, inflammation results in the development of
crypt abcesses in which neutrophils are in physical contact
with the apical intestinal epithelial membrane.I8 Neutrophils release 5'AMP that is converted by intestinal brush
border CD73/E5N to adenosine; the newly liberated adenosine triggers adenosine receptors on intestinal epithelial cells
and results in a secretory diarrhea."
Recent studies suggest that CD73/E5N may also participate in cell-cell or cell-matrix interaction^.^'^
Molecular Structure
The molecular structures of the human and rat CD73/
E5N cDNAs have recently been elucidated and the predicted amino acid sequences of the respective CD73/E5N
SUMMARY AND FUTURE DIRECTIONS
The identification of five membrane-associated enzymes
as hematopoietic differentiation antigens underscores the
potential importance of cell surface enzymes in the biology
of normal and malignant hematopoietic cells. Studies to
date suggest that certain membrane-associated enzymes
(CD 10/NEP, CD13/APN, BP- 1/6C3/APA, and CD26/
DPPIV) may function as part of a negative regulatory loop,
limiting cellular responses to a given peptide or protein substrate. These enzymes, which are coexpressed in a variety of
epithelial cells and expressed by overlapping subsets of hematopoietic cells, may also cooperate to limit some of the
same physiologic processes and hydrolyze many common
peptide substrates. For example, CDlO/NEP and CDl3/
APN cooperate to limit neutrophil-mediated inflammatory
responses triggered by peptides that are substrates for both
enzymes (eg, fMLP and met-enkephalin). The overlapping
patterns of expression of CD 1O/NEP and BP 1/6C3/APA
prompt speculation that these enzymes may have complementary roles in the regulation of stromal cell-dependent B
lymphopoiesis. That additional peptides such as substance
P are substrates for both CDlO/NEP and CD26/DPPIV
suggests that these enzymes may also cooperate in still-undefined processes. The fact that "neuropeptides" such as
substance P and met-enkephalin are substrates for enzymes
expressed by a restricted population of hematopoietic cells
has stimulated further investigation into the potential roles
for these peptides in immune function and inflammation.
Preliminary findings on the biologic functions of these
ectoenzymes have already begun to have therapeutic applications. In certain settings, enzyme inhibitors are being used
to potentiate the biologic activity of endogenous peptide
hormones. For example, oral inhibitors of CDlO/NEP,
which are being promoted as antidiarrheal medicat i o n ~ : ~ are
~ *also
~ ~ under
~
investigation as antihypertensive
agents. In other settings, the recombinant enzymes are being exploited to reduce local peptide concentrations, as illustrated by trials of aerosolized and systemic versions of
recombinant CDlO/NEP in reactive airways disease. The
growth-regulatoryeffects of CDlO/NEP on normal and malignant bronchial epithelial cells and normal lymphoid pro-
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
SHIPP AND LOOK
1064
genitors suggest additional uses for this recombinant enzyme.
Characterization of the relevant peptide substrates for
these enzymes may also have important clinical implications. For example, the identification of potential enzyme
cleavage sites in endogenous enkephalins and GRH has led
to the development of more effective synthetic analgesics
and GRH analogs. The characterization of bioactive substrates for membrane-associated enzymes expressed by hematopoietic cells is likely to be of similar benefit.
ACKNOWLEDGMENT
We thank Max Cooper, Ore Noren, Chikao Morimoto, Linda
Thompson, Richard Ashmun, and Linda Shapiro for their thoughtful review of the manuscript; John Gilbert for editorial suggestions;
and Donna Favreau for manuscript preparation.
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1993 82: 1052-1070
Hematopoietic differentiation antigens that are membrane-associated
enzymes: cutting is the key!
MA Shipp and AT Look
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