From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037
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ABSTRACT |
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MDC-15 (ADAM-15, metargidin), a membrane-anchored
metalloprotease/disintegrin/cysteine-rich protein, is expressed on the
surface of a wide range of cells and has an RGD tripeptide in its
disintegrin-like domain. MDC-15 is potentially involved in cell-cell
interactions through its interaction with integrins. We expressed a
recombinant MDC-15 disintegrin-like domain as a fusion protein with
glutathione S-transferase (designated D-15) in bacteria and
examined its binding function to integrins using mammalian cells
expressing different recombinant integrins. We found that D-15
specifically interacts with v
3 but not with the other integrins
tested (
2
1,
3
1,
4
1,
5
1,
6
1,
6
4,
v
1,
IIb
3, and
L
2). Mutation of the tripeptide RGD to
SGA totally blocked binding of D-15 to
v
3, suggesting that
D-15-
v
3 interaction is RGD-dependent. When the sequence RPTRGD is mutated to NWKRGD, D-15 is
recognized by both
IIb
3 and
v
3, suggesting that the
receptor binding specificity is mediated by the sequence flanking the
RGD tripeptide, as in snake venom disintegrins. These results indicate
that the disintegrin-like domain of MDC-15 functions as an
adhesion molecule and may be involved n
v
3-mediated
cell-cell interactions.
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INTRODUCTION |
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Metalloprotease/disintegrin/cysteine-rich proteins (MDCs, also called ADAMs)1 are membrane-anchored proteins with several domains including a metalloprotease domain, a disintegrin-like domain, a cysteine-rich sequence, an epidermal growth factor-like sequence, a transmembrane domain, and a short cytoplasmic domain (1). The biological functions of MDCs are not clear; however, we do know that fertilins (MDC-1 and -2) (2) are involved in sperm-egg binding and fusion (3), meltrins (MDC-12) (4) are involved in myoblast fusion during muscle development, and KUZ (a Drosophila MDC protein) (5) assists in neurogenesis. The MDC cytoplasmic domain has a proline-rich potential SH3 binding motif, suggesting that MDC-counter receptor interaction may induce signal transduction.
Integrins are a family of cell adhesion receptors that bind to a
variety of ligands, including extracellular matrix proteins and other
cell surface molecules (6-10). MDCs are potential ligands for
integrins, since most snake venom disintegrins interact with integrins
IIb
3 and
v
3 (reviewed in Ref. 11 and references therein).
However, little is known about the receptor specificity of MDCs, except
that mouse egg integrin
6
1 has been proposed as a receptor for
fertilin (2). Evans et al. (12) recently expressed
recombinant fertilin fragments in bacteria as fusion proteins with
maltose-binding protein (12). The recombinant fertilin-
fragment has
been shown to bind to the egg membrane to which sperm bind and to block
sperm from binding to the egg. These results suggest that the
disintegrin-like domains of MDCs may be properly folded in bacteria,
that glycosylation of the disintegrin-like domain may not be required
for interaction with receptors, and that a strategy using recombinant
MDC proteins is a viable alternative to those using purified materials
that are not easily available.
MDC-15 (metargidin) (13, 14) is the only known MDC that has an RGD
sequence in its disintegrin-like domain. MDC-15 is widely expressed in
various tissues and cells, including human umbilical vein endothelial
cells and smooth muscle cells (14). In this study, we determined the
receptor specificities of MDC-15 using the recombinant disintegrin-like
domain of MDC-15, which was expressed in bacteria. We determined the
receptor specificity of the recombinant protein using mammalian cells
expressing different recombinant human integrins. We discovered that
the recombinant disintegrin-like domain specifically interacts with
integrin v
3 but not with other RGD-dependent or
independent integrins tested, including
IIb
3. This is in contrast
to snake venom disintegrins, most of which bind to both
IIb
3 and
v
3 (Ref. 11 and references therein). We also found that the
integrin specificity of the recombinant disintegrin-like domain is
mediated by the RGD tripeptide and its flanking sequence, as in snake
venom disintegrins. These results suggest that MDC-15 may be involved
in
v
3-mediated cell-cell adhesion.
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EXPERIMENTAL PROCEDURES |
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Monoclonal Antibodies and Cell lines--
mAb 15 (15) was a kind
gift from M. H. Ginsberg (The Scripps Research Institute, La
Jolla, CA), 2G12 (16) was from V. Woods (University of California at
San Diego, La Jolla, CA), PL98DF6 (17) was from J. Ylanne (University
of Helsinki, Helsinki, Finland), PT25-2 (18) was from M. Handa and Y. Ikeda (Keio University, Tokyo, Japan), and LM142 (to human v) and
LM609 (to
v
3) (19) was from D. Cheresh (Scripps). Polyclonal
anti-
v cytoplasmic peptide antibody was purchased from Chemicon,
Temecula, CA. M-21 human melanoma cells were provided by D. Cheresh.
Preparation of the GST Fusion Protein of the Disintegrin-like
Domain of MDC-15 and of the 8-11th Type III Repeats of
Fibronectin--
A cDNA fragment of about 270 nucleotides that
encodes the disintegrin-like domain of MDC-15 (Met-420 to Glu-510) (13)
was amplified by polymerase chain reaction with a human erythroleukemia (K562) cell cDNA library as a template using
5'-CGGGATCCATGGCTGCTTTC-TGCGG and 5'-CGGGATCCTTACTCGCCATCCCCTAGGCTG as
primers. The cDNA fragment was subcloned into the BamHI
site of a pGEX-2T vector (Amersham Pharmacia Biotech). Synthesis of the
GST fusion protein of the disintegrin-like domain of MDC-15 was induced
in Escherichia coli DH5 by adding 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside in culture medium
as described previously (20). Protein was extracted from the bacterial
suspension by sonication and purified using glutathione-agarose (Sigma)
affinity chromatography.
Development of Chinese Hamster Ovary (CHO) Cells Expressing
Different Human Integrins--
We developed CHO cells that express
different human integrins. The cDNA constructs were transfected
into CHO cells together with a neomycine-resistant gene. Those cell
lines expressing human 2 (
2-CHO) (23), human
3 (
3-CHO)
(24), human
4 (
4-CHO) (25, 26), human
5 (
5-CHO) (26, 27),
human
L
2 (
L
2-CHO) (28), human
v (
v-CHO), human
3
(
3-CHO), both human
v and
3 (
v
3-CHO) (29), and human
IIb
3 (
IIb
3-CHO) (30) have been described in the cited
references. The cDNA constructs for
6 (
6-CHO) and
6 and
4 (for
6
4-CHO) were co-transfected into CHO cells with a
neomycine gene. After selection with G-418, cells stably expressing
human integrins were cloned by sorting to obtain high
expressors.2 The
2-,
3-,
4-,
5-,
6-, and
v-CHO cells homogeneously expressed human
2,
3,
4,
5, and
v/hamster
1 hybrids,
respectively. The
3-CHO cells expressed human
3/hamster
v
hybrid.
Adhesion Assays-- Wells of 96-well Immulon-2 microtiter plates (Dynatech Laboratories, Chantilly, VA) were coated with 100 µl of PBS (10 mM phosphate buffer, 0.15 M NaCl, pH 7.4) containing substrates at a concentration of 20 µg/ml and were incubated overnight at 4 °C. The remaining protein binding sites were blocked by incubating with 1% bovine serum albumin (Calbiochem) for 1 h at room temperature. Cells (105 cells/well) in 100 µl of Dulbecco's modified Eagle's medium were added to the wells and incubated at 37 °C for 1 h. After gently rinsing the wells three times with PBS to remove unbound cells, bound cells were quantified by measuring endogenous phosphatase activity (31).
Affinity Chromatography on D-15-- Purified D-15 was absorbed to glutathione-agarose (Sigma) after free glutathione was removed by gel filtration on a PD-10 column (Amersham). Cells were harvested with 3.5 mM EDTA in PBS and washed with PBS. Cells (about 5 × 106) were then surface-labeled with 125I using IODO-GEN (Pierce) (32), washed three times with PBS, and solubilized at 4 °C for 1 h in 1 ml of 10 mM Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl, 100 mM octylglucoside, 2.5 mM MnCl2, and 1 mM phenylmethylsulfonyl fluoride (Sigma). The insoluble materials were removed by centrifugation at 15,000 × g for 10 min. The supernatant was then incubated with a small amount of underivatized agarose at 4 °C for 15 min to remove nonspecific binding material. The supernatant was incubated at 4 °C for 1 h with 200-500 µl of packed D-15-glutathione-agarose that had been equilibrated with 10 mM Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl, 25 mM octylglucoside, 2.5 mM MnCl2, and 1 mM phenylmethylsulfonyl fluoride (washing buffer). The unbound materials were washed with 20 times the column volume of washing buffer, and the bound materials were eluted with 20 mM EDTA instead of 2.5 mM MnCl2 in washing buffer; then, 0.5-ml fractions were collected. Twenty-µl aliquots from each fraction were analyzed by SDS-polyacrylamide gel electrophoresis using 7% polyacrylamide gel under nonreducing conditions followed by autoradiography.
Binding Recombinant GST Fusion Protein and Fibrinogen to CHO Cells-- Recombinant GST fusion protein and fibrinogen (Chromogenix, Stockholm, Sweden) were labeled with fluorescein isothiocyanate (FITC) (33). Cells were incubated with mouse IgG or mAb PT25-2 at 10 µg/ml for 30 min at 4 °C in Dulbecco's modified Eagle's medium. Then, FITC-labeled fibrinogen was added at a final concentration of 100 µg/ml, and the mixture was further incubated for 30 min at room temperature. After washing the cells once with PBS to remove unbound labeled protein, bound protein was quantified by flow cytometry in FACSCan (Beckton-Dickinson).
Other Methods-- Site-directed mutagenesis was carried out using the unique site elimination method (34). The presence of mutations was verified by DNA sequencing.
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RESULTS |
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The Recombinant Disintegrin-like Domain of MDC-15-- To prove that MDC-15 mediates cell-cell adhesion through interaction with integrins, we expressed the disintegrin-like domain of MDC-15 (Met-420 to Glu-510), a putative integrin binding site (Fig. 1A), as a fusion protein with GST in bacteria (designated D-15). We obtained soluble D-15 and purified it using affinity chromatography on glutathione-agarose. Fig. 1B shows that the purified D-15 migrates as a monomer with a Mr of 36,000 under nonreducing conditions (lane 1), and the control, wt GST protein, migrates as a monomer with a Mr of approximately 26,000. The sizes of these proteins match the values calculated from the primary structures of these proteins (35,930 and 26,968, respectively). Although the D-15 preparation contained some degradation products, we used it for adhesion and binding assays without further purification.
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Adhesion to D-15 of CHO Cells Expressing Different Recombinant
Integrins--
We determined whether D-15 supports integrin-mediated
cell adhesion using CHO cells expressing different recombinant
integrins. Parent CHO cells express 5
1 as a major integrin but do
not express
2 or
3 integrins (29, 35). As shown in Fig.
2A, wt GST (the negative
control) does not support adhesion to any of the cells used, but GST-FN
(the positive control), which contains the central cell binding domain
of rat fibronectin (the 8-11th type III repeats), supported all of the
cell lines used. D-15 supported adhesion of
3-CHO cells (that
express
v
3) and
IIb
3-CHO cells (that express both
IIb
3 and
v
3) but not parent CHO cells and cells expressing
other exogenous integrins (including
2
1,
3
1,
4
1,
5
1,
6
1,
6
4,
v
1, and
L
2). These results
indicate that D-15 interacts either with
v
3 or with both
IIb
3 and
v
3. Fig. 2B shows that both D-15 and
GST-FN support maximum adhesion of
3-CHO cells at the coating
concentration of 10 µg of protein/ml, suggesting that D-15 and GST-FN
support adhesion of
3-CHO cells at comparable levels. Fig.
2C shows that adhesion to D-15 of
3-CHO cells is
completely blocked by function-blocking anti-
v
3 mAb LM609, but
adhesion to GST-FN is not. These results support the idea that adhesion
to D-15 is mediated exclusively by
v
3, whereas adhesion to GST-FN
is mediated by multiple receptors that include
v
3,
v
1, and
5
1.
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Affinity Chromatography of Solubilized IIb
3 and
v
3
Integrins on D-15 Immobilized to Agarose--
To determine whether
D-15 interacts with
v
3 or both
IIb
3 and
v
3, we
carried out affinity chromatography on immobilized D-15. Purified D-15
was coupled to glutathione-agarose and incubated with lysates of
125I-labeled
v
3,
IIb
3, or parent CHO cells.
Bound materials were eluted with 20 mM EDTA. Fig.
3 shows that two protein bands
corresponding in size to
v/
IIb and
3 were eluted with lysates
from
v
3- and
IIb
3-CHO cells, but very little radioactivity
was eluted with the lysate of parent CHO cells. We analyzed the
eluted materials by immunoprecipitation using antibodies specific to
v,
IIb, or
3.
IIb
3 was detected only in the unbound
fraction with
IIb
3-CHO cells.
v
3 was detected in both the
bound and unbound fractions with
v
3-CHO and
IIb
3-CHO cells.
These results indicate that D-15 binds to solubilized
v
3 but not
to solubilized
IIb
3.
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v
3-D-15 Interaction Is Dependent on the Tripeptide RGD and
the Flanking Sequence in the Putative Integrin Binding Site--
To
determine whether
v
3-D-15 interaction is
RGD-dependent, we mutated the D-15 RGD sequence to SGA. The
mutant protein (designated D-15/SGA) was expressed as a soluble monomer
(Fig. 1B), purified using affinity chromatography, and used
for adhesion assays. The mutation completely blocked adhesion of
3-CHO cells to the fusion protein (Fig.
4). Increasing the coating concentration
of the D-15/SGA mutant did not reverse the effects of the mutation.
These results indicate that D-15-
v
3 interaction is dependent on
the RGD tripeptide in the putative integrin binding site.
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Interaction between a Natural Human v
3 Heterodimer and
D-15--
Since we used a recombinant hamster
v/human
3 hybrid
integrin on CHO cells to study the receptor specificity of D-15, we determined whether the natural human
v
3 heterodimer recognize wt
and mutant D-15. As shown in Fig. 6, M-21
human melanoma cells expressing
v
3 adhered to wt D-15 and
D-15/NWK but not to D-15/SGA. Adhesion of M-21 cells to wt D-15 and
D-15/NWK was completely blocked by LM609. These results indicate that
the natural human
v
3 heterodimer, like a hybrid
v
3 on CHO
cells, specifically recognizes D-15 in an RGD-dependent
manner.
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DISCUSSION |
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Although MDC-15 and other members of its family have been proposed
to represent a new class of cell adhesion molecules based on deduced
amino acid sequences, their function is unclear in most cases (see the
Introduction). In this paper we have evaluated the receptor specificity
of MDC-15 by studying its recombinant disintegrin-like domain. Although
the recombinant fertilin- (MDC-2) fragment shows biological
functions (12), it is possible that the native disintegrin-like domains
of MDCs purified from mammalian cells or of recombinant MDCs expressed
in eukaryotic systems may fold or be modified differently and show
additional or different properties. In this study, we have shown that
the recombinant disintegrin-like domain of MDC-15 specifically
interacts with integrin
v
3 using adhesion assays and affinity
chromatography. We also present evidence that the receptor recognition
and specificity of the recombinant disintegrin-like domain of MDC-15 is
determined by the RGD tripeptide and flanking sequence. Substitution of
the RGD tripeptide to SGA completely blocks binding of D-15 to
v
3. Substitution of the sequence flanking the tripeptide RGD
changes the receptor specificity (from
v
3 to both
IIb
3 and
v
3). Thus, the binding specificity of the recombinant
disintegrin-like domain of MDC-15, like that of snake venom
disintegrins and dendroaspin, an RGD-containing neurotoxin variant
(38), is mediated by the RGD tripeptide and flanking sequence. NMR
structural studies of the snake venom disintegrins kistrin and
echistatin show that, in both cases, the region containing the RGD
sequence is a loop structure exposed to the outside of the disintegrin
molecule (40, 41). This peculiar structure probably supports the high
affinity interaction of snake venom disintegrins with integrins (36, 37). It would be interesting to determine whether the structure of the
recombinant disintegrin-like domain of MDC-15 is similar to those of
snake venom disintegrins.
It should be noted that the disintegrin-like domain of MDC-15 is unique
in its receptor specificity. Most snake venom disintegrins recognize
both v
3 and
IIb
3 (11). Barbourin is the only disintegrin that is specific to
IIb
3. There is no known snake venom
disintegrin that is specific to
v
3. Thus, the RGD tripeptide and
flanking sequence in the disintegrin-like domain of MDC-15 represent a unique
v
3-specific recognition sequence. The disintegrin-like domains of MDCs have additional Cys residues in the middle of the loops
in their putative integrin binding sites (RGDC in MDC-15,
for example). It has not been determined whether this Cys residue makes
a disulfide link with another Cys residue. The topology of this region
of the disintegrin-like domain is probably very different from that of
the snake venom class P II disintegrins in that its loop structure is
probably less flexible and its conformation more restricted, with an
increase in restriction if the Cys residues flanking the RGD tripeptide
are involved in a disulfide linkage (in the snake venom class P II
disintegrins, the RGD sequence is positioned within an extended,
flexible loop structure where there is only limited conformational
restriction of the RGD sequence; see Ref. 42 for review). It is
possible, therefore, that using short synthetic peptides (cyclic or
linear) derived from the RGD and flanking sequences of the
disintegrin-like domain of MDC-15 might provide different receptor
specificities than those obtained in this study using recombinant or
purified disintegrin-like domains.
The interaction of the disintegrin-like domain of MDC-15 with integrin
v
3 may be related to its biological functions. MDC-15 is not
expressed in vivo in normal vessels but is up-regulated in
lesions of atherosclerosis, where many macrophages are present (14).
MDC-15 on cultured endothelial cells undergoes proteolytic processing
(14), which appears to be associated with MDC activation (4, 43). It is
possible that activated MDC-15 on endothelial cells interacts with
v
3 on leukocytes during atherogenesis through its exposed
disintegrin-like domain.
v
3 has been shown to be involved in the
progression of melanoma and the induction of neo-vascularization by
tumor cells.
v
3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels (44, 45). It is possible that
activated MDC-15 and
v
3 on endothelial cells interact with each
other, leading to homotypic aggregation of endothelial cells during
angiogenesis. Based on the wide distribution of MDC-15, MDC-15-
v
3
interaction may mediate cell-cell interactions in many other instances
(e.g. metastasis). The snake venom
metalloprotease/disintegrin jararhagin is known to block
collagen-induced aggregation of platelets (46). It has been proposed
that the inhibition of platelet response to collagen by jararhagin is
mediated through the binding of jararhagin to the platelet
2-subunit
via the disintegrin domain followed by proteolysis of the
1 subunit
with loss of the integrin structure (conformation) necessary for the
binding of macromolecular ligands (47). It has been hypothesized that a
fragment of MDC-15 containing the metalloprotease and disintegrin-like
domains is released from cultured endothelial cells (14). It is
possible that the proteolytic fragment of MDC-15 containing the
metalloprotease and disintegrin-like domains interacts with
v
3,
as in the case of jararhagin and
2
1, and either modifies the
function of the integrin or promotes degradation of the matrix proteins
surrounding the cells that express
v
3. Therefore, the proposed
v
3-MDC-15 interaction may be of wide biological importance.
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ACKNOWLEDGEMENTS |
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We thank D. Cheresh, M. H. Ginsberg, M. Handa, Y. Ikeda, V. Woods, and J. Ylanne for valuable reagents.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM47157 and GM49899. This is publication 10574-VB from The Scripps Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dept. of Vascular Biology, VB-1, The Scripps Research Institute,
10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-784-7122; Fax: 619-784-7323; E-mail: takada{at}scripps.edu.
1 The abbreviations used are: MDC, metalloprotease/disintegrin/cysteine-rich protein; mAb, monclonal antibody; GST, glutathione S-transferase; CHO, Chinese hamster ovary; FN, fibronectin; FITC, fluorescent isothiocyanate; wt, wild-type; PBS, phosphate-buffered saline.
2 X.-P. Zhang, W. Puzon-McLaughlin, and Y. Takada, unpublished results.
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REFERENCES |
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