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Vol. 182, No. 3, 1992 February 14, 1992
THROMBOSPONDIN
Pages
SEQUENCE
MOTIF
(CSVrCG)
IS RESPONSIBLE
1208-1217
FOR CD36 BINDING
Adam S. Asch,‘Scott Silbiger, Edgar Heimer,*and Ralph L. Nachman
Specialized Center for Researchin Thrombosis, Division of Hematology-Oncology, Cornell University Medical College, New York, NY 10021 *Department of Molecular Genetics and Department of Peptide Chemistry, Hoffman-LaRoche, Nutley, New Jersey 07110
Received
December
17,
1991
To clarify the role of CD36 as a TSP receptor and to investigate the mechanismsof the TSPCD36 interaction, transfection studies were performed using CD36cDNA in a CDM8 plasmid. Jurkat cells transfected with CD36 cDNA express an 88kD membranesurface protein and acquire the ability to bind thrombospondin. The TSP amino acid sequence, CSVTCG, mediates the interaction of thrombospondin with CD36. CD36 transfectants but not control transfectants bind radiolabeled tyrosinated peptide (YCSVTCG). The hexapeptide inhibits thrombospondin expression on activated human platelets and results in diminishedplatelet aggregation. CSVTCG-albuminconjugates support CD36dependent adhesion of tumor cells, We conclude that the CSVTCG repeat sequence is a crucial determinant of CD36 thrombospondin binding. B 1992 Academic Press, Inc.
Thrombospondin is a cell and matrix protein with a wide tissue distribution and multiple biologic functions. Thrombospondin supports the secretion dependent phase of platelet aggregation by stabilizing the interaction of fibrinogen with its receptor (1). Thrombospondin binds to distinct sequenceswithin the A-alpha and B-beta chains of fibrinogen (2,3) and following the initiation of coagulation is incorporated into fibrin clot where it modulatesthe meshwork structure of fibrin filaments (4-8) and serves as a site for plasminogenlocalization and activation (7). Thrombospondin has a growth regulatory role for fibroblasts and smooth muscle cells (8,9). As an adhesive protein, thrombospondin supports the attachment of several cell types (10,ll) and may mediate cytoadhesion of P. falciparum infected erythrocytes to endothelium (12). Sequence homologieswith Malarial circumsporozoite protein, thrombospondin-relatedanonymous-protein (TRAP), properdin and complement components C8 and C9 suggest the importance of these conserved regions (13,14).
1
To whom correspondence
should
be addressed.
Abbreviations : TSP, thrombospondin; CS-protein, circumsporozoite surface-protein; DlT, dithiothreitol; FCS, fetal calf serum; KLH, keyhole limpet hemocyanin. 0006-291X/92 Copyright AU rights
$1.50
6 1992 by Academic Press, Inc. of reproduction in any form reserved.
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Identification of the specific cell binding domains of thrombospondin is critical to an understanding of the function of this protein and its receptors. Several cellular binding sites for thrombospondin have been described that interact via specific domains. The smaller, heparin binding NH2 terminal globular domain of thrombospondin appears to mediate the agglutination of fixed, activated human platelets (15,16), and heparin inhibits the binding of thrombospondin to one class of receptors on endothelial cells and Chinesehamster ovary cells (5,17). These cells and embryonic lung fibroblasts bind and endocytose thrombospondin in a heparin inhibitable fashion, and degrade it in a processthat is saturable (18). An integrin receptor for thrombospondin has been described (19) which functions in several cell linesto mediate attachment to the RGD sequence on thrombospondin coated surfaces and in platelets as one class of thrombospondin receptors (19-21). In addition, interactions of thrombospondin with immobilizedsulfated glycolipids are mediated by a domainthat exhibits homology with malarial circumsporozoite proteins (CS-protein)from several Plasmodiumspecies and with thrombospondin-related anonymous protein (22,23). A sequencewith homology to this region (VTCG) has recently been reported to support adhesion of several cell types (24,25), but the biologic relevance, mechanismsor receptors mediating adhesionto the peptide sequence have not been defined. CD36 (GPIV) is a specific cell surface receptor for thrombospondin (26) which is present on platelets, monocytes, endothelial cells, and the human HT1080fibrosarcoma and C32 melanoma cell lines. CD36 isolated from platelets binds thrombospondin in purified systems. Antibodies to CD36 inhibit the binding of fluid-phasethrombospondin to C32 melanomaand HT1080fibrosarcoma cells as well as thrombospondin binding to activated platelets (26). McGregor and Leung et al have demonstrated binding of thrombospondin to purified CD36 and inhibition of platelet aggregation and thrombospondin expressionwith anti-CD36 antibodies (27). These data have been corroborated by Kieffer et al in studies demonstrating crosslinking of thrombospondin to CD36 on surface labeled platelets (28). In these studies, CD36 was the predominant labeled species precipitated by monoclonal anti-thrombospondin, and antibodies to CD36 inhibited surface expression of thrombospondin. Beiso et al have also demonstrated thrombospondin binding to platelet derived CD36 (29), and more recently, Legrand et al have confirmed that anti-CD36 antibody inhibits TSP binding to activated platelets (30). Antibodies to CD36 inhibit the thrombospondin-mediatedcytoadherence of Plasmodium falciparum-parasitizederythrocytes to endothelial cells (31) and purified CD36 (32,33). However, the role of CD36 as a TSP receptor has remained somewhatcontroversial because of experiments reported by Oquendo et al. that showed that COS cells transfected with CD36 cDNA supported cytoadherence of Plasmodiumfalciparum parasitized erythrocytes but showed no specific increase in thrombospondin binding (34). In addition, studies by Aiken et al (35) showed no inhibition of the binding of a monoclonal anti-TSP to the platelet surface when they were activated in the presence of an anti-CD36 monoclonal. We now describe studies that clarify the role of CD36 as a TSP receptor and define a conserved amino acid motif (CSVrCG) in TSP that mediatesinteractions with CD36.
Materials and Methods N: C32 melanomacells were obtained from ATCC and maintained in MEM containing 10% FCS and supplementedwith 4 grams/liter glucose and penicillin/streptomycin. Human platelet rich plasmawas obtained from normal volunteers as previously described (26). Jurkat cells were a gift from D. Posnett (Cornell University Medical College, New York) and were maintained in RPM1containing 10% FCS. 1209
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Peptides and proteins: Purifiedthrombospondin was prepared and rgdiolabeled using chloramineT as previously described (7) to a specific activity of 8x10 cpm/ug. Purified recombinant thrombospondin heparin binding domainwas a kind gift of Dr. Jacob Hartman (Biotechnology General). Purity was assessedby SDS-PAGE/Westernblotting. Peptide domains (CSVrCG, YCSVTCG, TVSGCC and EGF-domain (AA 630-674)were synthesized by FMOC chemistry and purified to >95% by HPLC (36). Both carboxylic acid and carboamide forms were synthesized. For conjugation to KLH, a carboxyterminal amino-caprolylcysteine amide was added to the sequence to allow for selective conjugation in a favorable conformation (37). In some preparations cysteines were protected from oxidation by the addition of acetamidomethyl groups (ACM). RGDSwas purchased from PeninsulaLabs. Antipeotide antibodies Antibodies to KLH conjugated peptides were raised in nine rabbits with 50ug/immunization on a schedule of injection every three to four weeks. Titers were measured by ELISA against albumin-conjugatesof the corresponding peptides as well as thrombospondin. Western blotting confirmed the anti-thrombospondinand anti-CS protein reactivity of antiCSVTCG antibodies that were raisedto the ACM-blocked peptides. Antisera raised against the unprotected peptides (unblocked cysteines) showed no reactivity to either thrombospondin or CS-protein. Transfection studies Jurkat cells were transfected with CD36 in the CDM8 plasmid construct previously reported by Oquendo et al (34) with plasmid lacking an insert, or with CDB/CDMB (kind gift of B. Seed, Harvard Medical School, Boston). Transfection was performed according to the methods of Chen and Okayama (38) and Gorman vd Howard (39) with the following modifications. Cellswere transfected at a density of 2x10 /ml and then stimulated with 5mM sodium butyrate for 20 hours to enhance expressionof recombinant plasmids. Following transfection, the expression of CD36 was assessedby immunoperoxidasestaining of cytospin sampleswhich revealed a transfection efficiency of 30% at 48 hours. Expressionwas further confirmed by surface iodination of CD36transfectants and control transfectants, followed by immunoprecipitationwith anti-CD36 monoclonal antibody and SDS-PAGE autoradiography as previously described (34). In studies to control for the relative efficiency of transfection, CD8 expression in cells transfected with the CDMB plasmidcontaining a CD8 insert was equivalent to CD36 expression in CD36-transfectants. Figure 1 is an autoradiogram of the immunoprecipitates obtained from surface labeled, control transfectants (Lane 1) and from CD36-transfected Jurkat cells (Lane 2). The broad immunoprecipitateis similarto that previously seen in the COS cell transfection studies by Oquendo et al (34) and comigrates with platelet CD36.
88-
from transfected Jurkat cells. Cells were surface ~gg&g?L%Inobypreci pitation of CD36 lactoperoxidase and immunoprecipitated with anti-CD36 monoclonal
antibody.
Lane 1, control transfectants; lane 2, CD-36 transfected Jurkat cells.
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Bindina studies: Thrombospondin was labeledto a specific ytivity of 6x1O’cpm/ug for these studies, and cell binding was performed in triplicate with 5x10 cells in a total volume of 0.2 ml Tris-buffered salinewith 2mM CaC12at 4 degrees C for 40 minutes. Cell bound radioactivity was determined by centrifuging the cells through silicone oil and removing and counting the cell pellets. Nonspecific binding was determintd by cold inhibition studies. YCSVTCG(NH2) was radiolabeledto a specific activity of 4.6x10 cpm/ug by similarmethods, and the direct binding measuredas described above. Flow cvtometrv: Thrombospondin expressionwas measuredin resting and stimulated platelets in the presence of 200uM carboxyamidomethylated-CSVTCG(NH2),or TVSGCC(NH2) using a monospecific polyclonal anti-thrombospondinantibody followed by FIT&conjugated second antibody and an EPICSflow system. Platelet aaareaometrv: Platelet aggregation in responseto agonists was performed using a Payton aggregometer. Cell adhesion studies: CSVTCG peptide was crosslinked to BSA as previously described in a molar ratio of 25: 1. 1OOug/mlBSA in PBS was used to coat plastic as previously described (11). Cellswere allowed to adhere (100,000cells/disc) for two hours at 37 degrees C in serum free MEM. Following incubation the discs were washed in MEM and fixed with 1% glutaraldehyde. Photomicroscopy was performed using a Nikon diaphot inverted phase microscope. Results
Transfection studies. The binding of thrombospondin to CD36-transfectedcells was specific and saturable (Figure 2) with half maximalsaturation at 50 nM and with 30,000 sites per cell (100,000sites after correcting for expression efficiency). Greater than 80% of the binding was inhibited by 5mM EDTA, or anti-CD36 (not shown)). No saturable binding was observed to the control transfectants.
350
-
100
,
80 A CD36
10
20
30
40
50
TSP (pg/ml)
03
,rantf.cf.ntr
A + l ,i-CDJ*
.cWI GO-E z &Q 40 -
0 50
100
[CSVTCOI
150
200
~JM
Figure 2. Thrombospondin binding to transfected Jurkat cells. The binding of ’ 261thrombospondin to CD364ransfected (triangles) and control-transfected Jurkat cells (squares) was examined. Saturable binding to CD36 transfectants was observed that was half-maximal at 6OnM. No saturable binding was observed with the control transfectants. w Direct binding of YCSVTCG to transfected Jurkat cells. Carboxamidomethylated I-YCSVTCG(NH4 (cvsteines blocked bv ACM arouos) bound soecificallv to CD36 transfected Jurkat cdlls fipright solid triangles) &ith haEm&mal binding at 1 OOpM. No specific binding was observed to control transfectants (square&J r to CD36 transfectants preincubated with anti-CD36 antibody (open triangle) +/- SEM. I-YCSVTCG that was not protected from oxidation by ACM or did not demonstrate binding to CDSB-transfected Jurkat cells (inverted solid triangles). 1211
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Recent data by Rich et al. and by Prater suggest that a conserved sequence, CSVTCG, which is repeated multiple times within the TSP molecule and displays homology with the circumsporozoite surface (CS) protein of several Plasmodium species (13) and with thrombospondinrelated anonymous protein (TRAP) (14) mediates the attachment of cells through sulfated glycolipids (24,25). Previous studies from this laboratory suggested that the same domain might be responsible for the binding of thrombospondin to CD36. Acm-CSVTCG inhibited the binding of radiolabeled TSP to U937 cells, but a peptide derived from the EGF homologous domain had no effect, nor did a recombinant heparin binding domain (40). In contrast to many cell lines, Jurkat cells have been demonstrated to lack expression of sulfated glycolipid determinants (41). Therefore, we chose these cells to examine TSP binding to recombinantly expressed CD36 (Figure 2) and more specifically, for the study of the role of the CSVTCG sequence in mediating that binding. Direct binding of radiolabeled tyrosinated CSVTCG was measured in both CD36-transfectants and in control transfectants (Figure 3). Specific and saturable binding of YCSVTCG in the ACM-blocked conformation was observed with half maximal binding observed at 100 uM. No specific binding was observed to control transfectants containing no insert or CD6. No specific binding of the YCSVTCG peptide in the unprotected conformation was detected to either CD36-transfectants or to control transfectants. Additional controls included radiolabeled recombinant heparin-binding domain and EGF-domain which showed no increased affinity for CD36-transfectants compared with control (not shown). The observed stoichiometry of peptide binding suggested that aggregation of this relatively hydrophobic peptide occured leading to an overestimation of the number of bound peptide molecules. Since the size of the aggregates and therefore the true number of peptide molecules bound could not be determined, these data are expressed as percent of maximal binding. That this curve represents specific CD3Smediated binding is demonstrated by the inhibition obtained with anti-CD36 antibody, by the lack of binding to control transfectants expressing CD8 and by the lack of binding of control peptide to any of the transfected cells (Figure 3). Peptide aggregation has been observed previously by others (42-44) and is a likely explanation for the large number of bound molecules. Peptide aggregates may be an explanation for the extraordinary increase in surface fluorescence due to VTCG cell binding observed by Rich (24). Inhibition of platelet function. Thrombospondin is released from platelet alpha granule stores following platelet activation and binds to the platelet surface via a mechanism that is mediated in part by CD36. Thrombospondin diminishes the dissociation of fibrinogen from the GPllb/llla complex and antibodies to thrombospondin or CD36 inhibit platelet aggregation by blocking the association of thrombospondin with the platelet surface (1,26-30). The role of the CSVTCG domain in mediating thrombospondin surface expression on activated platelets was explored in studies measuring thrombospondin expression by flow cytometry. Thrombospondin surface expression on platelets stimulated by the calcium ionophore A231 87 was unaffected by the control peptide TVSGCC (200uM) but wasinhibited by CSVTCG (Figure 4a). The functional correlate of this inhibition of thrombospondin expression was seen in aggregometry experiments where CSVTCG (200uM) partially inhibited platelet aggregation in response to A231 87 (Figure 4b). Similar inhibition of platelet aggregation was seen with other platelet agonists including epinephrine, ADP and collagen but was not observed wlth aspirinated platelets (not shown). This effect is consistent with inhibition of platelet surface expression of thrombospondin by the CSVTCG peptide and is similar to the inhibition of aggregation that is observed with antibodies to thrombospondin and CD36 (1,27,30). 1212
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b
Figure 4. Inhibition of platelet thrombospondin expression. a) Flow cytometry reveals normal thrombospondin expression on the platelet surface following stimulation with the calcium ionophore A231 87 in the presence of the control peptide TVSGCC (20Of~M) and inhibition of thrombospondin surface expression in the presence of ACM-blocked CSVTCG(NH2) (200uM). b) Platelet aggregometry of platelet rich plasma reveals a diminished response to A231 87 in the presence of ACM-blocked CSVTCG(NH2) (20QM) compared with the control aggregation in the presence of TVSGCC (20OuM).
Adhesion studies. To investigate the receptor mechanisms by which this sequence mediates thrombospondin-cellutar interactions we examined the ability of this peptide sequence to support cell adhesion. CSVTCG-Albumin conjugates were coated on plastic as previously described (45) and their ability to support the adhesion of C32 melanoma cells was examined. Cell attachment was unaffected by RGDS, heparin, anti-beta3 integrin antibody, or the scrambled peptide, but was blocked by anti-CD36 monoclonal and by 200 uM carboxyamidomethylated CSVTCG (Figure 5). The failure of heparin to affect attachment to CSVTCG-albumin suggests that sulfated glycolipids on C32 melanoma cells are not necessary for this attachment, although their role in other cell types has been implicated by Prater et al.(25).
Discussion These studies demonstrate that recombinantly expressed CD36 functions as a TSP receptor and identify a domain of thrombospondin that mediates cellular interactions of this multifunctional protein with CD36. The hexapeptide sequence CSVTCG is part of a region of the thrombospondin molecule that displays homology with the CS-protein of several Plasmodium species. Thrombospondin contains nine CS-homologous repeats (three in each monomer) in a region that is filamentous by rotary shadowing, and is close to the regions that interact with fibrinogen and collagen. This area lies within the cysteine-rich region that forms the majority of disulfide links between thrombospondin monomers (46-49). The observation that thrombospondin mediates cytoadherence of the trophozoite stage (12) and that an antibody to an 88 kd glycoprotein on endothelial cells, monocytes, platelets, and C32 melanoma cells inhibited this phenomenon (31) led to our identification of CD36 as a cellular thrombospondin receptor (26) and as a mediator of cytoadherence (50). More recently, however, the description of a parasite-derived thrombospondin-related-anonymous-protein (TRAP) suggested that the conserved sequence expressed in these three proteins may be relevent to their adhesive
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Figure 5. Melanoma cell adhesion to ACM-blocked CSVTCG-albumin coated plastic. Melanoma cells adhere to CSVTCG-albumin coated plastic in the presence of the control
C32
peptide TVSGCC (A); adhesion is inhibited by anti-CD36 monoclonal antibody (B), and by ACM-blocked CSVTCG(NH ) (200uM) (c). No inhibition of adhesion is seen in the presence of heparin 1 OOug Pml (D), or RGDS (1 mM) (E), or antibody to beta-3 integrin complexes(F). CSVTCG peptide was crosslinked to BSA as previously described in a molar ratio of 25:l. 1 OOug/ml BSA in PBS was used to coat plastic as previously described by Roberts et al (Roberts et al.1 987b). Cells were allowed to adhere (100,000 Following incubation the cells/disc) for two hours at 37 degrees C in serum free MEM. discs were washed in MEM and fixed with I % glutaraldehyde. Photomicroscopy was performed using a Nikon diaphot inverted phase microscope. C32 melanoma cells were obtained from ATCC and maintained in MEM containing 10% FCS and supplemented with 4 grams/liter glucose and penicillin/streptomycin.
biologic functions (14). This concept was previously suggested by Born-stein et al (13). Recent data that cytoadherence to purified CD36 does not require exogenous thrombospondin (50) supports the possibility that TRAP--or some other parasite-induced protein (51).-interacts with CD36. The role of the same sequence in circumsporozoite surface proteins from several Plasmodium species is not clear, but our data and the recent report by Rich et al (24) suggests the possiblity that adhesive interactions of sporozoites might also be mediated by this domain with CD36. Other domains macromolecule
of thrombospondin via other receptor
clearly play a part in the cell surface systems. Heparin inhibitable binding 1214
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bospondin by fibroblasts, endothelial cells, and CHO cells has been reported previously (18); and RGD-dependentthrombospondin adhesion is mediated by an integrin receptor in some cell types (19). In many cell types, multiple cell receptors for thrombospondin coexist (5253) making interpretation of antibody inhibition studies difficult. In platelets, the role of CD36 as a thrombospondin binding site has been controversial, but the weight of evidence supports this finding. Several groups have presented data that support a role for platelet CD36 as a thrombospondin binding site (26-29). In contrast, Aiken et al recently reported that thrombospondin surface expressionthat was detected indirectly using Mab TSP-1 was not inhibited by anti-CD36 antibody (35). The latter finding raisesinteresting questions concerning the domain specificity of Mab TSP-1 and its effect on overall thrombospondin binding to platelets. More recently, additional confirmation of the role of platelet CD36 as a TSP binding site has been provided by Legrand et al, who showed inhibition of TSP surface expression by an anti-CD36 Mab (30). Our own data suggest that the domain CSVTCG is responsiblefor surface expressed TSP in activated platelets, and is consistent with data demonstrating inhibition of TSP surface expression by anti-CD36 antibodies. Alternative mechanismsfor TSP surface expression on activated platelets exist, and reports that patients with the Nak- platelet phenotype (who show altered reactivity with anti-CD36antibodies) still bind TSP confirm the complexity of the TSP-platelet interaction (54). The specific CD36-mediatedthrombospondin-and CSVTCG-binding that we observed are at odds with that reported by Oquendo et al (34) with CD36transfected COS cells. Several possible explanations may be relevant to the differences in observed CD36 function in these cell types. Jurkat cells were chosen for these studies becausethey lack CD36, do not express sulfated glycolipids (41), have a low background thrombospondin binding prior to transfection, grow unattached, and are human in origin. COS cells, by contrast, although they express no CD36, display a great deal of native thrombospondin binding that may obscure a relatively smallcomponent of binding that is secondary to recombinant protein expression. Further, since COS cells grow as monolayers,the measurementof fluid phase binding may be affected by matrix components or damaged and dead cells that are removed from the culture dish. Similar concerns apply if the studies are performed on cell monolayers. Finally, and perhaps most importantly, the cell in which the recombinant protein is expressed may be relevent to its function. The conformation or function of recombinantly expressed CD36 may be affected by the lipid composition as has been recently reported for some integrins (55) or by differences in glycosylation (56).\ Since no calcium was added in the binding studies, since the binding was performed in Tris buffered saline, and since EDTA had no effect on the binding of the peptide, these data are not influenced by the recently described calcium-phosphatemediated thrombospondin precipitation (57). These findings are consistent with the role of the calcium binding repeats in the observed calcium dependence of thrombospondin binding and suggest that the calcium dependence of thrombospondin-CD36interaction is a function of the conformational state of the ligand rather than that of CD36. In addition to its presence in thrombospondin, Malarial CS-proteins and in TRAP, the sequence CSVTCG appears in several other proteins that are of great biologic relevance. Properdin contains three such homologous repeat sequences (58), and shareswith thrombospondin the ability to bind to sulfated glycoconjugate proteins (23) raising the possibility that the same sequence might mediate interaction of these proteins with sulfated glycoconjugates or that properdin might share the ability to bind to CD36. Recent data by Prater et al (25) suggeststhat cell attachment to this sequence by cells lacking CD36, may be mediated
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by sulfated glycolipids. In contrast to the interaction of CSVTCG with 036, the interaction of this sequence with sulfated glycolipids is inhibited by reduction and alkylation (23) suggesting that specificity with respect to one or another binding sites depends on secondary structure considerations. In contrast to the findings of Holt et al (23) Prater’s group found no effect of alkylation of this sequence on cell attachment. The same conserved sequence is also found in antistasin, a salivary protein from the Mexican leech Haementeria officinalis that also binds sulfated glycoconjugates, and is an inhibitor of metastasis and coagulation (59). Our recent observations that C32 melanoma cells selected for high levels of CD36 expression exhibit a greater metastatic potential in nude mice than do cells that express low density CD36 (60) are consistent with the thrombospondin mediated enhancement of metastasis described in other tumor cell lines (61) and raise the possibility that this sequence might exert antimetastatic effects in vivo by blocking CD36-dependent thrombospondin adhesion and metastasis. Acknowledgments We wish to thank B. Ferris and S. Carson for expert technical help. CD36lCDM8 and CDB/CDMB were kind gifts from Dr. B. Seed (Harvard Medical School, Boston). Synthetic EGF domain(AA630-674) was a kind gift from Dr.J.Tam (Rockefeller University, NY). Jurkat cells were a kind gift from Dr. D. Posnett (Cornell University Medical College, NY). Whole circumsporozoite lysate was a kind gift from Dr. J. Barnwell (New York University, NY). This work was supported by grants from the NIH (AA and RN). AA is the recipient of a Stratton Foundation Award and the Mellon Teacher Scientist Award. References 1. Leung, L.L. (1984) J. Clin. lnvest.74, 1764-1772. 2. Bacon Baguley, T., Kudryk, B.J., and Walz, D.A. (1987) J. Biol. Chem.262, 1927-1930. 3. Bacon Baguley, T., Ogilvie, M.L., Gartner, T.K., and Walz, D.A. (1990) J. Biol. Chem.265, 2317-2323. 4. Bale, M.D. and Mosher, D.F. (1986) J. Biol. Chem.261,862-868. 5. Murphy Ullrich, J.E. and Mosher, D.F. (1985) Blood66, 1098-I 104. 6. Bale, M.D., Westrick, L.G., and Mosher, D.F. (1985) J. Biol. Chem.260, 7502-7508. 7. Silverstein, R.L., Leung, L.L., Harpel, P.C., and Nachman, R.L. (1984) J. Clin. lnvest.74, 1625-l 633. . 8. Majack, R.A., Cook, S.C., and Bornstein, P. (1986) Proc. Natl. Acad. Sci. USA83, 90509054. 9. Maiack. R.A.. Goodman, L.V.. and Dixit. V.M. (1988) J. Cell Biol.106. 415-422. 10. Tu’szynski, G.P., Rothman, V., Murphy,’ A., Siegler,‘K., Smith, L., Smith, S., Karczewski, J., and Knudsen, K.A. (1987) Science236, 1570-l 573. Il. Roberts, D.D., Sherwood, J.A., and Ginsburg, V. (1987) J. Cell Biol.104, 131-l 39. 12. Roberts, D.D., Sherwood, J.A., Spitalnik, S.L., Panton, L.J., Howard, R.J., Dixit, V.M., Frazier, W.A., Miller, L.H., and Ginsburg, V. (1985) Nature31 8, 64-66. 13. Kobayashi, S., Eden McCutchan, F., Framson, P., and Bornstein, P. (1986) Biochemistry.25, 8418-8425. 14. Robson, K.J., Hall, J.R., Jennings, M.W., Harris, T.J., Marsh, K., Newbold, C.I., Tate, V.E., and Weatherall, D.J. (1988) Nature335 79-82. 15. Gartner, T.K., Walz, D.A., Aiken, M., Starr Spires, L., and Ogilvie, M.L. (1984) Biochem. Biophys. Res. Commun.124, 290295. 16. Dixit, V.M., Haverstick, D.M., ORourke, K.M., Hennessy, S.W., Grant, G.A., Santoro, S.A., and Frazier, W.A. (1985) Biochemistry.24, 4270-4275. 17. Murphy Ullrich, J.E., Westrick, L.G., Esko, J.D., and Mosher, D.F. (1988) J. Biol. Chem.263, 64006406. 18. McKeown Longo, P.J., Hanning, R., and Mosher, D.F. (1984) J. Cell Biol.98, 22-28. 19. Lawler, J., Weinstein, R., and Hynes, R.O. (1988) J. Cell Biol.107, 1)-P 2351-6. 20. Lam, S.C., Plow, E.F., DSouza, S.E., Cheresh, D.A., Frelinger, A.L 3d., and Ginsberg, M.H. (1989) J. Biol. Chem.264, 3742-3749. 21. Lawler, J. and Hynes, R.O. (1989) Blood74,2022-2027. 1216
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22. Roberts, D.D., Haverstick, D.M., Dixit, V.M., Frazier, W.A., Santoro, S.A., and Ginsburg, V. (1985) J. Biol. Chem.260, 9405-9411. 23. Holt, G.D., Pangburn, M.K., and Ginsburg, V. (1990) J. Biol. Chem.265, 2852-2855. 24. Rich, K.A., George, F.W.,, Law, J.L., and Martin, J.W. (1990) Science249, 1574-1577. 25. Prater, C.A., Plotkin, J., Jaye, D., and Frazier, W.A. (1991) J. Cell Biol.112, 1031-l 040. 26. Asch, A.S., Barnwell, J., Silverstein, R.L., and Nachman, R.L. (1987) J. Clin. Invest.79, 1054-l 061. 27. McGregor, J.L., Catimel, B., Parmentier, S., Clezardin, P., Dechavanne, M., and Leung, L.L. (1989) J. Biol. Chem.264, 501-506. 28. Kieffer, N., Nurden, A.T., Hasitz, M., Titeux, M., and Breton-Gorius, J. (1988) Biochim. Biophys. Acta967,408-415. 29. Beiso, P., Pidard, D., Dubernard, V., Edelman, L., Kieffer, N., and Legrand, C. (1988) Blood72 Suppl, 1172a. 30. Legrand, C., Pidard, D., Beiso, P., Tenza, D., and Edelman, L. (1991) PlateletsP, 99-l 05. 31. Barnwell, J.M., Ockenhouse, CF., and Knowles, D.M.III (1985) J. Immunol.135, 3494-3497. 32. Barnwell, J.W., Asch, A.S., Nachman, R.L., Yamaya, M., Aikawa, M., and Ingravallo, P. (1989) J. Clin. Invest&t, 765-772. 33. Ockenhouse, C.F., Tandon, N.N., Magowan, C., Jamieson, G.A., and Chulay, J.D. (1989) Science243, 1469-l 471. 34. Oquendo, P., Hundt, E., Lawler, J., and Seed, B. (1989) Cell58, 95-101. 35. Aiken, M.L., Ginsberg, M.H., Byers Ward, V., and Plow, E.F. (1990) Blood76,2501-2509. 36. Huang, L.H., Ke, X.-H., Sweeney, W., and Tam, J.P. (1989) Biochem. Biophys. Res. Commun.160, 133-l 39. 37. Heath, W.F. and Merrifield, R.B. (1986) Proc. Natl. Acad. Sci. U. S. A.83, 6367-6371. 38. Chen, C. and Okayama, H. (1987) Mol. Cell. .Biol.7,2745-2752. 39. Gorman, C.M. and Howard, B.H. (1983) Nut. Acids. Res.l1,7631-7648. 40. Asch, AS., Heimer, E., and Nachman, R.L. (1990) Blood76, 445a.(Abstract) 41. Aruffo, A., Kolanus, W., Walz, G., Fredman, P., and Seed, 8. (1991) Cell67, 35-44. 42. Kirschner, D.A., Inouye, H., Duffy, L.K., Sinclair, A., Lind, M., and Selkoe, D.J. (1987) Proc. Natl. Acad. Sci. U. S. A.84, 6953-6957. 43. Homandberg, G.A. and Erickson, J.W. (1986) Biochemistry.25, 6917-6925. 44. Castano, E.M., Ghiso, J., Prelli, F., Gorevic, P.D., Migheli, A., and Frangione, B. (1986) Biochem. Biophys. Res. Commun.141, 782-789. 45. Danilov, Y.N. and Juliano, R.L. (1989) Exp. Cell Res.182, 186-196. 46. Dixit, V.M., Grant, G.A., Frazier, W.A., and Santoro, S.A. (1984) Biochem. Biophys. Res. Commun.119, 1075-l 081. 47. Galvin, N.J., Dixit, V.M., ORourke, K.M., Santoro, S.A., Grant, G.A., and Frazier, W.A. (1985) J. Cell Biol.101, 1434-l 441. 48. Frazier, W.A. (1987) J. Cell Biol.105, 625-632. 49. Dixit, V.M., Hennessy, S.W., Grant, G.A., Rotwein, P., and Frazier, W.A. (1986) Proc. Natl. Acad. Sci. USA83, 5449-5453. 50. Barnwell, J.W. (1989) Exp. Parasitol.69, 407-412. 51. Biggs, B.A., Gooze, L., Wycherley, K., Wilkinson, D., Boyd, A.W., Forsyth, K.P., Edelman, L., Brown, G.V., and Leech, J.H. (1990) J. Exp. Med.1 71, 1883-l 892. 52. Taraboletti, G., Roberts, D.D., and Liotta, L.A. (1987) J. Cell Biol.l05,2409-2415. 53. Asch, A.S., Tepler, J., Silbiger, S., and Nachman, R.L. (1991) J. Biol. Chem.266, 17401745. 54. Tandon, N.N., Ockenhouse, C.F., Greco, N.J., and Jamieson, G.A. (1991) Blood78,28092813. 55. Conforti, G., Zanetti, A., Pasquali-Ronchetti, I., Quaglino, D.,Jr., Neyroz, P., and Dejana, E. (1990) J. Biol. Chem.265, 401 l-401 9. 56. Rieber, M., Gross, A., and Rieber, M.S. (1987) Cancer Res.47, 5127-5131. 57. Sun, X. and Mosher, D.F. (1991) J. Clin. lnvest.87, 171-176. 58. Goundis, D. and Reid, K.B. (1988) Nature335, 82-85. 59. Holt, G.D., Krivan, H.C., Gasic, G.J., and Ginsburg, V. (1989) J. Biol. Chem.264, 12138121 40. 60. Wong, J.E.L. and Asch, AS. (1989) Blood (Abstract) 61. Tuszynski, G.P., Rothman, V.L., Murphy, A., and Knudsen, K.A. (1987) Semin. Thromb. Hemost. 3, 361-368.
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Vol. 182, No. 3, 1992 February 14, 1992
THROMBOSPONDIN
Pages
SEQUENCE
MOTIF
(CSVrCG)
IS RESPONSIBLE
1208-1217
FOR CD36 BINDING
Adam S. Asch,‘Scott Silbiger, Edgar Heimer,*and Ralph L. Nachman
Specialized Center for Researchin Thrombosis, Division of Hematology-Oncology, Cornell University Medical College, New York, NY 10021 *Department of Molecular Genetics and Department of Peptide Chemistry, Hoffman-LaRoche, Nutley, New Jersey 07110
Received
December
17,
1991
To clarify the role of CD36 as a TSP receptor and to investigate the mechanismsof the TSPCD36 interaction, transfection studies were performed using CD36cDNA in a CDM8 plasmid. Jurkat cells transfected with CD36 cDNA express an 88kD membranesurface protein and acquire the ability to bind thrombospondin. The TSP amino acid sequence, CSVTCG, mediates the interaction of thrombospondin with CD36. CD36 transfectants but not control transfectants bind radiolabeled tyrosinated peptide (YCSVTCG). The hexapeptide inhibits thrombospondin expression on activated human platelets and results in diminishedplatelet aggregation. CSVTCG-albuminconjugates support CD36dependent adhesion of tumor cells, We conclude that the CSVTCG repeat sequence is a crucial determinant of CD36 thrombospondin binding. B 1992 Academic Press, Inc.
Thrombospondin is a cell and matrix protein with a wide tissue distribution and multiple biologic functions. Thrombospondin supports the secretion dependent phase of platelet aggregation by stabilizing the interaction of fibrinogen with its receptor (1). Thrombospondin binds to distinct sequenceswithin the A-alpha and B-beta chains of fibrinogen (2,3) and following the initiation of coagulation is incorporated into fibrin clot where it modulatesthe meshwork structure of fibrin filaments (4-8) and serves as a site for plasminogenlocalization and activation (7). Thrombospondin has a growth regulatory role for fibroblasts and smooth muscle cells (8,9). As an adhesive protein, thrombospondin supports the attachment of several cell types (10,ll) and may mediate cytoadhesion of P. falciparum infected erythrocytes to endothelium (12). Sequence homologieswith Malarial circumsporozoite protein, thrombospondin-relatedanonymous-protein (TRAP), properdin and complement components C8 and C9 suggest the importance of these conserved regions (13,14).
1
To whom correspondence
should
be addressed.
Abbreviations : TSP, thrombospondin; CS-protein, circumsporozoite surface-protein; DlT, dithiothreitol; FCS, fetal calf serum; KLH, keyhole limpet hemocyanin. 0006-291X/92 Copyright AU rights
$1.50
6 1992 by Academic Press, Inc. of reproduction in any form reserved.
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Identification of the specific cell binding domains of thrombospondin is critical to an understanding of the function of this protein and its receptors. Several cellular binding sites for thrombospondin have been described that interact via specific domains. The smaller, heparin binding NH2 terminal globular domain of thrombospondin appears to mediate the agglutination of fixed, activated human platelets (15,16), and heparin inhibits the binding of thrombospondin to one class of receptors on endothelial cells and Chinesehamster ovary cells (5,17). These cells and embryonic lung fibroblasts bind and endocytose thrombospondin in a heparin inhibitable fashion, and degrade it in a processthat is saturable (18). An integrin receptor for thrombospondin has been described (19) which functions in several cell linesto mediate attachment to the RGD sequence on thrombospondin coated surfaces and in platelets as one class of thrombospondin receptors (19-21). In addition, interactions of thrombospondin with immobilizedsulfated glycolipids are mediated by a domainthat exhibits homology with malarial circumsporozoite proteins (CS-protein)from several Plasmodiumspecies and with thrombospondin-related anonymous protein (22,23). A sequencewith homology to this region (VTCG) has recently been reported to support adhesion of several cell types (24,25), but the biologic relevance, mechanismsor receptors mediating adhesionto the peptide sequence have not been defined. CD36 (GPIV) is a specific cell surface receptor for thrombospondin (26) which is present on platelets, monocytes, endothelial cells, and the human HT1080fibrosarcoma and C32 melanoma cell lines. CD36 isolated from platelets binds thrombospondin in purified systems. Antibodies to CD36 inhibit the binding of fluid-phasethrombospondin to C32 melanomaand HT1080fibrosarcoma cells as well as thrombospondin binding to activated platelets (26). McGregor and Leung et al have demonstrated binding of thrombospondin to purified CD36 and inhibition of platelet aggregation and thrombospondin expressionwith anti-CD36 antibodies (27). These data have been corroborated by Kieffer et al in studies demonstrating crosslinking of thrombospondin to CD36 on surface labeled platelets (28). In these studies, CD36 was the predominant labeled species precipitated by monoclonal anti-thrombospondin, and antibodies to CD36 inhibited surface expression of thrombospondin. Beiso et al have also demonstrated thrombospondin binding to platelet derived CD36 (29), and more recently, Legrand et al have confirmed that anti-CD36 antibody inhibits TSP binding to activated platelets (30). Antibodies to CD36 inhibit the thrombospondin-mediatedcytoadherence of Plasmodium falciparum-parasitizederythrocytes to endothelial cells (31) and purified CD36 (32,33). However, the role of CD36 as a TSP receptor has remained somewhatcontroversial because of experiments reported by Oquendo et al. that showed that COS cells transfected with CD36 cDNA supported cytoadherence of Plasmodiumfalciparum parasitized erythrocytes but showed no specific increase in thrombospondin binding (34). In addition, studies by Aiken et al (35) showed no inhibition of the binding of a monoclonal anti-TSP to the platelet surface when they were activated in the presence of an anti-CD36 monoclonal. We now describe studies that clarify the role of CD36 as a TSP receptor and define a conserved amino acid motif (CSVrCG) in TSP that mediatesinteractions with CD36.
Materials and Methods N: C32 melanomacells were obtained from ATCC and maintained in MEM containing 10% FCS and supplementedwith 4 grams/liter glucose and penicillin/streptomycin. Human platelet rich plasmawas obtained from normal volunteers as previously described (26). Jurkat cells were a gift from D. Posnett (Cornell University Medical College, New York) and were maintained in RPM1containing 10% FCS. 1209
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Peptides and proteins: Purifiedthrombospondin was prepared and rgdiolabeled using chloramineT as previously described (7) to a specific activity of 8x10 cpm/ug. Purified recombinant thrombospondin heparin binding domainwas a kind gift of Dr. Jacob Hartman (Biotechnology General). Purity was assessedby SDS-PAGE/Westernblotting. Peptide domains (CSVrCG, YCSVTCG, TVSGCC and EGF-domain (AA 630-674)were synthesized by FMOC chemistry and purified to >95% by HPLC (36). Both carboxylic acid and carboamide forms were synthesized. For conjugation to KLH, a carboxyterminal amino-caprolylcysteine amide was added to the sequence to allow for selective conjugation in a favorable conformation (37). In some preparations cysteines were protected from oxidation by the addition of acetamidomethyl groups (ACM). RGDSwas purchased from PeninsulaLabs. Antipeotide antibodies Antibodies to KLH conjugated peptides were raised in nine rabbits with 50ug/immunization on a schedule of injection every three to four weeks. Titers were measured by ELISA against albumin-conjugatesof the corresponding peptides as well as thrombospondin. Western blotting confirmed the anti-thrombospondinand anti-CS protein reactivity of antiCSVTCG antibodies that were raisedto the ACM-blocked peptides. Antisera raised against the unprotected peptides (unblocked cysteines) showed no reactivity to either thrombospondin or CS-protein. Transfection studies Jurkat cells were transfected with CD36 in the CDM8 plasmid construct previously reported by Oquendo et al (34) with plasmid lacking an insert, or with CDB/CDMB (kind gift of B. Seed, Harvard Medical School, Boston). Transfection was performed according to the methods of Chen and Okayama (38) and Gorman vd Howard (39) with the following modifications. Cellswere transfected at a density of 2x10 /ml and then stimulated with 5mM sodium butyrate for 20 hours to enhance expressionof recombinant plasmids. Following transfection, the expression of CD36 was assessedby immunoperoxidasestaining of cytospin sampleswhich revealed a transfection efficiency of 30% at 48 hours. Expressionwas further confirmed by surface iodination of CD36transfectants and control transfectants, followed by immunoprecipitationwith anti-CD36 monoclonal antibody and SDS-PAGE autoradiography as previously described (34). In studies to control for the relative efficiency of transfection, CD8 expression in cells transfected with the CDMB plasmidcontaining a CD8 insert was equivalent to CD36 expression in CD36-transfectants. Figure 1 is an autoradiogram of the immunoprecipitates obtained from surface labeled, control transfectants (Lane 1) and from CD36-transfected Jurkat cells (Lane 2). The broad immunoprecipitateis similarto that previously seen in the COS cell transfection studies by Oquendo et al (34) and comigrates with platelet CD36.
88-
from transfected Jurkat cells. Cells were surface ~gg&g?L%Inobypreci pitation of CD36 lactoperoxidase and immunoprecipitated with anti-CD36 monoclonal
antibody.
Lane 1, control transfectants; lane 2, CD-36 transfected Jurkat cells.
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Bindina studies: Thrombospondin was labeledto a specific ytivity of 6x1O’cpm/ug for these studies, and cell binding was performed in triplicate with 5x10 cells in a total volume of 0.2 ml Tris-buffered salinewith 2mM CaC12at 4 degrees C for 40 minutes. Cell bound radioactivity was determined by centrifuging the cells through silicone oil and removing and counting the cell pellets. Nonspecific binding was determintd by cold inhibition studies. YCSVTCG(NH2) was radiolabeledto a specific activity of 4.6x10 cpm/ug by similarmethods, and the direct binding measuredas described above. Flow cvtometrv: Thrombospondin expressionwas measuredin resting and stimulated platelets in the presence of 200uM carboxyamidomethylated-CSVTCG(NH2),or TVSGCC(NH2) using a monospecific polyclonal anti-thrombospondinantibody followed by FIT&conjugated second antibody and an EPICSflow system. Platelet aaareaometrv: Platelet aggregation in responseto agonists was performed using a Payton aggregometer. Cell adhesion studies: CSVTCG peptide was crosslinked to BSA as previously described in a molar ratio of 25: 1. 1OOug/mlBSA in PBS was used to coat plastic as previously described (11). Cellswere allowed to adhere (100,000cells/disc) for two hours at 37 degrees C in serum free MEM. Following incubation the discs were washed in MEM and fixed with 1% glutaraldehyde. Photomicroscopy was performed using a Nikon diaphot inverted phase microscope. Results
Transfection studies. The binding of thrombospondin to CD36-transfectedcells was specific and saturable (Figure 2) with half maximalsaturation at 50 nM and with 30,000 sites per cell (100,000sites after correcting for expression efficiency). Greater than 80% of the binding was inhibited by 5mM EDTA, or anti-CD36 (not shown)). No saturable binding was observed to the control transfectants.
350
-
100
,
80 A CD36
10
20
30
40
50
TSP (pg/ml)
03
,rantf.cf.ntr
A + l ,i-CDJ*
.cWI GO-E z &Q 40 -
0 50
100
[CSVTCOI
150
200
~JM
Figure 2. Thrombospondin binding to transfected Jurkat cells. The binding of ’ 261thrombospondin to CD364ransfected (triangles) and control-transfected Jurkat cells (squares) was examined. Saturable binding to CD36 transfectants was observed that was half-maximal at 6OnM. No saturable binding was observed with the control transfectants. w Direct binding of YCSVTCG to transfected Jurkat cells. Carboxamidomethylated I-YCSVTCG(NH4 (cvsteines blocked bv ACM arouos) bound soecificallv to CD36 transfected Jurkat cdlls fipright solid triangles) &ith haEm&mal binding at 1 OOpM. No specific binding was observed to control transfectants (square&J r to CD36 transfectants preincubated with anti-CD36 antibody (open triangle) +/- SEM. I-YCSVTCG that was not protected from oxidation by ACM or did not demonstrate binding to CDSB-transfected Jurkat cells (inverted solid triangles). 1211
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Recent data by Rich et al. and by Prater suggest that a conserved sequence, CSVTCG, which is repeated multiple times within the TSP molecule and displays homology with the circumsporozoite surface (CS) protein of several Plasmodium species (13) and with thrombospondinrelated anonymous protein (TRAP) (14) mediates the attachment of cells through sulfated glycolipids (24,25). Previous studies from this laboratory suggested that the same domain might be responsible for the binding of thrombospondin to CD36. Acm-CSVTCG inhibited the binding of radiolabeled TSP to U937 cells, but a peptide derived from the EGF homologous domain had no effect, nor did a recombinant heparin binding domain (40). In contrast to many cell lines, Jurkat cells have been demonstrated to lack expression of sulfated glycolipid determinants (41). Therefore, we chose these cells to examine TSP binding to recombinantly expressed CD36 (Figure 2) and more specifically, for the study of the role of the CSVTCG sequence in mediating that binding. Direct binding of radiolabeled tyrosinated CSVTCG was measured in both CD36-transfectants and in control transfectants (Figure 3). Specific and saturable binding of YCSVTCG in the ACM-blocked conformation was observed with half maximal binding observed at 100 uM. No specific binding was observed to control transfectants containing no insert or CD6. No specific binding of the YCSVTCG peptide in the unprotected conformation was detected to either CD36-transfectants or to control transfectants. Additional controls included radiolabeled recombinant heparin-binding domain and EGF-domain which showed no increased affinity for CD36-transfectants compared with control (not shown). The observed stoichiometry of peptide binding suggested that aggregation of this relatively hydrophobic peptide occured leading to an overestimation of the number of bound peptide molecules. Since the size of the aggregates and therefore the true number of peptide molecules bound could not be determined, these data are expressed as percent of maximal binding. That this curve represents specific CD3Smediated binding is demonstrated by the inhibition obtained with anti-CD36 antibody, by the lack of binding to control transfectants expressing CD8 and by the lack of binding of control peptide to any of the transfected cells (Figure 3). Peptide aggregation has been observed previously by others (42-44) and is a likely explanation for the large number of bound molecules. Peptide aggregates may be an explanation for the extraordinary increase in surface fluorescence due to VTCG cell binding observed by Rich (24). Inhibition of platelet function. Thrombospondin is released from platelet alpha granule stores following platelet activation and binds to the platelet surface via a mechanism that is mediated in part by CD36. Thrombospondin diminishes the dissociation of fibrinogen from the GPllb/llla complex and antibodies to thrombospondin or CD36 inhibit platelet aggregation by blocking the association of thrombospondin with the platelet surface (1,26-30). The role of the CSVTCG domain in mediating thrombospondin surface expression on activated platelets was explored in studies measuring thrombospondin expression by flow cytometry. Thrombospondin surface expression on platelets stimulated by the calcium ionophore A231 87 was unaffected by the control peptide TVSGCC (200uM) but wasinhibited by CSVTCG (Figure 4a). The functional correlate of this inhibition of thrombospondin expression was seen in aggregometry experiments where CSVTCG (200uM) partially inhibited platelet aggregation in response to A231 87 (Figure 4b). Similar inhibition of platelet aggregation was seen with other platelet agonists including epinephrine, ADP and collagen but was not observed wlth aspirinated platelets (not shown). This effect is consistent with inhibition of platelet surface expression of thrombospondin by the CSVTCG peptide and is similar to the inhibition of aggregation that is observed with antibodies to thrombospondin and CD36 (1,27,30). 1212
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b
Figure 4. Inhibition of platelet thrombospondin expression. a) Flow cytometry reveals normal thrombospondin expression on the platelet surface following stimulation with the calcium ionophore A231 87 in the presence of the control peptide TVSGCC (20Of~M) and inhibition of thrombospondin surface expression in the presence of ACM-blocked CSVTCG(NH2) (200uM). b) Platelet aggregometry of platelet rich plasma reveals a diminished response to A231 87 in the presence of ACM-blocked CSVTCG(NH2) (20QM) compared with the control aggregation in the presence of TVSGCC (20OuM).
Adhesion studies. To investigate the receptor mechanisms by which this sequence mediates thrombospondin-cellutar interactions we examined the ability of this peptide sequence to support cell adhesion. CSVTCG-Albumin conjugates were coated on plastic as previously described (45) and their ability to support the adhesion of C32 melanoma cells was examined. Cell attachment was unaffected by RGDS, heparin, anti-beta3 integrin antibody, or the scrambled peptide, but was blocked by anti-CD36 monoclonal and by 200 uM carboxyamidomethylated CSVTCG (Figure 5). The failure of heparin to affect attachment to CSVTCG-albumin suggests that sulfated glycolipids on C32 melanoma cells are not necessary for this attachment, although their role in other cell types has been implicated by Prater et al.(25).
Discussion These studies demonstrate that recombinantly expressed CD36 functions as a TSP receptor and identify a domain of thrombospondin that mediates cellular interactions of this multifunctional protein with CD36. The hexapeptide sequence CSVTCG is part of a region of the thrombospondin molecule that displays homology with the CS-protein of several Plasmodium species. Thrombospondin contains nine CS-homologous repeats (three in each monomer) in a region that is filamentous by rotary shadowing, and is close to the regions that interact with fibrinogen and collagen. This area lies within the cysteine-rich region that forms the majority of disulfide links between thrombospondin monomers (46-49). The observation that thrombospondin mediates cytoadherence of the trophozoite stage (12) and that an antibody to an 88 kd glycoprotein on endothelial cells, monocytes, platelets, and C32 melanoma cells inhibited this phenomenon (31) led to our identification of CD36 as a cellular thrombospondin receptor (26) and as a mediator of cytoadherence (50). More recently, however, the description of a parasite-derived thrombospondin-related-anonymous-protein (TRAP) suggested that the conserved sequence expressed in these three proteins may be relevent to their adhesive
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Figure 5. Melanoma cell adhesion to ACM-blocked CSVTCG-albumin coated plastic. Melanoma cells adhere to CSVTCG-albumin coated plastic in the presence of the control
C32
peptide TVSGCC (A); adhesion is inhibited by anti-CD36 monoclonal antibody (B), and by ACM-blocked CSVTCG(NH ) (200uM) (c). No inhibition of adhesion is seen in the presence of heparin 1 OOug Pml (D), or RGDS (1 mM) (E), or antibody to beta-3 integrin complexes(F). CSVTCG peptide was crosslinked to BSA as previously described in a molar ratio of 25:l. 1 OOug/ml BSA in PBS was used to coat plastic as previously described by Roberts et al (Roberts et al.1 987b). Cells were allowed to adhere (100,000 Following incubation the cells/disc) for two hours at 37 degrees C in serum free MEM. discs were washed in MEM and fixed with I % glutaraldehyde. Photomicroscopy was performed using a Nikon diaphot inverted phase microscope. C32 melanoma cells were obtained from ATCC and maintained in MEM containing 10% FCS and supplemented with 4 grams/liter glucose and penicillin/streptomycin.
biologic functions (14). This concept was previously suggested by Born-stein et al (13). Recent data that cytoadherence to purified CD36 does not require exogenous thrombospondin (50) supports the possibility that TRAP--or some other parasite-induced protein (51).-interacts with CD36. The role of the same sequence in circumsporozoite surface proteins from several Plasmodium species is not clear, but our data and the recent report by Rich et al (24) suggests the possiblity that adhesive interactions of sporozoites might also be mediated by this domain with CD36. Other domains macromolecule
of thrombospondin via other receptor
clearly play a part in the cell surface systems. Heparin inhibitable binding 1214
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bospondin by fibroblasts, endothelial cells, and CHO cells has been reported previously (18); and RGD-dependentthrombospondin adhesion is mediated by an integrin receptor in some cell types (19). In many cell types, multiple cell receptors for thrombospondin coexist (5253) making interpretation of antibody inhibition studies difficult. In platelets, the role of CD36 as a thrombospondin binding site has been controversial, but the weight of evidence supports this finding. Several groups have presented data that support a role for platelet CD36 as a thrombospondin binding site (26-29). In contrast, Aiken et al recently reported that thrombospondin surface expressionthat was detected indirectly using Mab TSP-1 was not inhibited by anti-CD36 antibody (35). The latter finding raisesinteresting questions concerning the domain specificity of Mab TSP-1 and its effect on overall thrombospondin binding to platelets. More recently, additional confirmation of the role of platelet CD36 as a TSP binding site has been provided by Legrand et al, who showed inhibition of TSP surface expression by an anti-CD36 Mab (30). Our own data suggest that the domain CSVTCG is responsiblefor surface expressed TSP in activated platelets, and is consistent with data demonstrating inhibition of TSP surface expression by anti-CD36 antibodies. Alternative mechanismsfor TSP surface expression on activated platelets exist, and reports that patients with the Nak- platelet phenotype (who show altered reactivity with anti-CD36antibodies) still bind TSP confirm the complexity of the TSP-platelet interaction (54). The specific CD36-mediatedthrombospondin-and CSVTCG-binding that we observed are at odds with that reported by Oquendo et al (34) with CD36transfected COS cells. Several possible explanations may be relevant to the differences in observed CD36 function in these cell types. Jurkat cells were chosen for these studies becausethey lack CD36, do not express sulfated glycolipids (41), have a low background thrombospondin binding prior to transfection, grow unattached, and are human in origin. COS cells, by contrast, although they express no CD36, display a great deal of native thrombospondin binding that may obscure a relatively smallcomponent of binding that is secondary to recombinant protein expression. Further, since COS cells grow as monolayers,the measurementof fluid phase binding may be affected by matrix components or damaged and dead cells that are removed from the culture dish. Similar concerns apply if the studies are performed on cell monolayers. Finally, and perhaps most importantly, the cell in which the recombinant protein is expressed may be relevent to its function. The conformation or function of recombinantly expressed CD36 may be affected by the lipid composition as has been recently reported for some integrins (55) or by differences in glycosylation (56).\ Since no calcium was added in the binding studies, since the binding was performed in Tris buffered saline, and since EDTA had no effect on the binding of the peptide, these data are not influenced by the recently described calcium-phosphatemediated thrombospondin precipitation (57). These findings are consistent with the role of the calcium binding repeats in the observed calcium dependence of thrombospondin binding and suggest that the calcium dependence of thrombospondin-CD36interaction is a function of the conformational state of the ligand rather than that of CD36. In addition to its presence in thrombospondin, Malarial CS-proteins and in TRAP, the sequence CSVTCG appears in several other proteins that are of great biologic relevance. Properdin contains three such homologous repeat sequences (58), and shareswith thrombospondin the ability to bind to sulfated glycoconjugate proteins (23) raising the possibility that the same sequence might mediate interaction of these proteins with sulfated glycoconjugates or that properdin might share the ability to bind to CD36. Recent data by Prater et al (25) suggeststhat cell attachment to this sequence by cells lacking CD36, may be mediated
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by sulfated glycolipids. In contrast to the interaction of CSVTCG with 036, the interaction of this sequence with sulfated glycolipids is inhibited by reduction and alkylation (23) suggesting that specificity with respect to one or another binding sites depends on secondary structure considerations. In contrast to the findings of Holt et al (23) Prater’s group found no effect of alkylation of this sequence on cell attachment. The same conserved sequence is also found in antistasin, a salivary protein from the Mexican leech Haementeria officinalis that also binds sulfated glycoconjugates, and is an inhibitor of metastasis and coagulation (59). Our recent observations that C32 melanoma cells selected for high levels of CD36 expression exhibit a greater metastatic potential in nude mice than do cells that express low density CD36 (60) are consistent with the thrombospondin mediated enhancement of metastasis described in other tumor cell lines (61) and raise the possibility that this sequence might exert antimetastatic effects in vivo by blocking CD36-dependent thrombospondin adhesion and metastasis. Acknowledgments We wish to thank B. Ferris and S. Carson for expert technical help. CD36lCDM8 and CDB/CDMB were kind gifts from Dr. B. Seed (Harvard Medical School, Boston). Synthetic EGF domain(AA630-674) was a kind gift from Dr.J.Tam (Rockefeller University, NY). Jurkat cells were a kind gift from Dr. D. Posnett (Cornell University Medical College, NY). Whole circumsporozoite lysate was a kind gift from Dr. J. Barnwell (New York University, NY). This work was supported by grants from the NIH (AA and RN). AA is the recipient of a Stratton Foundation Award and the Mellon Teacher Scientist Award. References 1. Leung, L.L. (1984) J. Clin. lnvest.74, 1764-1772. 2. Bacon Baguley, T., Kudryk, B.J., and Walz, D.A. (1987) J. Biol. Chem.262, 1927-1930. 3. Bacon Baguley, T., Ogilvie, M.L., Gartner, T.K., and Walz, D.A. (1990) J. Biol. Chem.265, 2317-2323. 4. Bale, M.D. and Mosher, D.F. (1986) J. Biol. Chem.261,862-868. 5. Murphy Ullrich, J.E. and Mosher, D.F. (1985) Blood66, 1098-I 104. 6. Bale, M.D., Westrick, L.G., and Mosher, D.F. (1985) J. Biol. Chem.260, 7502-7508. 7. Silverstein, R.L., Leung, L.L., Harpel, P.C., and Nachman, R.L. (1984) J. Clin. lnvest.74, 1625-l 633. . 8. Majack, R.A., Cook, S.C., and Bornstein, P. (1986) Proc. Natl. Acad. Sci. USA83, 90509054. 9. Maiack. R.A.. Goodman, L.V.. and Dixit. V.M. (1988) J. Cell Biol.106. 415-422. 10. Tu’szynski, G.P., Rothman, V., Murphy,’ A., Siegler,‘K., Smith, L., Smith, S., Karczewski, J., and Knudsen, K.A. (1987) Science236, 1570-l 573. Il. Roberts, D.D., Sherwood, J.A., and Ginsburg, V. (1987) J. Cell Biol.104, 131-l 39. 12. Roberts, D.D., Sherwood, J.A., Spitalnik, S.L., Panton, L.J., Howard, R.J., Dixit, V.M., Frazier, W.A., Miller, L.H., and Ginsburg, V. (1985) Nature31 8, 64-66. 13. Kobayashi, S., Eden McCutchan, F., Framson, P., and Bornstein, P. (1986) Biochemistry.25, 8418-8425. 14. Robson, K.J., Hall, J.R., Jennings, M.W., Harris, T.J., Marsh, K., Newbold, C.I., Tate, V.E., and Weatherall, D.J. (1988) Nature335 79-82. 15. Gartner, T.K., Walz, D.A., Aiken, M., Starr Spires, L., and Ogilvie, M.L. (1984) Biochem. Biophys. Res. Commun.124, 290295. 16. Dixit, V.M., Haverstick, D.M., ORourke, K.M., Hennessy, S.W., Grant, G.A., Santoro, S.A., and Frazier, W.A. (1985) Biochemistry.24, 4270-4275. 17. Murphy Ullrich, J.E., Westrick, L.G., Esko, J.D., and Mosher, D.F. (1988) J. Biol. Chem.263, 64006406. 18. McKeown Longo, P.J., Hanning, R., and Mosher, D.F. (1984) J. Cell Biol.98, 22-28. 19. Lawler, J., Weinstein, R., and Hynes, R.O. (1988) J. Cell Biol.107, 1)-P 2351-6. 20. Lam, S.C., Plow, E.F., DSouza, S.E., Cheresh, D.A., Frelinger, A.L 3d., and Ginsberg, M.H. (1989) J. Biol. Chem.264, 3742-3749. 21. Lawler, J. and Hynes, R.O. (1989) Blood74,2022-2027. 1216
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