Abstract
Thrombospondin (TS) mediates attachment, spreading, and motility of several cell types through at least four cell binding domains: the amino-terminal heparin binding domain, the type I repeats containing the CSVTCG sequence, the RGDA sequence in the last of the type III calcium binding repeats and the carboxyl-terminal cell or platelet binding domain (CBD). The attachment of human melanoma cells (G361) to the COOH-terminal domain is independent of the RGDA sequence and is inhibited by the monoclonal antibody C6.7. To define the cell binding site(s) within this 212-residue COOH-terminal domain, we have synthesized eight overlapping peptides (seven 30-mers and a final 37-mer) representing the entire sequence of the CBD. Several of these peptides are insoluble in aqueous buffers at high concentration. Cell adhesion assays have been devised which employ covalent coupling of peptides in chaotropic solvents to chemically derivatized plastic 96-well plates. Three synthetic peptides, two of which are nonadjacent in the linear sequence, are potent attachment factors for G361 cells. C6.7 blocks adhesion to one of these peptides, whereas sulfated glycoconjugates inhibit adhesion of cells to all three. Polyclonal antibodies raised against the peptides inhibit cell adhesion to the peptides, the recombinant CBD, and to intact TS. The peptides GRGDSP and VTCG are not inhibitory. These sites are thus independent from the type I repeats and the RGDA sequence of TS. Each of the active peptides inhibits cell attachment to the other active peptides as well as to the CBD and to intact TS. This mutual inhibition suggests that the peptides share a common cellular receptor which may contain an associated glycoconjugate chain. These data indicate that the COOH-terminal cell binding domain of TS contains at least two peptide sequences which contribute to the attachment of a wide variety of cells.
Highlights
Four cell binding sites within the modular TS structure have been identified as shown in Fig. 1: 1)the globular NH,terminal heparin binding domain of TS (Dixit et al, 1984), which is responsible for chemotaxis and spreading of certain human melanomas on TS (Taraboletti, 1987);the heparin binding domain binds to heparin, heparan sulfate proteoglycans (Sun et al, 1989), fucoidin, and sulfaattachment to the other active peptides as well as to tides (Roberts et al, 1985); 2) the CSVTCG sequence in the the cell or platelet binding domain (CBD) and to intact TS
Synthetic peptides were solubilized with 6 N guanidine HCl because five of the eight peptides were insoluble at high concentration in Trisbuffered saline (TBS)
Using the same chemistry that is employed in making protein conjugates of synthetic peptides, each peptide in guanidine HC1 solution was coupled to COOH-derivatized plates with a water-soluble carbodiimide (l-ethyl-343-dimethylaminopropy1)carbodiimide) or through its NH,terminal cysteine residue to amino-derivatized plates using the succinimidyl 3-(2-pyridy1dithio)propionate (SPDP) reagent
Summary
The generation and characterization ofmAbC6.7 have been described and itsepitope localized to theCBD (Dixit et al, 1985; Kosfeld et al, 1991). G361 cells were obtained from the ATCC (1424) and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum at 5% Con(Roberts et al, 1987). All reagents were obtained from Sigma unless specified otherwise. Was from Pharmacia LKB Biotechnology Inc. PeptidesGRGDSP and GRGESP were from Immuno-Dynamics, Inc. Human platelet T S was purified as described (Dixit etal., 1984).VTCG was synthesized and purified by the Washington University Protein Chemistry Facility
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