Identification of active peptide sequences in the carboxyl-terminal cell binding domain of human thrombospondin-1.
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.
Kosfeld
M D
MD
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Frazier
W A
WA
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D006801:Humans; D037102:Lectins / Q000378:metabolism; D046911:Macromolecular Substances; D008545:Melanoma; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D019699:Thrombospondins
D000911:Antibodies, Monoclonal; D037102:Lectins; D046911:Macromolecular Substances; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; D019699:Thrombospondins