HEPARIN-BINDING DOMAIN, TYPE-1 AND TYPE-2 REPEATS OF THROMBOSPONDIN MEDIATE ITS INTERACTION WITH HUMAN BREAST-CANCER CELLS

Thrombospondin is an adhesive glycoprotein that promotes breast cancer cell adhesion to human vascular endothelial cells (Incardona et al., 1995). In this study, we have identified the molecular domains of thro mbospondin that mediate its binding to specific receptors on the human breast adenocarcinoma cell line, MDA-MB-231. Two recombinant fragment s from the amino-terminus (TSPN18 and TSPN28), and the fusion proteins of the type 1 and type 2 repeats of human thrombospondin, inhibited b inding of radiolabeled thrombospondin to MDA-MB-231 cells in suspensio n by 40-60% at 50 mu g/ml whereas the type 3 repeat, carboxy-terminus and unfused glutathione-S-transferase as well as the synthetic peptide Gly-Arg-Gly-Asp-Ser (500 mu g/ml) had little or no effect. Heparin an d various glycosaminoglycans as heparan sulfate, chondroitin sulfates A, B or C, and fucoidan inhibited thrombospondin binding to MDA-MB-231 cells by more than 60% whereas dextran sulfate had only little effect . Treatment of cells with heparitinase, chondroitinase ABC, and hyalur onidase, but not with neuraminidase, induced 30-50% inhibition of thro mbospondin binding suggesting the participation of both heparan sulfat e and chondroitin sulfate cell surface-associated molecules. Inhibitio n of proteoglycan sulfation by chlorate or inhibition of glycosaminogl ycan chain formation by two beta-D-xylosides also led to a substantial inhibition of thrombospondin binding. Our results indicate that sever al domains within the thrombospondin molecule, namely the amino-termin us, type 1 and type 2 repeats, participate in its binding to specific receptors bearing sulfated glycosaminoglycans on MDA-MB-231 cells. Bio logical assays have indicated that, in addition to these domains, the peptide Cly-Arg-Gly-Asp-Ser inhibited MDA-MB-231 cell attachment to th rombospondin suggesting that the last type 3 repeat of the molecule ma y also contribute to its cell adhesive activity. (C) 1996 Wiley-Liss, Inc.