PERLECAN IS RESPONSIBLE FOR THROMBOSPONDIN-1 BINDING ON THE CELL-SURFACE OF CULTURED PORCINE ENDOTHELIAL-CELLS

Thrombospondin 1 (TSP1), a high molecular weight glycoprotein of the e xtracellular matrix, interacts with glucosaminoglycan at the cell surf ace of porcine endothelial cells (Schon et al, Eur. J, Cell Biol, 59, 329-339 (1992)), Ln this stud! we identified and characterized the hep aran sulfate proteoglycan (HSPG) responsible for TSP1 binding and upta ke in endothelial cells and investigated some properties of the TSP1-p roteoglycan interaction, Porcine endothelial cells synthesize proteogl ycans containing heparan sulfate (HS) or chondroitin/dermatan sulfate (CS/DS), CS/DS-containing compounds are present predominantly in the c ulture medium, On Sepharose CL-4B the cellular proteoglycan fraction y ielded two HS-containing compounds with a K-av=0.18 and K-av=0.55. Onl y the larger HS-containing component was sensitive to alkaline treatme nt and,vas also found in the medium fraction, Trypsin treatment of end othelial cells revealed that the large HS-containing component represe nts a cell surface-associated proteoglycan, whereas the smaller fracti on represents a pool of intracellular HS-chains, The cellular HSPG is partially localized at the spiral cell surface but also incorporated a nd tightly bound to the subendothelial matrix. Deglycosylation of the high molecular weight HSPG resulted in the identification of a core pr otein of about 400 kDa. Using specific antibodies, in ELISA assays and in immunoblot analysis we observed that the large HSPG is identical t o the extracellular matrix proteoglycan, perlecan, Immunohistochemical studies confirmed the location of perlecan on tile epical cell surfac e and additionally as a dense fibrillar network surrounding tile cells , Purified perlecan bound to TSP1 in a dose-dependent manner and the b inding was mediated by its glycosaminoglycan side chains, In competiti on assays using various sulfated polysaccharides, heparin potently inh ibited binding of perlecan to TSP1 immobilized on nitro cellulose, Der matan sulfate was a less effective inhibitor, Calcium bound to TSP1, w as found to influence its capacity for binding perlecan, The present d ata provide evidence that perlecan is required for binding and concent rating TSP1 at the spiral surface of vascular endothelial cells during receptor-mediated endocytosis.