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 extracellular matrix, interacts with glycosaminoglycan at the cell surface of porcine endothelial cells (Schön et al., Eur.J. Cell Biol. 59, 329-339 (1992)). In this study we identified and characterized the heparan sulfate proteoglycan (HSPG) responsible for TSP1 binding and uptake in endothelial cells and investigated some properties of the TSP1-proteoglycan interaction. Porcine endothelial cells synthesize proteoglycans containing heparan sulfate (HS) or chondroitin/dermatan sulfate (CS/DS). CS/DS-containing compounds are present predominantly in the culture medium. On Sepharose CL-4B the cellular proteoglycan fraction yielded two HS-containing compounds with a Kav = 0.18 and Kav = 0.55. Only the larger HS-containing component was sensitive to alkaline treatment and was also found in the medium fraction. Trypsin treatment of endothelial cells revealed that the large HS-containing component represents a cell surface-associated proteoglycan, whereas the smaller fraction represents a pool of intracellular HS-chains. The cellular HSPG is partially localized at the apical cell surface but also incorporated and tightly bound to the subendothelial matrix. Deglycosylation of the high molecular weight HSPG resulted in the identification of a core protein of about 400 kDa. Using specific antibodies, in ELISA assays and in immunoblot analysis we observed that the large HSPG is identical to the extracellular matrix proteoglycan, perlecan. Immunohistochemical studies confirmed the location of perlecan on the apical cell surface and additionally as a dense fibrillar network surrounding the cells. Purified perlecan bound to TSP1 in a dose-dependent manner and the binding was mediated by its glycosaminoglycan side chains. In competition assays using various sulfated polysaccharides, heparin potently inhibited binding of perlecan to TSP1 immobilized on nitrocellulose. Dermatan sulfate was a less effective inhibitor. Calcium bound to TSP1 was found to influence its capacity for binding perlecan. The present data provide evidence that perlecan is required for binding and concentrating TSP1 at the apical surface of vascular endothelial cells during receptor-mediated endocytosis.
Vischer
P
P
Institut für Arterioskleroseforschung an der Universität, Münster/Germany.
Feitsma
K
K
Schön
P
P
Völker
W
W
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D002808:Chondroitin Lyases; D003871:Dermatan Sulfate / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D005109:Extracellular Matrix / Q000737:chemistry; D006025:Glycosaminoglycans / Q000032:analysis / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008970:Molecular Weight; D011485:Protein Binding; D011509:Proteoglycans / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine; D019699:Thrombospondins
D002118:Calcium; D015815:Cell Adhesion Molecules; D002808:Chondroitin Lyases; D003871:Dermatan Sulfate; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin; D006497:Heparan Sulfate; D008562:Membrane Glycoproteins; D011509:Proteoglycans; D019699:Thrombospondins; C071927:perlecan