title: Exposure of the cryptic Arg-Gly-Asp sequence in thrombospondin-1 by protein disulfide isomerase.
abstract: Thrombospondin-1 is a matrix protein that inhibits proliferation, motility and sprouting of endothelial cells in vitro and angiogenesis in vivo. One mechanism by which thrombospondin-1 may influence endothelial cell biology is through interaction with the endothelial cell alphav beta3 integrin receptor. This interaction is mediated via a cryptic Arg-Gly-Asp sequence in the C-terminal Ca2+-binding region of thrombospondin-1. Exposure of the Arg-Gly-Asp sequence is controlled by disulfide interchange events in the Ca2+-binding loops and C-globular domain. Limited reduction of thrombospondin-1 by dithiothreitol exposes the Arg-Gly-Asp sequence which can bind to the alphav beta3 integrin receptor and support endothelial cell spreading (X. Sun, K. Skorstengaard, D.F. Mosher, J. Cell Biol. 118 (1992) 693-701). Our aim was to identify possible physiological reductants that can mediate Arg-Gly-Asp exposure. We now report that protein disulfide isomerase, which is known to catalyze disulfide interchange in thrombospondin-1 and change its enzyme inhibitory properties and its binding to monoclonal antibodies, was secreted by bovine aortic endothelial cells and deposited on the cell surface. There was an average of approximately 2.2 fg of protein disulfide isomerase on the surface of a bovine aortic endothelial cell. Treatment of thrombospondin-1 with purified protein disulfide isomerase enhanced adhesion of endothelial cells to thrombospondin-1 in an Arg-Gly-Asp-dependent manner through the alphav beta3 integrin receptor and supported cell spreading. Both Ca2+-depleted and Ca2+-replete thrombospondin-1 were substrates for protein disulfide isomerase. These results suggest that endothelial cell derived protein disulfide isomerase may regulate Arg-Gly-Asp-dependent binding of thrombospondin-1.
authors.affiliation: Centre for Thrombosis and Vascular Research, School of Pathology, University of New South Wales, Sydney, NSW 2052, Australia.
authors.forename: K A
authors.initials: KA
authors.lastname: Hotchkiss
authors.forename: L J
authors.initials: LJ
authors.lastname: Matthias
authors.forename: P J
authors.initials: PJ
authors.lastname: Hogg
mesh_terms: D000818:Animals; D001011:Aorta / Q000201:enzymology; D001792:Blood Platelets / Q000378:metabolism; D002417:Cattle; D002448:Cell Adhesion / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008858:Microscopy, Phase-Contrast; D009842:Oligopeptides / Q000737:chemistry*; D019704:Protein Disulfide-Isomerases / Q000032:analysis*; D019700:Thrombospondin 1 / Q000737:chemistry*
chemical_list: D004220:Disulfides; D009842:Oligopeptides; D019700:Thrombospondin 1; C047981:arginyl-glycyl-aspartic acid; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D004229:Dithiothreitol