α-Fucose-mediated binding and degradation of tissue-type plasminogen activator by HepG2 cells

Abstract

The glycoprotein tissue-type plasminogen activator (t-PA) is subject to hepatic clearance in humans. Here, the interaction of t-PA with a well- differentiated hepatoma cell line (HepG2) was examined. Suspended HepG2 cells bound 125I-t-PA in a specific, saturable, and reversible fashion through a Ca2+-dependent, active site-independent mechanism. Binding isotherms indicated a high affinity system with a single class of saturable binding sites (K(d) 39 nM; maximum binding capacity 493,000 sites per cell). Bound t- PA was rapidly degraded at 37°C in a manner inhibited by lysosomotropic agents or metabolic inhibitors. Pretreatment of t-PA with monoclonal antibodies against the EGF/fibronectin finger domain, but not kringle 2 or kringle 1, reduced total binding by 86%. Binding of 125I-t-PA to HepG2 cells was inhibited by monosaccharides fucose and galactose and by the neoglycoprotein fucosyl-albumin. Enzymatic removal of α-fucose residues, but not α-galactose, high mannose, or complex oligosaccharide from 125I-t- PA, reduced specific binding by 60±5%. Binding was also inhibited by high, but not low, molecular weight urokinase, which contains an EGF-based threonine-linked α-fucose homologous to that of t-PA. These data suggest that EGF-associated O-linked α-fucose may mediate t-PA binding and degradation by HepG2 cells. This mechanism may be relevant to other proteins with analogous structures.