Alpha-Fucose-mediated Binding and Degradation of Tissue-type Plasminogen Activator by HepG2 Cells

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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 Iodine-125-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 (Kd 39 nM; maximum binding capacity 493,000 sites per cell). Bound t-PA was rapidly degraded at 37 degrees 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 Iodine-125-t-PA to HepG2 cells was inhibited by monosaccharides fucose and galactose and by the neoglycoprotein fucosyl-albumin. Enzymatic removal of alpha-fucose residues, but not alpha-galactose, high mannose, or complex oligosaccharide from Iodine-125-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 alpha-fucose homologous to that of t-PA. These data suggest that EGF-associated O-linked alpha-fucose may mediate t-PA binding and degradation by HepG2 cells. This mechanism may be relevant to other proteins with analogous structures. (J. Clin. Invest. 1994. 93:703-710.) Key words: tissue plasminogen activator. thrombolysis. hepatic clearance. alpha-fucose. epidermal growth factor domain

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