alpha-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 '25I-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°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 a-fucose residues, but not a-galactose, high mannose, or complex oligosaccharide from '25I-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 a-fucose homologous to that of t-PA. These data suggest that EGF-associated 0-linked a-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.)

Regulation of Tissue Plasminogen Activator Activity by Cells

“…In competition experiments including monosaccharides up to 20 mM or 10 mg/ml ovalbumin in kinetic assays, normal patterns of stimulation were observed with no detectable inhibition over a range of cell densities from 0 to 3 ϫ 10 7 cells/ml. These levels of monosaccharides have previously been shown to inhibit binding of tPA to glycosylation sitespecific cell surface receptors (27). Our results indicate that lack of glycosylation has an indirect effect on stimulation of tPA activity by cells.…”

Macrophage foam cell lipoprotein(a)/apoprotein(a) receptor. Cell-surface localization, dependence of induction on new protein synthesis, and ligand specificity.

“…Lp(a) is also found in rhesus monkeys, although the apo(a) moiety of this Lp(a) differs in several respects from human apo(a), including an inactive lysine-binding domain at K4 37 and a modification of the RGD peptide sequence at K4 35 of human apo(a). 21 - 32 The data in Fig 3 show that foam cells degraded rhesus monkey Lp(a) to the same degree as human (fast isoform) Lp(a).…”

“…21 - 32 The data in Fig 3 show that foam cells degraded rhesus monkey Lp(a) to the same degree as human (fast isoform) Lp(a). Thus, neither an active K4 37 lysine-binding domain nor an intact K4 35 RGD sequence appears to be necessary for interaction with the foam cell Lp(a) receptor activity.…”

“…In an additional experiment to test the possible role of other sugar moieties known to be present on apo(a), 3234 we followed the strategy of Hajjar and Reynolds. 35 These investigators demonstrated that a 250-fold molar excess of D-fucose or D-galactose inhibited the binding of Last, we sought to determine whether the interaction of the foam cell receptor activity with apo(a), which has multiple disulfide bonds, ylated, and this ligand was compared with native l25 I-rapo(a) for uptake and degradation by control and foam cell macrophages. The data in Fig 6 demonstrate two points: first, whereas the degradation of native 125 I-rapo(a) was increased by =450% in foam cells, the degradation of reduced and denatured 125 I-r-apo(a) was increased by only 28% in foam cells; second, the total amount of reduced and denatured 125 I-r-apo(a) degraded by the foam cells was approximately half that of native 125 I-r-apo(a).…”

IL-13, Asthma and Glycosylation in Airway Epithelial Repair

“…Recent studies have suggested that annexin II (AII) is also capable of binding carbohydrates however this work is still in preliminary stages. Previously, the binding of tissue-type plasminogen activator (t-PA) was shown to bind HepG2 cells, however, following enzymatic removal of -fucose residues on t-PA, this binding was dramatically decreased [100]. Subsequently, AII has been shown to be a cell surface receptor for t-PA [101].…”