Characterization of the interaction between urinary urokinase and the asialoglycoprotein receptor of liver cells

Urokinase-type plasminogen activator (u-PA) is rapidly cleared from the circulation with a half-life of a few minutes. We have previously shown that non-glycosylated recombinant u-PA is recognized by LRP, whereas urinary u-PA is recognized by the asialoglycoprotein receptor (ASGPr) on liver parenchymal cells. In the present study we biochemically characterized the latter interaction. The ASGPr was isolated and purified from rat liver tissue and a binding assay was developed. In this assay urinary uPA specifically bound to the receptor with apparent Kd ≈ 30 nM. Specific antibodies against the receptor completely blocked the binding of u-PA. In line with the known carbohydrate specificity of the ASGPr, D-GalNAc proved to be the most effective inhibitor of u-PA binding from a series of monosaccharides, followed by D-Gal and L-Fuc, whereas D-GlcNAc was ineffective. Recent literature showed that the N-linked oligosaccharides of urinary u-PA do not terminate with the common Gal-GlcNAc element but with a GalNAc-GlcNAc element, which is partially sulfated (Eur. J. Biochem. 1995, 228:1009). Sulfated forms of u-PA were separated from non-sulfated forms by using the lectin Wisteria floribunda agglutinin. Only the non-sulfated forms of u-PA (30 % of the total) appeared to bind to the ASGPr. We conclude that a fraction of urinary u-PA bears oligosaccharides which are specifically recognized by the asialoglycoprotein receptor on liver cells