The Carbohydrate Moiety of Human Urinary Kallikrein

抄録

Highly purified human urinary kallikrein (HUK) [EC 3.4.21.35] was subjected to hydrazinolysis in order to release the carbohydrate moieties. The released oligosaccharides were N-acetylated with acetic anhydride and reduced with sodium borohydride (NaB³H₄). The radioactive oligosaccharides were applied to a column of Hitachi Custom Resin #2630. One neutral (N) and five acidic (AI-AV) peaks were observed, indicating that the HUK was heterogeneous as regards the charge of the oligosaccharide moieties. Sialic acid residues of tritium-labelled AI and AII oligosaccharides were separately removed by neuraminidase treatment, and the asialo oligosaccharides of AI and AII thus obtained were separated by successive chromatographies on columns of Bio-Gel P-4, concanavalin A-Sepharose 4B and Ricinus communis agglutinin-Sepharose 4B, and their structures were investigated by a combination of endo- and exo-glycosidase digestions. Tritium labelled asialo-oligosaccharides AI and AII were each applied to a column of Bio-Gel P-4, and several peaks (AI-1, AI-2 and AI-3 from AI, and AII-1, AII-2 and AII-3 from AII) were obtained. The main asialo-oligosaccharides, AI-2 and AII-2, were each digested with a mixture of β-galactosidase and β-N-acetylhexosaminidase, and yielded small oligosaccharides having the structures aMan₂-βMan-βGlcNAc-GlcNAc and aMan₂-βMan-βGlcNAc-(Fuc-)-GlcNac, respectively. These results indicate that a major portion of the HUK oligosaccharide has a common core structure, and that heterogeneity arises from 1) variation of the charge of the oligosaccharides, 2) variation of the molecular weights of the oligosaccharides.