Solubilization of mannosyltransferase activities for the biosynthesis of mammary glycoproteins

Abstract

The microsomal preparation from the lactating bovine mammary tissue was solubilized by treatment with nonionic detergent, NP-40, at a protein/detergent ratio of 1.5:1 and a detergent concentration of 0.5%. Following centrifugation at 147,000 .times. g for 120 min, the supernatant fration was incubated with labeled sugar nucleotides, GDP-Man [mannose] and UDP-GlcNAc [N-acetylglucosamine]. It synthesized a series of lipid-linked saccharides up to (Man)5-(GlcNAc)2. The solubilized glycosyltransferases retained up to about 60% of the activity after 2 wk storage at 4.degree. C. The biosynthesis of glycolipids was stimulated by a mixture of lipids obtained by extracting the mammary microsomes with CHCl3/CH3OH (2:1). A labeled lipid-linked tetrasaccharide of the structure Man.alpha.1 .fwdarw. 3Man.beta. .fwdarw. GlcNAc.beta. .fwdarw. GlcNAc was isolated by labeling baby hamster kidney cells with [2-3H]mannose under conditions of glucose starvation followed by extraction of the cells with CHCl3/CH3OH (2:1) and separation of the lipids by high-performance liquid chromatography. When this lipid-linked tetrasaccharide was incubated with the solubilized bovine mammary microsomes and GDP-Man, it was elongated to a lipid-linked heptasaccharide having the structure Man.alpha.1 .fwdarw. 2Man.alpha.1 .fwdarw. 2Man.alpha.1 .fwdarw. 3(Man.alpha.1 .fwdarw. 6) Man.beta. .fwdarw. GlcNAc.beta. .fwdarw. GlcNAc. The kinetics of the elongation reaction also revealed the intermediary formation of smaller amounts of lipid-linked pentasaccharide and hexasaccharide. The elongation reaction did not require any divalent metal ion and had a broad pH optimum between 6.8 and 7.6. The lack of inhibition of the elongation reaction by EDTA or amphomycin supports earlier studies that DGP-Man, rather than mannosylphosphoryldolichol, is the direct donor of mannosyl residues for the biosynthesis of glycolipids up to (Man)5(GlcNAc)2. Mannosylphosphorylretinol was ineffective as mannosyl donor for the elongation reaction.