Asparagine-linked glycoprotein biosynthesis in rat brain: Identification of glucosidase I, glucosidase II, and an endomannosidase (glucosyl mannosidase)

Abstract

Previous studies from this laboratory provided evidence, largely based upon the presence of a novel α- d-mannosidase, suggesting that the biosynthesis of N-linked glycoproteins may be different in brain as compared to other tissues (Tulsiani, D. R. P., and Touster, O. (1985) J. Biol. Chem. 260, 13,081–13,087). In the present report we describe studies on the enzymes involved in early processing reactions. These studies indicate that the brain, like other tissues, contains glucosidases I and II. The two glucosidases were separated as distinct activities with some overlapping by chromatography on a DE-52 column. The differential inhibition studies and substrate specificity studies support our conclusion that, as in other tissues, rat brain glucosidase I cleaves α1,2-linked terminal glucosyl residues, whereas glucosidase II prefers α1,3-linked glucosyl residues. In addition to these two processing glucosidases, we have characterized an endo enzyme (glucosyl mannosidase) in rat brain. The endomannosidase cleaves a disaccharide (glucosyl α1,3-mannose) from monoglucosylated oligosaccharides (GlcMan 7–9GlcNAc). Little or no activity was observed when di- or triglucosylated oligosaccharide was used as a substrate. The pH optimum of the glucosyl mannosidase is 6.2–6.8. The enzyme appears to be an intrinsic microsomal membrane component, since washing of the microsomal membranes with salt solution did not release the enzyme in soluble form. A mixture of Triton X-100 and sodium deoxycholate is required for complete solubilization of the enzyme. The solubilized enzyme is eluted from a Bio-Gel A-1.5m column as a single peak with an apparent molecular weight of 380,000.