Asparagine-linked glycoprotein biosynthesis in rat brain: identification of glucosidase I, glucosidase II, and and endomannosidase (glucosyl mannosidase).
Previous studies from this laboratory provided evidence, largely based upon the presence of a novel alpha-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 alpha 1,2-linked terminal glucosyl residues, whereas glucosidase II prefers alpha 1,3-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 alpha 1,3-mannose) from monoglucosylated oligosaccharides (GlcMan7-9GlcNAc). 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.
Tulsiani
D R
DR
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235.
Coleman
V D
VD
Touster
O
O
D000818:Animals; D001216:Asparagine; D001921:Brain / Q000201:enzymology*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation; D005947:Glucose / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D065089:Glycoside Hydrolase Inhibitors; D007700:Kinetics; D008297:Male; D008358:Mannose / Q000378:metabolism; D008361:Mannosidases / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000302:isolation & purification / Q000378:metabolism*
D002241:Carbohydrates; D003902:Detergents; D006023:Glycoproteins; D065089:Glycoside Hydrolase Inhibitors; D011092:Polyethylene Glycols; D001216:Asparagine; D017830:Octoxynol; C034251:4-nitrophenyl-alpha-glucosidase; D008361:Mannosidases; C042059:glucosidase I; C063015:glycoprotein endo-alpha-1,3-mannosidase; D000520:alpha-Glucosidases; D005947:Glucose; D008358:Mannose