Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant. II. Structure of novel Man6-10GlcNAc2 processing intermediates on secreted invertase.
Alg3 yeast mutants synthesize endoglycosidase H-resistant oligosaccharides whose precursor for elongation is Man1 alpha-->2Man1 alpha-->2Man1 alpha-->3(Man1 alpha-->6)Man1 beta-->4GlcNAc2 (Verostek, M.F., Atkinson, P.H., and Trimble, R. B. (1991) J. Biol. Chem. 266, 5547-5551). To characterize alg3 glycan elongation in vivo, oligosaccharides on alg3,sec18 invertase synthesized and secreted at 26 degrees C were released with peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase and purified by Bio-Gel P-4 chromatography. Large (Man > 30GlcNAc2) and intermediate (Man5-10GlcNAc2) sized oligosaccharides were pooled separately, and the smaller ones were exchanged with 2H2O for one- and two-dimensional DQF-COSY 1H NMR analyses at 500 MHz. Although there was no detectable substitution of the terminal alpha 1,6-core-linked mannose, addition of alpha 1,6-, alpha 1,2-, and alpha 1,3-mannoses to the alpha 1,3-linked core branch of a majority of the Man5 precursor was analogous to core-filling reactions seen on wild type invertase glycans (Trimble, R.B., and Atkinson, P.H. (1986) J. Biol. Chem. 261, 9815-9824). Two additional types of oligosaccharide structures were found; those which retained glucose and those consistent with mannan elongation. Glucose retention appeared to be due to inefficient trimming from minor glucosylated intermediates, while mannan elongation was by extension of a new alpha 1,6-linked branch from the alpha 1,3-core-linked residue as seen in wild-type core oligosaccharides (Hernandez, L.M., Ballou, L., Alvarado, E., Gillece-Castro, B.L., Burlingame, A.L., and Ballou, C. E. (1989) J. Biol. Chem. 264, 11849-11856) or mnn1,mnn2,mnn10 processing intermediates (Ballou, L., Alvarado, E., Tsai, P-k., Dell, A., and Ballou, C.E. (1989) J. Biol. Chem. 264, 11857-11864). Thus, the alpha 1,6-linked branch additions which form Man9GlcNAc2-PP-dolichol from Man5GlcNAc2-PP-dolichol appear to provide important structural information enabling efficient recognition by the endoplasmic reticulum-glucosyltransferases forming oligosaccharide-lipid as well as the glucosidases involved in early trimming reactions, but the alg3 mutant documents that they are unnecessary for normal yeast mannan elongation.
Verostek
M F
MF
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
Atkinson
P H
PH
Trimble
R B
RB
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D043324:beta-Fructofuranosidase
D006023:Glycoproteins; D009844:Oligosaccharides; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase