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.
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
M F
MF
Verostek
P H
PH
Atkinson
R B
RB
Trimble
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