title: Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris.
abstract: Saccharomyces SUC2 invertase, secreted by the methylotrophic yeast Pichia pastoris and purified to homogeneity from the growth medium by DE-52 chromatography, appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a diffuse ladder of species at 85-90 kDa, while the secreted Saccharomyces form migrated as a broad band from 100 to 150 kDa. Endo-beta-N-acetylglucosaminidase H released the Pichia invertase carbohydrate generating a 60-kDa protein with residual Asn-linked GlcNAcs and oligosaccharides separated on Bio-Gel P-4 into Man8-11GlcNAc. Nearly 75% of the oligosaccharides were equally distributed between Man8,9GlcNAc, while 17% were Man10GlcNAc and 8% were Man11GlcNAc. Oligosaccharide pools were analyzed for homogeneity by high-pH anion-exchange chromatography, and structures were assigned using 500 MHz one- and two-dimensional 1H NMR spectroscopy. Pichia Man8GlcNAc was the same isomer as found in Saccharomyces, which arises by removing the alpha 1,2-linked terminal mannose from the middle arm of the lipid-oligosaccharide Man9GlcNAc (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). The Man9GlcNAc pool was 5% lipid-oligosaccharide precursor and 95% Man8GlcNAc isomer with a terminal alpha 1,6-linked mannose on the lower-arm alpha 1,3-core-linked residue (Hernández, L. M., Ballou, L., Alvarado, E., Gillece-Castro, B. L., Burlingame, A. L., and Ballou, C. E. (1989) J. Biol. Chem. 264, 11849-11856). An alpha 1,2-linked mannose on the new alpha 1,6-linked branch in Man9GlcNAc provided 80% of the Man10GlcNAc, which is the structure on Saccharomyces invertase (Trimble, R. B., and Atkinson, P. H. (1986) J. Biol. Chem. 261, 9815-9824). A minor Man10GlcNAc (12%) and the principal Man11GlcNAc (82%) were the major Man9,10GlcNAc with novel alpha 1,2-linked mannoses on the preexisting alpha 1,2-linked termini. Although Pichia glycans did not have terminal alpha 1,3-linked mannoses as found on Saccharomyces core oligosaccharides, over 60% of the structures were isometric configurations unique to lower eukaryotes.
authors.affiliation: New York State Department of Health, Wadsworth Center for Laboratories & Research, Albany, New York 12201-0509.
authors.forename: R B
authors.initials: RB
authors.lastname: Trimble
authors.forename: P H
authors.initials: PH
authors.lastname: Atkinson
authors.forename: J F
authors.initials: JF
authors.lastname: Tschopp
authors.forename: R R
authors.initials: RR
authors.lastname: Townsend
authors.forename: F
authors.initials: F
authors.lastname: Maley
mesh_terms: D002240:Carbohydrate Sequence; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010843:Pichia / Q000201:enzymology / Q000378:metabolism*; D012440:Saccharomyces / Q000201:enzymology*; D014169:Transformation, Bacterial; D043324:beta-Fructofuranosidase
chemical_list: D009844:Oligosaccharides; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase