Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris

R. B. Trimble, P. H. Atkinson, J. F. Tschopp, R. R. Townsend, F. Maley

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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-β-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 Man8-11 GlcNAc. 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 α1,2-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 α1,6-linked mannose on the lower-arm α1,3-core-linked residue (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). An α1,2-linked mannose on the new α1,6-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 α1,2-linked mannoses on the preexisting α1,2-linked termini. Although Pichia glycans did not have terminal α1,3-linked mannoses as found on Saccharomyces core oligosaccharides, over 60% of the structures were isometric configurations unique to lower eukaryotes.

Original languageEnglish
Pages (from-to)22807-22817
Number of pages11
JournalJournal of Biological Chemistry
Volume266
Issue number34
StatePublished - Dec 1 1991
Externally publishedYes

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