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

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 Man_8-11GlcNAc. Nearly 75% of the oligosaccharides were equally distributed between Man_8,9GlcNAc, 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 (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 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 Man_9,10GlcNAc 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.