Structural elucidation of an -1,2-mannosidase resistant oligosaccharide produced in Pichia pastoris

• Published in: Glycobiology (Oxford) Vol. 21; no. 12; pp. 1606 - 1615
• Main Authors: Gomathinayagam, Sujatha, Mitchell, Teresa, Zartler, Edward R, Heiss, Christian, Azadi, Parastoo, Zha, Dongxing, Houston-Cummings, Nga Rewa, Jiang, Youwei, Li, Fang, Giaccone, Erin, Porambo, Richard J, Anderson, Carrie L, Sethuraman, Natarajan, Li, Huijuan, Stadheim, Terrance A
• Format: Journal Article
• Language: English
• Published: Oxford University Press 01.12.2011
• Subjects: mannosyltransferase; linked mannose; NMR spectroscopy; N-glycosylation
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwr082

The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P. pastoris strains may contain residual fungal-type high-mannose structures. In a pool of N-linked glycans enzymatically released by protein N-glycosidase from a reporter glycoprotein expressed in a developmental glycoengineered P. pastoris strain, an oligosaccharide with a mass consistent with a Hexose9GlcNAc2 oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man9GlcNAc2 standard. However, this Hexose9GlcNAc2 oligosaccharide was found to be resistant to -1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal -linked glucose. To further characterize this oligosaccharide, we purified the Hexose9GlcNAc2 oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) 1H NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D 1H and 13C NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide -Man-(1 → 2)- -Man-(1 → 2)- -Man-(1 → 2)- -Man-(1 → 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple -linkages on the -1,3 arm of the Man9GlcNAc2 oligosaccharide.