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

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 Hexose(9)GlcNAc(2) oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man(9)GlcNAc(2) standard. However, this Hexose(9)GlcNAc(2) oligosaccharide was found to be resistant to alpha-1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal alpha-linked glucose. To further characterize this oligosaccharide, we purified the Hexose(9)GlcNAc(2) oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) H-1 NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D H-1 and C-13 NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide alpha-Man-(1 -> 2)-beta-Man-(1 -> 2)-beta-Man-(1 -> 2)-alpha-Man-(1 -> 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple beta-linkages on the alpha-1,3 arm of the Man(9)GlcNAc(2) oligosaccharide.