Characterization of glycosylated variants of β-lactoglobulin expressed in Pichia pastoris

Glycosylated variants of β-lactoglobulin (BLG) were produced in the methylotrophic yeast Pichia pastoris to mimic the glycosylation pattern of glycodelin, a homologue of BLG found in humans. Glycodelin has three sites for glycosylation, corresponding to amino acids 63-65 (S1), 85-87 (S2) and 28-30 (S3) of BLG. These three sites were engineered into BLG to produce the variants S2, S12 and S123, which carried one, two and three glycosylation sites, respectively. The oligosaccharides on these BLG variants ranged from (mannose)₉(N-acetylglucosamine)₂ (Man₉GN₂) to Man₁₅GN₂ and were of the α-linked high mannose type. The variant S123 exhibited highest levels of glycosylation, with the range of glycans being Mang_9-14GN₂. Digestion of S123 with α-1,2 linkage specific mannosidase resulted in a single product corresponding to Man₆GN₂. These results indicated a glycosylation pattern consisting of a Man₅GN₂ structure extended by 4-9 mannose residues attached mainly by α-1,2 linkages. The results also indicated extension of the Man₅GN₂ structure by a single α-1,6-linked mannose. The N-linked glycosylation pathway in P.pastoris is significantly different from that in Saccharomyces cerevisiae, with the addition of shorter outer chains to the core and no α-1,3 outer extensions.