Production of heterologous glycoproteins by a glycosylation-defective alg3och1 mutant of Schizosaccharomyces pombe

The early stages of N-linked glycosylation are highly conserved between fungal and mammalian cells. Such N-linked oligosaccharides are synthesized through the ordered assembly of a dolichyl pyrophosphate (Dol-PP)-linked Glc(3)Man(9)ClcNAc(2) structure by the sequential actions of several glycosyltransferases located in the endoplasmic reticulum (ER). Of the glycosyltransferase genes, Saccharomyces cerevisiae ALG3 has been identified to encode the Dol-P-Man: Man(5)GlcNAc(2)-PP-Dol alpha 1,3-mannosyltransferase, and an alg3 mutant has been shown to accumulate an Endo H-resistant M5B (Man alpha 1,2-Man alpha 1,2-Man alpha 1,3(Man alpha 1,6-)-Man beta 1,4-GlcNAc beta 1,4-GlcNAc) structure. Although Schizosaccharomyces pombe contains a homolog of the ALG3 gene (SPAC7D4.06c), the role of this gene in oligosaccharide biosynthesis is not at all clear. In this study, we deleted the alg3(+) gene in the och1 Delta mutant and analyzed the detailed oligosaccharide structures in alg3 Delta och1 Delta double mutant. The oligosaccharides were prepared from cell-surface glycoproteins by hydrazinolysis and fluorescent labeling with 2-aminopyridine. The labeled oligosaccharides were analyzed by high performance liquid chromatography, in combination with sequential glycosidase digestion and methylation analysis. These analyses revealed that the N-linked oligosaccharides of S. pombe alg3 Delta och1 Delta cells mainly consisted of two or three a-galactose-capped M5B structures. Finally, western blot analysis of recombinant human transferin suggested that heterologously expressed glycoproteins in alg3 Delta och1 Delta cells have Endo H-resistant N-linked oligosaccharide structures similar to those of alg3 Delta och1 Delta cell-surface glycoproteins. (C) 2010 Elsevier B.V. All rights reserved.