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

Paper ID Volume ID Publish Year Pages File Format Full-Text
23977 43486 2010 9 PDF Available
Title
Production of heterologous glycoproteins by a glycosylation-defective alg3och1 mutant of Schizosaccharomyces pombe
Abstract

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 Glc3Man9GlcNAc2 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:Man5GlcNAc2-PP-Dol α1,3-mannosyltransferase, and an alg3 mutant has been shown to accumulate an Endo H-resistant M5B (Manα1,2-Manα1,2-Manα1,3(Manα1,6-)-Manβ1,4-GlcNAcβ1,4-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 och1Δ mutant and analyzed the detailed oligosaccharide structures in alg3Δoch1Δ 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 alg3Δoch1Δ cells mainly consisted of two or three α-galactose-capped M5B structures. Finally, western blot analysis of recombinant human transferrin suggested that heterologously expressed glycoproteins in alg3Δoch1Δ cells have Endo H-resistant N-linked oligosaccharide structures similar to those of alg3Δoch1Δ cell-surface glycoproteins.

Keywords
Schizosaccharomyces pombe; Alg3; Och1; N-Linked oligosaccharide; Galactosylation
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Production of heterologous glycoproteins by a glycosylation-defective alg3och1 mutant of Schizosaccharomyces pombe
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 150, Issue 3, November 2010, Pages 348–356
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us