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

Takao Ohashi, Shin ichi Nakakita, Wataru Sumiyoshi, Kaoru Takegawa

Research output: Contribution to journalArticle

10 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)348-356
Number of pages9
JournalJournal of Biotechnology
Volume150
Issue number3
DOIs
Publication statusPublished - Nov 1 2010

Fingerprint

Glycosylation
Oligosaccharides
Glycoproteins
Schizosaccharomyces
Genes
Glycosyltransferases
Membrane Glycoproteins
Mannosyltransferases
Naphazoline
Methylation
Glycoside Hydrolases
Biosynthesis
High performance liquid chromatography
Transferrin
Galactose
Endoplasmic Reticulum
Yeast
Labeling
Saccharomyces cerevisiae
Digestion

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Applied Microbiology and Biotechnology

Cite this

Production of heterologous glycoproteins by a glycosylation-defective alg3och1 mutant of Schizosaccharomyces pombe. / Ohashi, Takao; Nakakita, Shin ichi; Sumiyoshi, Wataru; Takegawa, Kaoru.

In: Journal of Biotechnology, Vol. 150, No. 3, 01.11.2010, p. 348-356.

Research output: Contribution to journalArticle

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