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)GlcNAc(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 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.
Ohashi
Takao
T
Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
Nakakita
Shin-ichi
S
Sumiyoshi
Wataru
W
Takegawa
Kaoru
K
D015153:Blotting, Western; D002236:Carbohydrate Conformation; D049109:Cell Proliferation; D048430:Cell Shape; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008364:Mannosyltransferases / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D019887:Peanut Agglutinin; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism
D006023:Glycoproteins; D008565:Membrane Proteins; D009844:Oligosaccharides; D019887:Peanut Agglutinin; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; D014168:Transferrin; D008364:Mannosyltransferases; C539723:Och1 protein, S pombe; D006026:Glycoside Hydrolases