Glycobiology, 2001, Vol. 11, No. 10 813-820
© 2001 Oxford University Press
Galactosylation of N-linked oligosaccharides by human ß-1,4-galactosyltransferases I, II, III, IV, V, and VI expressed in Sf-9 cells
3Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0015, Japan; 4Department of Pathology, Beijing Medical University, Beijing 100083, P.R. China; and 5Nisshin Flour Milling Co., Ltd., Saitama 356-8511, Japan
Several studies showed that Sf-9 cells can synthesize the galactosylated N-linked oligosaccharides if ß-1,4-galactosyltransferase (ß-1,4-GalT) is supplied. The full-length human ß-1,4-GalT I, II, III, IV, V, and VI cDNAs were independently transfected into Sf-9 cells, and the galactosylation of endogenous membrane glycoproteins was examined by lectin blot analysis using Ricinus communis agglutinin-I (RCA-I), which preferentially interacts with oligosaccharides terminated with Galß1
4GlcNAc group. Several RCA-I-reactive bands appeared in all of the gene-transfected cells, and disappeared on treatment of blots with ß-1,4-galactosidase or N-glycanase prior to incubation with lectin. Introduction of the antisense ß-1,4-GalT II and V cDNAs separately into human colorectal adenocarcinoma SW480 cells, in which ß-1,4-GalT I, II, and V genes were expressed, resulted in the reduction of RCA-I binding toward N-linked oligosaccharides of the membrane glycoproteins. Differences were found in their Km values toward UDP-Gal and GlcNAcß-S-pNP and in their acceptor specificities toward oligosaccharides with the GlcNAcß14(GlcNAcß12)Man branch and with the GlcNAcß16(GlcNAcß12)Man branch. These results indicate that ß-1,4-GalTs II, III, IV, V, and VI are involved in the N-linked oligosaccharide biosynthesis cooperatively but not in a redundanat manner with ß-1,4-GalT I within cells.
1 These authors contributed equally to this article.
2 To whom correspondence should be addressed
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