Galactosylation of N-linked oligosaccharides by human beta-1,4-galactosyltransferases I, II, III, IV, V, and VI expressed in Sf-9 cells.
Several studies showed that Sf-9 cells can synthesize the galactosylated N-linked oligosaccharides if beta-1,4-galactosyltransferase (beta-1,4-GalT) is supplied. The full-length human beta-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 Galbeta1-->4GlcNAc group. Several RCA-I-reactive bands appeared in all of the gene-transfected cells, and disappeared on treatment of blots with beta-1,4-galactosidase or N-glycanase prior to incubation with lectin. Introduction of the antisense beta-1,4-GalT II and V cDNAs separately into human colorectal adenocarcinoma SW480 cells, in which beta-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 K(m) values toward UDP-Gal and GlcNAcbeta-S-pNP and in their acceptor specificities toward oligosaccharides with the GlcNAcbeta1-->4(GlcNAcbeta1-->2)Man branch and with the GlcNAcbeta1-->6(GlcNAcbeta1-->2)Man branch. These results indicate that beta-1,4-GalTs II, III, IV, V, and VI are involved in the N-linked oligosaccharide biosynthesis cooperatively but not in a redundant manner with beta-1,4-GalT I within cells.
Guo
S
S
Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0015, Japan.
Sato
T
T
Shirane
K
K
Furukawa
K
K
D000230:Adenocarcinoma / Q000201:enzymology; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D015179:Colorectal Neoplasms / Q000201:enzymology; D017931:DNA Primers; D018076:DNA, Complementary; D005690:Galactose / Q000378:metabolism*; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D020014:K562 Cells; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D018411:Spodoptera
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D009844:Oligosaccharides; D005700:Galactosyltransferases; D005690:Galactose