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 V I cDNAs were independently transfected into Sf-9 cells, and the galactosyla tion of endogenous membrane glycoproteins was examined by lectin blot analy sis using Ricinus communis agglutinin-I (RCA-I), which preferentially inter acts with oligosaccharides terminated with Gal beta1 --> 4GlcNAc group. Sev eral RCA-I-reactive bands appeared in all of the gene-transfected cells, an d disappeared on treatment of blots with beta -1,4-galactosidase or N-glyca nase prior to incubation with lectin. Introduction of the antisense beta -1 ,4-GalT II and V cDNAs separately into human colorectal adenocarcinoma SW48 0 cells, in which beta -1,4-GalT I, II, and V genes were expressed, resulte d 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 GlcNAc beta -S-pNP and in their acceptor specificities toward oligosaccharides with the GlcNAc beta1 -->4(GlcNAc beta1 -->2)Man branch an d with the GlcNAc beta1 -->6(GlcNAc beta1 -->2)Man branch. These results in dicate that beta -1,4-GalTs II, III, IV, V, and VI are involved in the N-li nked oligosaccharide biosynthesis cooperatively but not in a redundanat man ner with beta -1,4-GalT I within cells.