Cloning of a Novel Member of the UDP-Galactose:β-N-Acetylglucosamine β1,4-Galactosyltransferase Family, β4Gal-T4, Involved in Glycosphingolipid Biosynthesis

Abstract

A novel putative member of the human UDP-galactose:␤-N-acetylglucosamine ␤1,4-galactosyltransferase family, designated ␤4Gal-T4, was identified by BLAST analysis of expressed sequence tags. The sequence of ␤4Gal-T4 encoded a type II membrane protein with significant sequence similarity to other ␤1,4-galactosyltransferases. Expression of the full coding sequence and a secreted form of ␤4Gal-T4 in insect cells showed that the gene product had ␤1,4-galactosyltransferase activity. Analysis of the substrate specificity of the secreted form revealed that the enzyme catalyzed glycosylation of glycolipids with terminal ␤-GlcNAc; however, in contrast to ␤4Gal-T1, -T2, and -T3, this enzyme did not transfer galactose to asialo-agalacto-fetuin, asialo-agalactotransferrin, or ovalbumin. The catalytic activity of ␤4Gal-T4 with monosaccharide acceptor substrates, Nacetylglucosamine as well as glucose, was markedly activated in the presence of ␣-lactalbumin. The genomic organization of the coding region of ␤4Gal-T4 was contained in six exons. All intron/exon boundaries were similarly positioned in ␤4Gal-T1, -T2, and -T3. ␤4Gal-T4 represents a new member of the ␤4-galactosyltransferase family. Its kinetic parameters suggest unique functions in the synthesis of neolactoseries glycosphingolipids.A family of human UDP-galactose:␤-N-acetylglucosamine ␤1,4-galactosyltransferases (␤4Gal-Ts) 1 was recently identified (1-3). Four genes within this family encode ␤4-galactosyltransferases, which form the Gal␤1-4GlcNAc linkage (2, 4 -6). The kinetic parameters and expression patterns of these enzymes differ and they are predicted to show some degree of overlap in biological function (2, 3, 6). Two ␤4-galactosyltransferases, ␤4Gal-T1 and -T2, can function as lactose synthases in the presence of ␣-lactalbumin (2, 3, 7), whereas ␤4Gal-T3 and ␤4Gal-T5 2 are largely insensitive to ␣-lactalbumin modulation (2, 6, 8). ␤4Gal-T1, -T2, and -T3 catalyze transfer of galactose to lactoseries glycosphingolipids, but ␤4Gal-T3 only efficiently catalyzes synthesis of the first N-acetyllactosamine unit in lactoseries glycolipids (2). In contrast, ␤4Gal-T5 was reported to be inactive with a glycolipid substrate (Lc 3 Cer) 2 as well as with the glycoprotein acceptor, asialo-agalacto-transferrin (6). A rat lactosylceramide synthase was recently purified and cloned by Nomura et al. (9), and it appears to represent the ortholog of the human member of the gene ␤4-galactosyltransferase family designated ␤4Gal-T6 (10). Thus, the formation of Gal␤1-4Glc(NAc) linkages in different glycoconjugates and their varying oligosaccharide structures may be catalyzed by different ␤4-galactosyltransferases.Analysis of ESTs suggested the existence of additional members of the human ␤4Gal-T gene family (1-3), and recently, Lo et al. (10) compared the full coding sequences of six homologous human genes and suggested a nomenclature based on sequence similarity: ␤4Gal-T1 (5, 11, 12), ␤4Gal-T2 (2), ␤4Gal-T3 (2), ␤4Gal-T4, ␤4Gal-T5 (6), and ␤4Gal-T6 (9). So far, all genes except one, ...

Poly-N-acetyllactosamine Extension inN-Glycans and Core 2- and Core 4-branchedO-Glycans Is Differentially Controlled by i-Extension Enzyme and Different Members of the β1,4-Galactosyltransferase Gene Family

“…This is probably due to the intrinsic nature of ␤4Gal-TIV, which acts less efficiently on longer poly-Nacetyllactosamine units, as shown in synthetic substrates utilized in the present study and lacto-series glycolipids (28). In the present study, we demonstrated that core 4-branched Oglycans are galactosylated by ␤4Gal-TI but are even less efficient acceptors for poly-N-acetyllactosamine formation than core 2-branched O-glycans (Figs.…”

“…Expression of cDNAs Encoding ␤4Gal-TII, -TIII, -TIV, and -TV␤4Gal-TII, -TIII, and -TIV were expressed in insect cells, and the supernatants from these transfected insect cells were used as an enzyme source as described previously (27,28,36). .0 nmol/h/ml (for ␤4Gal-Ts), determined using 0.5 mM GlcNAc␤13p-nitrophenol, or 380.0 nmol/h/ml (for iGnT), determined using 0.5 mM Gal␤134Glc␤13p-nitrophenol, was present in these experiments.…”

Molecular Cloning and Characterization of a Novel Human β1,4-N-Acetylgalactosaminyltransferase, β4GalNAc-T3, Responsible for the Synthesis of N,N′-Diacetyllactosediamine, GalNAcβ1–4GlcNAc

“…␤4Gal-T1 to -T6 exhibit activity to transfer Gal to GlcNAc with a ␤1,4-linkage in N-and O-glycans and glycolipids (1)(2)(3)(4). ␤4Gal-T7 synthesizes the Gal␤1-4Xyl structure that occurs in the linkage portion of glycosaminoglycans (5,6).…”

Cloning and Expression of a Proteoglycan UDP-Galactose:β-Xylose β1,4-Galactosyltransferase I

“…The unmarked cross-peaks are all intraresidue correlations. cells, which exhibited only about 2% of the normal level of galactosyltransferase I activity, could prime glycosaminoglycan synthesis on exogenously added ␤-xylosides (30), although this activity may stem from ␤Glc(NAc) ␤4GalTs (7,31).…”

“…The six ␤4Gal-Ts have highly conserved sequence motifs in the putative catalytic domain including four conserved cysteine residues. The genomic organization of the first four genes is similar and includes conservation of spacing for five intron/exon boundaries in the coding regions (4,7,9,10). This suggests that these genes arose late in evolutionary terms as a result of gene duplication and subsequent sequence divergence.…”