We have isolated three types of cDNAs encoding novel ␤1,3-N-acetylglucosaminyltransferases (designated ␤3Gn-T2, -T3, and -T4) from human gastric mucosa and the neuroblastoma cell line SK-N-MC. These enzymes are predicted to be type 2 transmembrane proteins of 397, 372, and 378 amino acids, respectively. They share motifs conserved among members of the ␤1,3-galactosyltransferase family and a ␤1,3-N-acetylglucosaminyltransferase (designated ␤3Gn-T1), but show no structural similarity to another type of ␤1,3-N-acetylglucosaminyltransferase (iGnT). Each of the enzymes expressed by insect cells as a secreted protein fused to the FLAG peptide showed ␤1,3-N-acetylglucosaminyltransferase activity for type 2 oligosaccharides but not ␤1,3-galactosyltransferase activity. These enzymes exhibited different substrate specificity. Transfection of Namalwa KJM-1 cells with ␤3Gn-T2, -T3, or -T4 cDNA led to an increase in poly-N-acetyllactosamines recognized by an anti-i-antigen antibody or specific lectins. The expression profiles of these ␤3Gn-Ts were different among 35 human tissues. ␤3Gn-T2 was ubiquitously expressed, whereas expression of ␤3Gn-T3 and -T4 was relatively restricted. ␤3Gn-T3 was expressed in colon, jejunum, stomach, esophagus, placenta, and trachea. ␤3Gn-T4 was mainly expressed in brain. These results have revealed that several ␤1,3-Nacetylglucosaminyltransferases form a family with structural similarity to the ␤1,3-galactosyltransferase family. Considering the differences in substrate specificity and distribution, each ␤1,3-N-acetylglucosaminyltransferase may play different roles.A family of human ␤1,3-galactosyltransferases (␤3Gal-Ts) 1 consisting of five members (␤3Gal-T1, -T2, -T3, -T4, and -T5) was recently identified (1-4). The first ␤1,3-galactosyltransferase (␤3Gal-T1), which catalyzes the formation of type 1 oligosaccharides, was isolated by us using an expression cloning approach (1). Expression patterns of ␤3Gal-T1 and type 1 oligosaccharides strongly suggested the existence of ␤3Gal-T1 homologs. For instance, type 1-derived oligosaccharides such as sialyl-Le a were known to be expressed in colon and pancreatic cancer cell lines, whereas expression of ␤3Gal-T1 was detected in brain, but not in cancer cells. Our early approach using Southern hybridization failed to detect the existence of ␤3Gal-T1 homologous genes. However, recent accumulation of nucleotide sequence information on human cDNAs and genes such as expressed sequence tags (ESTs) enabled us to search homologous genes that do not have high similarity as detected by hybridization, but show significant similarity. A homology search based on the nucleotide or amino acid sequence of ␤3Gal-T1 led to the isolation of ␤3Gal-T2, -T3, and -T4, indicating that ␤3Gal-Ts form a family (1-3).␤3Gal-T2 catalyzed a similar reaction, but showed different substrate specificity compared with ␤3Gal-T1. The activity of ␤3Gal-T3 has not been detected, whereas the corresponding mouse enzyme exhibits weak ␤3Gal-T activity for both GlcNAc and GalNAc (5). On the other...

A new member of the UDP-N-acetylglucosamine:␤-galactose ␤1,3-N-acetylglucosaminyltransferase (␤3Gn-T) family having the ␤3Gn-T motifs was cloned from rat and human cDNA libraries and named ␤3Gn-T5 based on its position in a phylogenetic tree. We concluded that ␤3Gn-T5 is the most feasible candidate for lactotriaosylceramide (Lc 3 Cer) synthase, an important enzyme which plays a key role in the synthesis of lacto-or neolacto-series carbohydrate chains on glycolipids. ␤3Gn-T5 exhibited strong activity to transfer GlcNAc to glycolipid substrates, such as lactosylceramide (LacCer) and neolactotetraosylceramide (nLc 4 Cer; paragloboside), resulting in the synthesis of Lc 3 Cer and neolactopentaosylceramide (nLc 5 Cer), respectively. A marked decrease in LacCer and increase in nLc 4 Cer was detected in Namalwa cells stably expressing ␤3Gn-T5. This indicated that ␤3Gn-T5 exerted activity to synthesize Lc 3 Cer and decrease LacCer, followed by conversion to nLc 4 Cer via endogenous galactosylation. The following four findings further supported that ␤3Gn-T5 is Lc 3 Cer synthase. 1) The ␤3Gn-T5 transcript levels in various cells were consistent with the activity levels of Lc 3 Cer synthase in those cells. 2) The ␤3Gn-T5 transcript was presented in various tissues and cultured cells. 3) The ␤3Gn-T5 expression was up-regulated by stimulation with retinoic acid and down-regulated with 12-O-tetradecanoylphorbol-13-acetate in HL-60 cells. 4) The changes in ␤3Gn-T5 transcript levels during the rat brain development were determined. Points 2, 3, and 4 were consistent with the Lc 3 Cer synthase activity reported previously.To date, three members of the human ␤1,3-N-acetylglucosaminyltransferase (␤3Gn-T) 1 family (␤3Gn-T2, -T3, and -T4) (1, 2) and five members of the human ␤1,3-galactosyltransferase (␤3Gal-T) family (␤3Gal-T1, -T2, -T3, -T4, and -T5) have been identified (3-6). All of them share amino acid motifs (␤3Gn-T motifs or ␤3Gal-T motifs) in three regions of the catalytic domain. The first, ␤3Gn-T, was cloned by an expression cloning method using an anti-i antibody (7). However, this enzyme is unique in that it does not have the ␤3Gn-T motifs although it transfers GlcNAc to Gal with an ␤1,3-linkage, resulting in the synthesis of polylactosamine chains. It was named iGn-T (7). Thereafter, ␤3Gn-T1 was isolated based on structural similarity with the ␤3Gal-T family (2). We previously reported three additional ␤3Gn-Ts, ␤3Gn-T2, -T3, and -T4, which are also structurally related to the ␤3Gn-T family (1). However, the cDNA sequence of ␤3Gn-T1 was recently corrected by Zhou et al. (see Ref. 2). The corrected sequence of ␤3Gn-T1 was identical to that of ␤3Gn-T2 which was isolated * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank TM /EBI Data Bank with the accession num...

No abstract

No abstract