Molecular Cloning and Characterization of UDP-GlcNAc:Lactosylceramide β1,3-N-Acetylglucosaminyltransferase (β3Gn-T5), an Essential Enzyme for the Expression of HNK-1 and Lewis X Epitopes on Glycolipids

Abstract

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...

A novel β1,3‐N‐acetylglucosaminyltransferase (β3Gn‐T8), which synthesizes poly‐N‐acetyllactosamine, is dramatically upregulated in colon cancer

“…elongation of polylactosamine chains, however they exhibit a different substrate specificity dependent on the length of the polylactosamine chain [1,3]. Our in vitro analysis previously demonstrated that b3Gn-T2 is most active toward polylactosamine substrates having 2-5 LacNAc repeats [1].…”

“…Three b3-glycosyltransferase (b3-GT) motifs, XIRX(S/T)W(G/L/M), (F/Y)-XXXXDXD and (E/D)DVXXGX, which we found in a previous study, are commonly encoded in b3-GTs that combine two sugars with a b1,3-linkage [1][2][3][4]. These specific motifs are shared by b3Gal-Ts, b3GalNAc-Ts and b3Gn-Ts.…”

“…The oligosaccharide substrates having the polylactosamine structures, containing repeats of lactosamine (Galb1-4GlcNAc; LN), were labeled with 2-aminobenzamide (2AB) and used as acceptor substrates [1,14]. But no activity toward any polylactosamine oligosaccharide was detected in b3Gn-T8 (data not shown).…”

“…Our in vitro analysis previously demonstrated that b3Gn-T2 is most active toward polylactosamine substrates having 2-5 LacNAc repeats [1]. Polylactosamine chains are often modified to express differentiation antigens and functional oligosaccharides.…”

“…In the past, we succeeded in the molecular cloning of a series of b3-GT genes. Thirteen human b3-GTs, i.e., five b3Gal-Ts, two b3GalNAc-Ts and six b3Gn-Ts, have been identified and reported [1][2][3][4][5][6][7][8].…”