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

A new member of the UDP-N-acetylglucosamine:beta -galactose beta1,3-N-acetylglucosaminyltransferase (beta 3Gn-T) family having the beta 3Gn-T motifs was cloned from rat and human cDNA libraries and named beta 3Gn-T5 based on its position in a phylogenetic tree. We concluded that beta 3Gn-T5 is the most feasible candidate for lactotriaosylceramide (Lc,Cer) synthase, an important enzyme which plays a key role in the synthesis of lacto- or neolacto-series carbohydrate chains on glycolipids. beta 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 beta 3Gn-T5. This indicated that beta 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 beta 3Gn-T5 is Lc(3)Cer synthase. 1) The beta 3Gn-T5 transcript levels in various cells were consistent with the activity levels of Lc(3)Cer synthase in those cells. 2) The beta 3Gn-T5 transcript was presented in various tissues and cultured cells. 3) The beta 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 beta 3Gn-T5 transcript levels during the rat brain development were determined. Points 2, 3, and 4 were consistent with the Lc,Cer synthase activity reported previously.