Togayachi A, Akashima T, Ookubo R, Kudo T, Nishihara S, Iwasaki H, Natsume A, Mio H, Inokuchi J, Irimura T, Sasaki K, Narimatsu H
Division of Cell Biology, Institute of Life Science, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan.
J Biol Chem. 2001 Jun 22;276(25):22032-40. doi: 10.1074/jbc.M011369200. Epub 2001 Mar 30.
A new member of the UDP-N-acetylglucosamine:beta-galactose beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T) family having the beta3Gn-T motifs was cloned from rat and human cDNA libraries and named beta3Gn-T5 based on its position in a phylogenetic tree. We concluded that beta3Gn-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. beta3Gn-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 beta3Gn-T5. This indicated that beta3Gn-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 beta3Gn-T5 is Lc(3)Cer synthase. 1) The beta3Gn-T5 transcript levels in various cells were consistent with the activity levels of Lc(3)Cer synthase in those cells. 2) The beta3Gn-T5 transcript was presented in various tissues and cultured cells. 3) The beta3Gn-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 beta3Gn-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.
从大鼠和人类cDNA文库中克隆出UDP-N-乙酰葡糖胺：β-半乳糖β1,3-N-乙酰葡糖胺基转移酶（β3Gn-T）家族的一个具有β3Gn-T基序的新成员，并根据其在系统发育树中的位置将其命名为β3Gn-T5。我们得出结论，β3Gn-T5是乳糖三糖神经酰胺（Lc(3)Cer）合酶最有可能的候选者，该酶在糖脂上乳糖或新乳糖系列碳水化合物链的合成中起关键作用。β3Gn-T5对将GlcNAc转移到糖脂底物上表现出很强的活性，如乳糖神经酰胺（LacCer）和新乳糖四糖神经酰胺（nLc(4)Cer；副球蛋白），分别导致Lc(3)Cer和新乳糖五糖神经酰胺（nLc(5)Cer）的合成。在稳定表达β3Gn-T5的Namalwa细胞中检测到LacCer显著减少而nLc(4)Cer增加。这表明β3Gn-T5具有合成Lc(3)Cer并减少LacCer的活性，随后通过内源性半乳糖基化转化为nLc(4)Cer。以下四个发现进一步支持β3Gn-T5是Lc(3)Cer合酶。1）各种细胞中β3Gn-T5转录水平与这些细胞中Lc(3)Cer合酶的活性水平一致。2）β3Gn-T5转录本存在于各种组织和培养细胞中。3）在HL-60细胞中，视黄酸刺激可上调β3Gn-T5表达，而12-O-十四烷酰佛波醇-13-乙酸酯可下调其表达。4）测定了大鼠脑发育过程中β3Gn-T5转录水平的变化。第2、3和4点与先前报道的Lc(3)Cer合酶活性一致。
