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
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
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DOI：
10.1074/jbc.m011369200
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发表时间：
2001-06-22
影响因子：
4.8
通讯作者：
Narimatsu, H
中科院分区：
文献类型：
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作者：
Togayachi, A;Akashima, T;Narimatsu, H
从大鼠和人cDNA文库中克隆出具有β 3Gn-T基序的β -半乳糖β a1,3- n-乙酰氨基葡萄糖氨基转移酶（β 3Gn-T）家族新成员，并根据其在系统发育树上的位置命名为β 3Gn-T5。我们得出结论，β 3Gn-T5是最可行的候选乳三聚神经酰胺（Lc,Cer）合成酶，这是一种重要的酶，在糖脂上合成乳或新乳系列碳水化合物链中起关键作用。β 3Gn-T5表现出较强的将GlcNAc转移到糖脂底物，如乳糖神经酰胺（LacCer）和新乳酸四糖神经酰胺（nLc(4)Cer）；合成了Lc(3)Cer和nLc(5)Cer。在稳定表达β 3Gn-T5的Namalwa细胞中，LacCer明显减少，nLc(4)Cer明显增加。这表明β 3Gn-T5具有合成Lc(3)Cer和降低LacCer的活性，然后通过内源性半乳糖基化转化为nLc(4)Cer。以下四项发现进一步支持β 3Gn-T5为Lc(3)Cer合酶。1) β 3Gn-T5在不同细胞中的转录水平与Lc(3)Cer合成酶的活性水平一致。2) β 3Gn-T5转录物在多种组织和培养细胞中均有表达。3) HL-60细胞β 3Gn-T5在维甲酸刺激下表达上调，在12- o -十四烷醇-13-乙酸刺激下表达下调。4)测定大鼠脑发育过程中β 3Gn-T5转录物水平的变化。第2、3、4点与之前报道的Lc、Cer合成酶活性一致。
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.