Molecular cloning, gene organization and expression of the human UDP-GalNAc:Neu5Acα2-3Galβ-R βl,4-N-acetylgalactosaminyltransferase responsible for the biosynthesis of the blood group Sda/Cad antigen:: evidence for an unusual extended cytoplasmic domain
Molecular cloning, gene organization and expression of the human UDP-GalNAc:Neu5Acα2-3Galβ-R βl,4-N-acetylgalactosaminyltransferase responsible for the biosynthesis of the blood group Sda/Cad antigen:: evidence for an unusual extended cytoplasmic domain
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DOI：
10.1042/bj20021892
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发表时间：
2003-07-15
影响因子：
4.1
通讯作者：
Harduin-Lepers, A
中科院分区：
文献类型：
--
作者：
Montiel, MD;Krzewinski-Recchi, MA;Harduin-Lepers, A
人Sd(a)抗原是通过在被α 2,3链唾液酸残基取代的亚末端半乳糖残基上加一个β 1,4-链的n -乙酰半乳糖胺残基形成的。我们利用先前克隆的小鼠UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4- n -乙酰半乳糖胺基转移酶（Sd(a) beta1,4GalNAc转移酶）cDNA序列筛选人类EST和基因组数据库，并鉴定相应的人类基因。该序列在17号染色体上跨越超过35kb的基因组DNA，包括至少12个外显子。通过反转录PCR判断，人类基因表达广泛，因为它在几乎所有研究的细胞类型中都检测到不同的量。Northern blot分析显示，Sd(a) beta1、4GalNAc转移酶转录本8.8、6.1、4.7、3.8和1.65 kb在结肠中高表达，在肾脏、胃、回肠和直肠中表达较少。从Caco-2细胞中扩增出完整的编码核苷酸序列。有趣的是，两个前外显子的交替使用，分别命名为El(s)和El，导致产生两个转录本。这些核苷酸序列可能产生506和566个氨基酸残基的两种蛋白质，除了它们的细胞质尾部外，它们的序列相同。短形式与小鼠酶高度相似（74%的同一性），而长形式显示出一个不寻常的由66个氨基酸残基组成的细胞质长尾，这在任何其他哺乳动物糖基转移酶中都没有描述过。瞬时转染到具有共同催化结构域的Cos-7细胞中，获得了该蛋白的可溶性形式，该蛋白催化GalNAc残基转移到α 2,3-唾液化受体底物上，形成Sd(a)和Cad抗原共有的GalNAcbeta1-4[Neu5Acalpha2-3]Gal 1-R三糖。
The human Sd(a) antigen is formed through the addition of an N-acetylgalactosamine residue via a beta1,4-linkage to a sub-terminal galactose residue substituted with an alpha2,3-linked sialic acid residue. We have taken advantage of the previously cloned mouse cDNA sequence of the UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4-N-acetylgalactosaminyltransferase (Sd(a) beta1,4GalNAc transferase) to screen the human EST and genomic databases and to identify the corresponding human gene. The sequence spans over 35 kb of genomic DNA on chromosome 17 and comprises at least 12 exons. As judged by reverse transcription PCR, the human gene is expressed widely since it is detected in various amounts in almost all cell types studied. Northern blot analysis indicated that five Sd(a) beta1,4GalNAc transferase transcripts of 8.8, 6.1, 4.7, 3.8 and 1.65 kb were highly expressed in colon and to a lesser extent in kidney, stomach, ileum and rectum. The complete coding nucleotide sequence was amplified from Caco-2 cells. Interestingly, the alternative use of two first exons, named El(s) and El, leads to the production of two transcripts. These nucleotide sequences give rise potentially to two proteins of 506 and 566 amino acid residues, identical in their sequence with the exception of their cytoplasmic tail. The short form is highly similar (74 % identity) to the mouse enzyme whereas the long form shows an unusual long cytoplasmic tail of 66 amino acid residues that is as yet not described for any other mammalian glycosyltransferase. Upon transient transfection in Cos-7 cells of the common catalytic domain, a soluble form of the protein was obtained, which catalysed the transfer of GalNAc residues to alpha2,3-sialylated acceptor substrates, to form the GalNAcbeta1-4[Neu5Acalpha2-3]Gal 1-R trisaccharide common to both Sd(a) and Cad antigens.