Expression Cloning of Human Globoside Synthase cDNAs
Expression Cloning of Human Globoside Synthase cDNAs
复制标题
人球苷酸合成酶 cDNA 的表达克隆
DOI：
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发表时间：
2000
影响因子：
4.8
通讯作者：
Kazuro Furukawa
中科院分区：
文献类型：
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作者：
T. Okajima;Yoko Nakamura;M. Uchikawa;D. Haslam;S. Numata;Keiko Furukawa;T. Urano;Kazuro Furukawa
通过真核细胞表达克隆系统，我们分离到了葡糖苷合成酶（β1,3- n -乙酰半乳糖氨基转移酶）基因的cdna。将转染SV40大T抗原和先前克隆的Gb3/CD77合成酶cDNA的小鼠成纤维细胞L细胞与从人肾mRNA制备的cDNA文库和Forssman合成酶cDNA共转染，用抗Forssman单克隆抗体筛选Forssman抗原阳性细胞。分离的cdna包含一个单一的开放阅读框，预测具有351个氨基酸的II型膜蛋白。令人惊讶的是，cDNA克隆结果与先前报道的β3Gal-T3相同，β3Gal-T3已与其他半乳糖转移酶序列同源性克隆。对转染cdna的L细胞提取物进行底物特异性分析，证实该基因产物实际上是β1,3- n -乙酰半乳糖胺基转移酶，特异性催化n -乙酰半乳糖胺转移到globotriaosylceramide上。从转染dna的细胞中提取的中性糖脂的TLC免疫染色结果也支持新合成的成分为环球苷的身份。结果表明，明显属于单一糖基转移酶家族的糖基转移酶不一定催化利用相同受体甚至相同糖供体的反应。球苷合成酶基因在心脏、脑、睾丸等组织中均有表达。我们建议将克隆的葡糖苷合酶基因命名为β3GalNAc-T1。
By using a eukaryocytic cell expression cloning system, we have isolated cDNAs of the globoside synthase (β1,3-N-acetylgalactosaminyltransferase) gene. Mouse fibroblast L cells transfected with SV40 large T antigen and previously cloned Gb3/CD77 synthase cDNAs were co-transfected with a cDNA library prepared from mRNA from human kidney together with Forssman synthase cDNA, and Forssman antigen-positive cells were panned using an anti-Forssman monoclonal antibody. The isolated cDNAs contained a single open reading frame predicting a type II membrane protein with 351 amino acids. Surprisingly, the cDNA clones turned out to be identical with previously reported β3Gal-T3, which had been cloned by sequence homology with other galactosyltransferases. Substrate specificity analysis with extracts from cDNA-transfected L cells confirmed that the gene product was actually β1,3-N-acetylgalactosaminyltransferase that specifically catalyzes the transfer of N-acetylgalactosamine onto globotriaosylceramide. Results of TLC immunostaining of neutral glycolipids from the cDNA-transfected cells also supported the identity of the newly synthesized component as globoside. The results show that glycosyltransferases apparently belonging to a single glycosyltransferase family do not necessarily catalyze reactions utilizing the same acceptor or even the same sugar donor. The globoside synthase gene was expressed in many tissues, such as heart, brain, testis, etc. We propose the designation β3GalNAc-T1 for the cloned globoside synthase gene.
确定 GM2 和 GD2 神经节苷脂表达的 β 1,4 N-乙酰半乳糖胺基转移酶 cDNA 的表达克隆。
DOI：
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发表时间：
1992
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Nagata,Y;Yamashiro,S;Yodoi,J;Lloyd,KO;Shiku,H;Furukawa,K
通讯作者：
Furukawa,K
影响因子：
4.8
作者：
Amado, M;Almeida, R;Clausen, H
通讯作者：
Clausen, H