Characterization of a novel galactose β1,3-N-acetylglucosaminyltransferase (β3Gn-T8):: the complex formation of β3Gn-T2 and β3Gn-T8 enhances enzymatic activity
Characterization of a novel galactose β1,3-N-acetylglucosaminyltransferase (β3Gn-T8):: the complex formation of β3Gn-T2 and β3Gn-T8 enhances enzymatic activity
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DOI：
10.1093/glycob/cwi082
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发表时间：
2005-10-01
期刊：
影响因子：
4.3
通讯作者：
Yamashita, K
中科院分区：
文献类型：
--
作者：
Seko, A;Yamashita, K
我们表征了β 1，3-N-乙酰葡糖胺转移酶（β 3Gn-T）基因家族的新成员β 3Gn-T8。在巴斯德毕赤酵母（P. pastoris）中表达β 3Gn-T8的重组可溶形式，并将其底物特异性与β 3Gn-T2的底物特异性进行比较。这两种酶具有相似的底物特异性，并且识别四触角N-聚糖和2，6-分支三触角聚糖优先于2，4-分支三触角聚糖、双触角聚糖和乳糖-N-新四糖（LNnT），表明它们对2，6-分支结构，例如[Gal β 1 -> 4GlcNAc β 1 -> 2（Gal β 1 -> 4GlcNAc β 1 -> 6）Man α 1 -> 6 Man]。有趣的是，当可溶性重组β 3Gn-T2和β 3Gn-T8混合时，混合物的Vmax/Km值分别比单独的β 3Gn-T2和β 3Gn-T8高9.3倍和160倍。酶的Sephacryl S-300凝胶过滤显示，每种β 3Gn-T2、β 3Gn-T8和混合物的表观分子量分别为90-160、45-65和110-210 kDa，表明β 3Gn-T2和β 3Gn-T8可以形成具有增强的酶活性的复合物。这是第一份报告表明，在体外混合糖基转移酶显示增强的酶活性，通过形成一个heterocomplex。这些结果表明，β 3 Gn-T8和β 3 Gn-T2协同参与多触角N-聚糖特异性分支结构的延伸。
We characterized a novel member of the beta 1,3-N-acetylglucosaminyltransferase (beta 3Gn-T) gene family, beta 3Gn-T8. A recombinant soluble form of beta 3Gn-T8 was expressed in Pichia pastoris (P. pastoris), and its substrate specificity was compared with that of beta 3Gn-T2. The two enzymes had similar substrate specificities and recognized tetraantennary N-glycans and 2,6-branched triantennary glycans in preference to 2,4-branched triantennary glycans, biantennary glycans, and lacto-N-neotetraose (LNnT), indicating their specificity for 2,6-branched structures such as [Gal beta 1 -> 4GlcNAc beta 1 -> 2(Gal beta 1 -> 4GlcNAc beta 1 -> 6)Man alpha 1 -> 6Man]. Interestingly, when soluble recombinant beta 3Gn-T2 and beta 3Gn-T8 were mixed, the Vmax/Km value of the mixture was 9.3- and 160-fold higher than those of individual beta 3Gn-T2 and -T8, respectively. Sephacryl S-300 gel filtration of the enzymes revealed that apparent molecular weights of each beta 3Gn-T2, beta 3Gn-T8, and the mixture were 90-160, 45-65, and 110-210 kDa, respectively, suggesting that beta 3Gn-T2 and -T8 can form a complex with enhanced enzymatic activity. This is the first report demonstrating that in vitro mixed glycosyltransferases show enhanced enzymatic activity through the formation of a heterocomplex. These results suggested that beta 3Gn-T8 and beta 3Gn-T2 are cooperatively involved in the elongation of specific branch structures of multiantennary N-glycans.