trans-sialidase of Trypanosoma cruzi: location of galactose-binding site(s).
trans-sialidase of Trypanosoma cruzi: location of galactose-binding site(s).
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克氏锥虫的转唾液酸酶：半乳糖结合位点的位置。
DOI：
10.1006/bbrc.1999.1154
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Taylor,G
中科院分区：
文献类型：
--
作者：
Chuenkova,M;Pereira,M;Taylor,G
克氏锥虫在其表面上表达转唾液酸酶，其催化唾液酸从哺乳动物宿主聚糖转移到其自身表面糖蛋白。有人提出，该酶由三个结构域之前的长的C-末端重复序列，这是不需要的酶活性。这些结构域中的第一个与催化唾液酸水解的细菌唾液酸酶具有显著的序列同一性。本文报道了TS 19 γ转唾液酸酶基因的N端结构域的序列，该基因在细菌中表达，具有与T.克鲁兹已经克隆和表达了TS 19 γ的各种缺失突变体，没有C-末端串联重复序列，并测量了它们的转唾液酸酶和唾液酸酶活性。这些实验表明，所有三个N-末端结构域都是完全转唾液酸酶活性所必需的，尽管只有第一个是唾液酸酶活性所必需的。然而，即使使用包含第一N-末端结构域的最短构建体，也观察到一些转移酶活性。缺失突变体，以探测的N-末端残基的第一个结构域的作用表明，前33个残基也需要转唾液酸酶活性，但不是唾液酸酶活性。基于我们的细菌唾液酸酶和定点突变的结构，TS 19 y的第一个N-末端结构域的分子建模表明该结构域内半乳糖结合位点的位置。
Trypanosoma cruzi expresses a trans-sialidase on its surface, which catalyzes the transfer of sialic acid from mammalian host glycans to its own surface glycoproteins. It has been proposed that the enzyme consists of three domains prior to a long C-terminal repeating sequence that is not required for enzyme activity. The first of these domains shares significant sequence identity with bacterial sialidases which catalyse the hydrolysis of sialic acid. Here we report the sequence of the N-terminal domains of the TS19y trans-sialidase gene, which was expressed in bacteria with the same specific activity as natural enzyme of T. cruzi. Various deletion mutants of TS19y, without the C-terminal tandem repeat, have been cloned and expressed and their trans-sialidase and sialidase activities measured. These experiments show that all three N-terminal domains are required for full trans-sialidase activity, though only the first is necessary for sialidase activity. Some transferase activity is observed, however, even with the shortest construct comprising the first N-terminal domain. Deletion mutants to probe the role of the N-terminal residues of the first domain suggest that the first 33 residues are also required for trans-sialidase activity, but not for sialidase activity. Molecular modelling of the first N-terminal domain of TS19y based on our structures of bacterial sialidases and site-directed mutations suggests the location of a galactose-binding site within this domain.