A nucleotidase with unique catalytic properties is secreted by Trichinella spiralis
A nucleotidase with unique catalytic properties is secreted by Trichinella spiralis
复制标题
DOI：
10.1016/j.molbiopara.2004.04.008
复制
发表时间：
2004-08-01
影响因子：
1.5
通讯作者：
Sadeghi, SJ
中科院分区：
文献类型：
--
作者：
Gounaris, K;Selkirk, ME;Sadeghi, SJ
我们已经从寄生线虫旋毛虫中分离并表达了编码一种新的分泌型核苷酸酶的cDNA，该核苷酸酶催化核苷5-二磷酸和5 '-单磷酸的水解，但不催化核苷5-三磷酸的水解。全长cDNA编码具有N-末端信号肽的550个氨基酸的蛋白质，但缺乏用于添加糖基磷脂酰肌醇（GPI）锚的C-末端特征序列。在巴斯德毕赤酵母中的表达导致具有Mg 2+依赖性二磷酸水解酶/腺苷三磷酸双磷酸酶和5 '-核苷酸酶的催化性质的活性酶的分泌。该蛋白质序列与来自多种生物体的5 '-核苷酸酶同源，但不包含在腺苷三磷酸双磷酸酶中特异性保守的序列，这表明它是一类新的分泌型核苷酸酶的代表。该酶基本上对核苷5 '-一磷酸中的AMP具有单特异性，并以UDP >> ADP的顺序催化核苷5'-二磷酸的水解。二磷酸酶活性依赖于镁离子和还原剂的存在下，而5-核苷酸酶的活性增强，通过这些添加。动力学分析表明，该酶具有变构行为。活性位点的数目的测定表明，催化的两个不同的反应发生在相同的活性位点。数据进行了讨论，在感染过程中的主机嘌呤能信号的调节。(C)2004 Elsevier B. V.保留所有权利。
We have isolated and expressed a cDNA from the parasitic nematode Trichinella spiralis encoding a novel secreted nucleotidase which catalyses the hydrolysis of nucleoside 5-diphosphates and 5'-monophosphates, but not 5-triphosphates. The full length cDNA encodes a protein of 550 amino acids with an N-terminal signal peptide, but lacking a C-terminal signature sequence for addition of a glycosyl phosphatidylinositol (GPI) anchor. Expression in Pichia pastoris resulted in the secretion of an active enzyme with the catalytic properties of both a Mg2+-dependent diphosphohydrolase/apyrase and a 5'-nucleotidase. The protein sequence is homologous to 5'-nucleotidases from a wide variety of organisms but contains no sequences specifically conserved in apyrases, suggesting that it is a representative of a new class of secreted nucleotidase. The enzyme was essentially monospecific for AMP among the nucleoside 5'-monophosphates and catalysed the hydrolysis of nucleoside 5'-diphosphates in the order of UDP >> ADP. The diphosphatase activity was dependent on the presence of magnesium ions and a reducing agent, while the 5-nucleotidase activity was enhanced by these additions. Kinetic analyses indicated that the enzyme exhibits allosteric behaviour. Determination of the number of active sites suggested that catalysis of the two different reactions occurs at the same active site. The data are discussed in terms of regulation of host purinergic signalling during infection. (C) 2004 Elsevier B.V. All rights reserved.