Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis
Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis
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DOI：
10.1128/jb.182.19.5425-5432.2000
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发表时间：
2000-10-01
影响因子：
3.2
通讯作者：
Ullrich, A
中科院分区：
文献类型：
--
作者：
Koul, A;Choidas, A;Ullrich, A
从结核分枝杆菌H(37)RV基因组DNA中克隆了两个与蛋白酪氨酸磷酸酶基因序列同源的基因。两种推定的酪氨酸磷酸酶分别命名为MPtpA和MPtpB，其分子量分别为17.5和30 kDa。MPtpA和MPtpB在大肠杆菌中以谷胱甘肽S-转移酶融合蛋白的形式表达，亲和纯化的蛋白能使髓鞘碱性蛋白的磷酸酪氨酸残基去磷酸化，但不能去磷酸化髓鞘碱性蛋白的丝氨酸/苏氨酸残基。酪氨酸磷酸酶的特异性抑制剂原钒酸钠可抑制这些磷酸酶的活性，但丝氨酸/苏氨酸磷酸酶的抑制剂冈田酸、催化位点突变MPtpA的半胱氨酸11和MPtpB的半胱氨酸160不能抑制酶的活性。Southern杂交分析表明，虽然mptpA存在于生长缓慢的分枝杆菌物种以及快速生长的腐生植物中，但mptpB仅限于结核分枝杆菌复合体的成员。在结核分枝杆菌的全细胞裂解液和培养滤液中都存在这些磷酸酶，这表明这些蛋白被分泌到细胞外培养液中。由于酪氨酸磷酸酶对几种致病菌的毒力是必不可少的，mptpB的有限分布使其成为结核分枝杆菌毒力基因的一个很好的候选基因。
Two genes with sequence homology to those encoding protein tyrosine phosphatases were cloned from genomic DNA of Mycobacterium tuberculosis H(37)Rv. The calculated molecular masses of these two putative tyrosine phosphatases, designated MPtpA and MPtpB, were 17.5 and 30 kDa, respectively. MPtpA and MPtpB were expressed as glutathione S-transferase fusion proteins in Escherichia coli, The affinity-purified proteins dephosphorylated the phosphotyrosine residue of myelin basic protein (MBP), but they failed to dephosphorylate serine/threonine residues of MBP. The activity of these phosphatases was inhibited by sodium orthovanadate, a specific inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine/threonine phosphatases, Mutations at the catalytic site motif, cysteine 11 of MPtpA and cysteine 160 of MPtpB, abolished enzyme activity. Southern blot analysis revealed that, while mptpA is present in slow-growing mycobacterial species as well as fast-growing saprophytes, mptpB was restricted to members of the M tuberculosis complex. These phosphatases were present in both whole-cell lysates and culture filtrates of M tuberculosis, suggesting that these proteins are secreted into the extracellular medium. Since tyrosine phosphatases are essential for the virulence of several pathogenic bacteria, the restricted distribution of mptpB makes it a good candidate for a virulence gene of M. tuberculosis.