Characterization and kinetic analysis of protein tyrosine phosphatase-H2 from Microplitis demolitor bracovirus
Characterization and kinetic analysis of protein tyrosine phosphatase-H2 from Microplitis demolitor bracovirus
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DOI：
10.1016/j.ibmb.2010.07.003
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发表时间：
2010-09-01
影响因子：
3.8
通讯作者：
Strand, Michael R.
中科院分区：
文献类型：
--
作者：
Eum, Jai-Hoon;Bottjen, Rachel C.;Strand, Michael R.
多DNA病毒Microplitis demolitor bracovinis（MdBV）编码13个与经典蛋白酪氨酸磷酸酶（PTP）具有同源性的基因。先前的序列分析表明，MdBV PTP基因家族的五个成员（ptp-H2、-H3、-H5、-N1和-N2）编码PTP，七个家族成员编码假磷酸酶，并且一个家族成员是假基因。先前的实验研究进一步暗示PTP-H2在MdBV感染后使宿主血细胞的功能丧失。在这里，我们报告的表达PTP-H2和大肠杆菌细胞中选择的突变体作为非融合或硫氧还蛋白融合蛋白。通过镍亲和层析纯化后，全长和突变体蛋白在还原条件下在SDS-PAGE凝胶上以预测大小的单一条带运行。非融合形式的PTP-H2使用磷酸肽END（pY）INASL和二氟-4-甲基石蒜酰磷酸（DiFMUP）作为底物表现出经典的Michaelis-Menten动力学。如预期的，非融合突变体PTP-H2（C236 S）没有酶活性，而PTP-H2的硫氧还蛋白融合形式具有低水平的活性。PTP-H2在pH 4.0和26 ℃的醋酸钠缓冲液中表现出最佳活性，其活性随缓冲液离子强度的增加而降低。铜、肝素和经典PTP抑制剂钒酸盐的存在也大大降低了活性。使用抗PTP-H2抗体，免疫印迹和免疫细胞化学研究仅检测到FTP-H2的血细胞从MdBV感染的Pseudoplusia includens。总之，我们的研究结果表明，FTP-H2是一种功能性酪氨酸磷酸酶，特异性表达于MdBV感染的血细胞。(C)2010爱思唯尔有限公司版权所有。
The polydnavirus Microplitis demolitor bracovinis (MdBV) encodes 13 genes that share homology with classical protein tyrosine phosphatases (PTPs). Prior sequence analysis suggested that five members of the MdBV PTP gene family (ptp-H2, -H3, -H5, -N1 and -N2) encode PTPs, seven family members encode pseudophosphatases, and one family member is a pseudogene. Prior experimental studies further implicated PTP-H2 in disabling the function of host hemocytes following infection by MdBV. Here we report expression of PTP-H2 and selected mutants in Escherichia coli cells as non-fusion or thioredoxin-fusion proteins. Following purification by nickel affinity chromatography, the full-length and mutant proteins ran as single bands of predicted size on SDS-PAGE gels under reducing conditions. The non-fusion form of PTP-H2 exhibited classical Michaelis-Menten kinetics using the phosphopeptide END(pY) INASL and difluoro-4-methylumbiliferyl phosphate (DiFMUP) as substrates. As expected, the non-fusion mutant PTP-H2(C236S) had no enzymatic activity, while the thioredoxin-fusion form of PTP-H2 had low levels of activity. PTP-H2 exhibited optimal activity at pH 4.0 and 26 degrees C in sodium acetate buffer, and its activity was diminished by increasing buffer ionic strength. Activity was also greatly reduced by the presence of copper, heparin, and the classical PTP inhibitor vanadate. Using an anti-PTP-H2 antibody, immunoblotting and immunocytochemical studies only detected FTP-H2 in hemocytes from MdBV-infected Pseudoplusia includens. Overall, our results indicate that FTP-H2 is a functional tyrosine phosphatase that is specifically expressed in MdBV-infected hemocytes. (C) 2010 Elsevier Ltd. All rights reserved.