Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del I.P.N., Ciudad de México, Mexico.
Programa de Doctorado en Ciencias Biomédicas, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de, Mexico.
Biochimie. 2021 Jan;180:43-53. doi: 10.1016/j.biochi.2020.10.015. Epub 2020 Oct 26.
Entamoeba histolytica is an intestinal protozoan parasite of humans and is endemic in developing countries. E. histolytica has two low molecular weight protein tyrosine phosphatase (LMW-PTP) genes, EhLMW-PTP1 and EhLMW-PTP2, which are expressed in cultured trophozoites, clinical isolates, and cysts. The amino acid sequences of proteins EhLMW-PTP1 and EhLMW-PTP2 showed only one amino acid difference between them at position A85V, respectively. Both genes are expressed in cultured trophozoites, mainly EhLMW-PTP2, and in trophozoites recovered from amoebic liver abscess, the expression of EhLMW-PTP1 is downregulated. We cloned the two genes and purified the corresponding recombinant (rEhLMW-PTPs) proteins. Antibodies anti-rEhLMW-PTP2 showed that during red blood cells uptake by E. histolytica, the EhLMW-PTPs were found in the phagocytic cups based on analysis of fluorescence signals. On the other hand, rEhLMW-PTPs showed an optimum phosphatase activity at pH 6.0 with p-nitrophenyl phosphate as the substrate. They dephosphorylate phosphotyrosine and 3-O-methylfluorescein phosphate, but not phosphoserine or phosphothreonine, and the enzymatic activity is inhibited by orthovanadate. rEhLMW-PTP1 and rEhLMW-PTP2 exhibited optimum temperatures of activities at 60 °C and 58 °C, respectively, with high thermal stability at 50 °C. Also, the rEhLMW-PTPs showed high specific activities and specific km value with pNPP or OMFP as the substrates at the physiological temperature (37 °C).
溶组织内阿米巴是一种人类肠道原生动物寄生虫，在发展中国家流行。E. histolytica 有两个低分子量蛋白酪氨酸磷酸酶（LMW-PTP）基因，EhLMW-PTP1 和 EhLMW-PTP2，它们在培养的滋养体、临床分离株和包囊体中表达。EhLMW-PTP1 和 EhLMW-PTP2 蛋白的氨基酸序列在位置 A85V 处只有一个氨基酸差异。这两个基因在培养的滋养体中均有表达，主要是 EhLMW-PTP2，在从阿米巴肝脓肿中恢复的滋养体中，EhLMW-PTP1 的表达下调。我们克隆了这两个基因，并纯化了相应的重组（rEhLMW-PTPs）蛋白。抗 rEhLMW-PTP2 的抗体表明，在溶组织内阿米巴摄取红细胞的过程中，EhLMW-PTPs 存在于吞噬杯中，基于荧光信号分析。另一方面，rEhLMW-PTPs 在 pH6.0 时具有最佳的磷酸酶活性，以对硝基苯磷酸酯作为底物。它们可以去磷酸化磷酸酪氨酸和 3-O-甲基荧光素磷酸酯，但不能去磷酸化磷酸丝氨酸或磷酸苏氨酸，并且酶活性被正钒酸盐抑制。rEhLMW-PTP1 和 rEhLMW-PTP2 的最佳活性温度分别为 60°C 和 58°C，在 50°C 时具有较高的热稳定性。此外，rEhLMW-PTPs 在生理温度（37°C）下以 pNPP 或 OMFP 作为底物时具有较高的比活性和比 km 值。
