Heterologous expression and biochemical characterization of the recombinant nucleoside triphosphate diphosphohydrolase 2 (LbNTPDase2) from Leishmania (Viannia) braziliensis.

IF 3 4区医学 Q2 NEUROSCIENCES Purinergic Signalling Pub Date : 2024-10-01 Epub Date: 2023-11-24 DOI:10.1007/s11302-023-09980-9

Nancy da Rocha Torres Pavione, João Victor Badaró de Moraes, Isadora Cunha Ribeiro, Raissa Barbosa de Castro, Walmir da Silva, Anna Cláudia Alves de Souza, Victor Hugo Ferraz da Silva, Raphael de Souza Vasconcellos, Gustavo da Costa Bressan, Juliana Lopes Rangel Fietto

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Abstract

Leishmania braziliensis is a pathogenic protozoan parasite that causes American Tegumentary Leishmaniasis (ATL), an important tropical neglected disease. ENTPDases are nucleotidases that hydrolyze intracellular and/or extracellular nucleotides. ENTPDases are known as regulators of purinergic signalling induced by extracellular nucleotides. Leishmania species have two isoforms of ENTPDase, and, particularly, ENTPDase2 seems to be involved in infectivity and virulence. In this study, we conducted the heterologous expression and biochemical characterization of the recombinant ENTPDase2 of L. braziliensis (rLbNTPDase2). Our results show that this enzyme is a canonical ENTPDase with apyrase activity, capable of hydrolysing triphosphate and diphosphate nucleotides, and it is dependent on divalent cations (calcium or magnesium). Substrate specificity was characterized as UDP>GDP>ADP>GTP>ATP=UTP. The enzyme showed optimal activity at a neutral to basic pH and was partially inhibited by suramin and DIDS. Furthermore, the low apparent Km for ADP suggests that the enzyme may play a role in adenosine-mediated signalling. The biochemical characterization of this enzyme can open new avenues for using LbNTPDase2 as a drug target.

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巴西利什曼原虫重组核苷三磷酸二磷酸水解酶2 (LbNTPDase2)的异源表达及生化特性研究。

巴西利什曼原虫是一种致病的原生动物寄生虫，可引起美洲背生利什曼病(ATL)，这是一种重要的热带被忽视疾病。entpase是水解细胞内和/或细胞外核苷酸的核苷酸酶。entpases被认为是细胞外核苷酸诱导的嘌呤能信号的调节因子。利什曼原虫有两种entpase亚型，特别是entpase 2似乎与传染性和毒力有关。在本研究中，我们对巴西乳杆菌重组ENTPDase2 (rLbNTPDase2)进行了异源表达和生化鉴定。我们的研究结果表明，该酶是一种典型的具有apyrase活性的entpase，能够水解三磷酸和二磷酸核苷酸，并且依赖于二价阳离子(钙或镁)。底物特异性表现为UDP>GDP>ADP>GTP>ATP=UTP。该酶在中性至碱性pH下表现出最佳活性，并被苏拉明和DIDS部分抑制。此外，ADP的低表观Km表明该酶可能在腺苷介导的信号传导中发挥作用。该酶的生化特性为利用LbNTPDase2作为药物靶点开辟了新的途径。

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来源期刊

Purinergic Signalling 医学-神经科学

CiteScore

6.60

自引率

17.10%

发文量

审稿时长

6-12 weeks

期刊介绍： Nucleotides and nucleosides are primitive biological molecules that were utilized early in evolution both as intracellular energy sources and as extracellular signalling molecules. ATP was first identified as a neurotransmitter and later as a co-transmitter with all the established neurotransmitters in both peripheral and central nervous systems. Four subtypes of P1 (adenosine) receptors, 7 subtypes of P2X ion channel receptors and 8 subtypes of P2Y G protein-coupled receptors have currently been identified. Since P2 receptors were first cloned in the early 1990’s, there is clear evidence for the widespread distribution of both P1 and P2 receptor subtypes in neuronal and non-neuronal cells, including glial, immune, bone, muscle, endothelial, epithelial and endocrine cells.