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
{"title":"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。","authors":"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","doi":"10.1007/s11302-023-09980-9","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":20952,"journal":{"name":"Purinergic Signalling","volume":" ","pages":"509-520"},"PeriodicalIF":3.0000,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11377403/pdf/","citationCount":"0","resultStr":"{\"title\":\"Heterologous expression and biochemical characterization of the recombinant nucleoside triphosphate diphosphohydrolase 2 (LbNTPDase2) from Leishmania (Viannia) braziliensis.\",\"authors\":\"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\",\"doi\":\"10.1007/s11302-023-09980-9\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":20952,\"journal\":{\"name\":\"Purinergic Signalling\",\"volume\":\" \",\"pages\":\"509-520\"},\"PeriodicalIF\":3.0000,\"publicationDate\":\"2024-10-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11377403/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Purinergic Signalling\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1007/s11302-023-09980-9\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2023/11/24 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"NEUROSCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Purinergic Signalling","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1007/s11302-023-09980-9","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2023/11/24 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"NEUROSCIENCES","Score":null,"Total":0}
Heterologous expression and biochemical characterization of the recombinant nucleoside triphosphate diphosphohydrolase 2 (LbNTPDase2) from Leishmania (Viannia) braziliensis.
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.
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.