title: The expression of NTPDase1 and -2 of Leishmania infantum chagasi in bacterial and mammalian cells: Comparative expression, refolding and nucleotidase characterization.
abstract: Visceral Leishmaniasis (VL) represents an important global health problem in several warm countries around the world. The main targets in this study are the two nucleoside triphosphate diphosphohydrolases (NTPDases) from Leishmania infantum chagasi that are the main etiologic agent of VL in the New World. These enzymes, called LicNTPDase1 and -2, are homologous to members 5 and 6 of the mammalian E-NTPDase/CD39 superfamily of enzymes. These enzymes hydrolyze nucleotides and accordingly can participate in the purine salvage pathways and in the modulation of purinergic signaling through the extracellular nucleotide-dependent host immune responses. They can therefore affect adhesion and infection of host cells and the parasite virulence. To further characterize these enzymes, in this work, we expressed LicNTPDase1 and -2 in the classical bacterial system Escherichia coli and mammalian cell system COS-7 cells. Our data demonstrate that changes in refolding after expression in bacteria can increase the activity of recombinant (r) rLicNTPDase2 up to 20 times but has no significant effect on rLicNTPDase1. Meanwhile, the expression in COS-7 led to a significant increase in activity for rLicNTPDase1.
authors.affiliation: Biochemistry and Molecular Biology Department, Universidade Federal de Viçosa, Viçosa, MG, Brazil; Instituto Nacional de Biotecnologia Estrutural e Química Medicinal em Doenças Infecciosas- INBEQMeDI, Brazil; Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université Laval, Québec City, QC G1V 0A6, Canada.
authors.forename: M S
authors.initials: MS
authors.lastname: Bastos
authors.affiliation: Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université Laval, Québec City, QC G1V 0A6, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Tremblay
authors.affiliation: Biochemistry and Molecular Biology Department, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
authors.forename: J M
authors.initials: JM
authors.lastname: Agripino
authors.affiliation: Biochemistry and Molecular Biology Department, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
authors.forename: I L A
authors.initials: ILA
authors.lastname: Rabelo
authors.affiliation: Biochemistry and Molecular Biology Department, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
authors.forename: L P
authors.initials: LP
authors.lastname: Barreto
authors.affiliation: Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université Laval, Québec City, QC G1V 0A6, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Pelletier
authors.affiliation: Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université Laval, Québec City, QC G1V 0A6, Canada; Centre de recherche du CHU de Québec - Université Laval, Québec City, QC G1V 4G2, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Lecka
authors.affiliation: Veterinary Department, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
authors.forename: A
authors.initials: A
authors.lastname: Silva-Júnior
authors.affiliation: Biochemistry and Molecular Biology Department, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
authors.forename: G C
authors.initials: GC
authors.lastname: Bressan
authors.affiliation: Biochemistry and Molecular Biology Department, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
authors.forename: M R
authors.initials: MR
authors.lastname: Almeida
authors.affiliation: Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université Laval, Québec City, QC G1V 0A6, Canada; Centre de recherche du CHU de Québec - Université Laval, Québec City, QC G1V 4G2, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Sévigny
authors.affiliation: Biochemistry and Molecular Biology Department, Universidade Federal de Viçosa, Viçosa, MG, Brazil; Instituto Nacional de Biotecnologia Estrutural e Química Medicinal em Doenças Infecciosas- INBEQMeDI, Brazil. Electronic address: jufietto@ufv.br.
authors.forename: J L R
authors.initials: JLR
authors.lastname: Fietto
mesh_terms: D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001081:Apyrase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019556:COS Cells; D002522:Chlorocebus aethiops; D004926:Escherichia coli; D015870:Gene Expression; D018314:Leishmania infantum / Q000201:enzymology / Q000235:genetics*; D058849:Protein Refolding; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D015703:Antigens, CD; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D001081:Apyrase; C067869:CD39 antigen
keywords: Bacterial system; Heterologous expression; Leishmania infantum; Mammalian system; Parasitic NTPDases