Maia Ana Carolina Ribeiro Gomes, Porcino Gabriane Nascimento, Detoni Michelle de Lima, Emídio Nayara Braga, Marconato Danielle Gomes, Faria-Pinto Priscila, Fessel Melissa Regina, Reis Alexandre Barbosa, Juliano Luiz, Juliano Maria Aparecida, Marques Marcos José, Vasconcelos Eveline Gomes
Departamento de Bioquímica, Laboratório de Estrutura e Função de Proteínas, Pós-Graduação em Imunologia e DIP/Genética e Biotecnologia, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil.
Parasitol Int. 2013 Feb;62(1):44-52. doi: 10.1016/j.parint.2012.09.004. Epub 2012 Sep 18.
We identified a shared B domain within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived from the potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived from the Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83-122). By immunoprecipitation assays and Western blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87-99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.
我们在植物和寄生虫的核苷三磷酸二磷酸水解酶（NTPDases）中鉴定出一个共享的B结构域。现在，在婴儿利什曼原虫前鞭毛体中检测到一种不受腺苷酸激酶和ATP酶抑制剂影响的NTPDase活性。通过对去污剂匀浆的前鞭毛体制剂进行非变性凝胶电泳，可见一条水解核苷二磷酸和三磷酸的活性条带，经SDS-PAGE和银染后鉴定为一条50kDa的单一多肽。通过蛋白质印迹法，它被针对马铃薯焦磷酸酶（SA）、r-pot B结构域（SB）、源自马铃薯焦磷酸酶的重组多肽、以及LbB1LJ（SC）或LbB2LJ（SD）（源自利什曼原虫NTPDase 1的合成肽）产生的免疫血清所识别，并且也被来自内脏利什曼病犬的血清样本所识别，从而鉴定出具有抗原性的婴儿利什曼原虫NTPDase 1及其保守的B结构域（r83 - 122）。通过免疫沉淀试验和蛋白质印迹法，免疫血清SA和SB在鞭毛体制剂中鉴定出具有催化活性的NTPDase 1。此外，免疫血清SB（44%）和SC或SD（87 - 99%）抑制了其活性，表明对B结构域有直接影响。通过ELISA检测，38只感染犬中分别有37%、45%或50%对r-pot B结构域、LbB1LJ和LbB2LJ呈血清阳性，证实了B结构域的抗原性。
