The UAP of Rhodnius prolixus

SIEBRA KRUG MONIQUE; FRUTTERO LEONARDO; SOMMER NATALIA; MOYETTA NATALIA; COSTE GRAHL MATHEUS; CARLINI CELIA REGINA; STANISÇUASKI FERNANDA

Porto Alegre

Otro; XVI Encontro do Programa de Pós-Graduação em Biologia Celular e Molecular (PPGBCM) da Universidade Federal do Rio Grande do Sul; 2015

Jaburetox (Jbtx), a 10 kDa peptide derived from a Canavalia ensiformis urease isoform (JBUreII), has shown entomotoxic effects in several insect species. Previous results from our group, demonstrated an abnormal movement in antennae and legs when Triatoma infestans was injected with Jbtx. Immunoprecipitation assays revealed that an unknown protein was bound to Jbtx, and it was later identified by MS/MS as the enzyme UDP-N-acetylglucosamine pyrophosphorylase (UAP - EC 2.7.7.23). UAP catalyzes the synthesis of UDP-N-acetylglucosamine, a precursor for protein glycosylation, chitin biosynthesis and glycoinositolphospholipids (GIPLs) biosynthesis. In this study, we will investigate the possible role of UAP in urease and Jbtx entomotoxicity by i) analyzing the expression profile of UAP in different organs of the model insect Rhodnius prolixus fed with Jbtx and JBU, ii) cloning, expressing and characterizing the recombinant UAP of R. prolixus, and iii) analyzing phylogenetically the evolution of insects? UAP enzymes. For the expression analysis, fifth instar insects were fed with saline solution, Jbtx or JBU (0.1 µg of protein/mg of insect weight) and dissected 6 or 18 hours after feeding. The organs analyzed for UAP and inositol-transferase (enzyme in the GIPLs biosynthesis pathway) expression were the anterior and posterior midguts, malpighian tubules, salivary glands, central nervous system, fat body and epidermis. These organs were macerated in a saline solution, the RNA from these homogenates was extracted and the cDNA was synthesized, followed by RT-qPCR. The RT-qPCR results showed that the UAP expression is heterogeneous among distinct organs and that it is increased at specific organs when insects were fed with Jbtx. Analysis of JBU fed insects are under way. For the recombinant expression of UAP, several organs from R. prolixus were macerated in a saline solution and RNA was extracted from this homogenate. Then, cDNA was synthesized and used as template to amplify the UAP gene by standard PCR, using specific primers. This amplicon was cleaved with the enzymes NdeI and BamHI and successfully inserted in pET15b vector. Sequencing of this construct is in progress. For the phylogenetic analysis, protein sequences of UAP were searched by name in VectorBase (https://www.vectorbase.org/), where only the Aedes aegypti UAP was retrieved. After that, a domain pBLAST on pHmmer (http://www.ebi.ac.uk/Tools/hmmer/search/phmmer) was performed and the results were analyzed in MEGA 6 software, following the Maximmum-Likelihood, bootstrap 1000 method, generating a phylogenetic tree that is consistent with insects? orders evolution.