RESUMO
Two proteins from the eggshell of Rhodnius prolixus were isolated, characterized and named Rp30 and Rp45 according to their molecular masses. Purified proteins were used to obtain specific antiserum which was later used for immunolocalization. The antiserum against Rp30 and Rp45 detected their presence inside the follicle cells, their secretion and their association with oocyte microvilli. Both proteins are expressed during the final stage of vitellogenesis, preserved during embryogenesis and discarded together with the eggshell. The amino terminals were sequenced and both proteins were further cloned using degenerated primers. The amino acid sequences appear to have a tripartite arrangement with a highly conserved central domain which presents a repetitive motif of valine-proline-valine (VPV) at intervals of 15 amino acid residues. Their amino acid sequence showed no similarity to any known eggshell protein. The expression of these proteins was also investigated; the results demonstrated that this occurred strictly in choriogenic follicles. Antifungal activity against Aspergillus niger was found to be associated with Rp45 but not with Rp30. A. niger exposed to Rp45 protein induced growth inhibition and several morphological changes such as large vacuoles, swollen mitochondria, multi-lamellar structures and a disorganized cell wall as demonstrated by electron microscopy analysis.
Assuntos
Proteínas do Ovo/metabolismo , Proteínas de Insetos/metabolismo , Rhodnius/metabolismo , Sequência de Aminoácidos , Animais , Antifúngicos/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Aspergillus niger/efeitos dos fármacos , Clonagem Molecular , Proteínas do Ovo/química , Proteínas do Ovo/farmacologia , Desenvolvimento Embrionário , Proteínas de Insetos/química , Proteínas de Insetos/farmacologia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Óvulo/metabolismo , Rhodnius/embriologia , Rhodnius/crescimento & desenvolvimento , Alinhamento de Sequência , Análise de Sequência de Proteína , VitelogêneseRESUMO
The insect Rhodnius prolixus is a hematophagous hemipteran that has five nymphal instars. Fifth instar nymphs contain, in their salivary glands, four nitrophorins which have already been described in the literature (NP1, NP2, NP3 and NP4). Two new hemeproteins were isolated and partially characterized from first instar nymphs. NP2, that shows an anticoagulant activity, was also identified, but NP1, NP3 and NP4 were not found. As these new hemeproteins have amino-terminal sequences clearly homologous to already described nitrophorins and were capable of binding nitric oxide, they were named nitrophorins 5 and 6, although they showed an unusual Soret band at 412 nm. In each subsequent nymphal stage, a new nitrophorin emerges. In the second instar, NP4 comes into view, in the third instar NP1 appears, and NP3 is only found in fifth instar nymphs and adults, showing that the nitrophorin profile of R. prolixus saliva is stage-specific.
Assuntos
Hemeproteínas/metabolismo , Rhodnius/metabolismo , Saliva/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Hemeproteínas/química , Estágios do Ciclo de Vida , Dados de Sequência Molecular , Rhodnius/crescimento & desenvolvimento , Proteínas e Peptídeos Salivares/química , Homologia de Sequência de AminoácidosRESUMO
Rhodnius prolixus oocyte extracts were chromatographed on an ion exchange column in order to purify vitellin (VT). Three VT heterogeneous populations were identified and named VT(1), VT(2), and VT(3) according to their order of elution from the column. The phosphate content of each population was determined, after lipid extraction, and a heterogeneous distribution was found: VT(1) being the less phosphorylated (50 mol P/mol protein) and VT(3) the heavily phosphorylated population (281 mol P/mol protein). Analysis of radioactivity associated with each VT population purified from animals fed with (32)Pi showed the same phosphorylation profile. Due to the fact that vitellogenin is the known precursor of VT, we have also chromatographed 32P-VG in the same way as we purified VT. Only one VG's population was detected and resembled to VT(3) with respect to its elution profile. All VT populations contain the same neutral lipids, but they were heterogeneous with respect to phospholipid composition. VT(1) presents phosphatidylcholine and phosphatidylethanolamine whereas VT(2) and VT(3) also showed cardiolipin and probably phosphatidylserine. Sugar composition of VT(2) and VT(3) includes mannose as the main associated carbohydrate but VT(1) also contains glucose resembling VG. Although VG and VT are similar with respect to the elution profile, their sugar composition is different. These results suggest a post-endocytosis processing on VG molecule. The possible biological function of VT heterogeneous populations is discussed.
