Cisteína-proteinases em promastigotas de Leishmania (Viannia) braziliensis.
Cysteine-proteinases in promastigotes of Leishmania (Viannia) braziliensis.
It was detected cysteine-proteinases (CPs) in infective Leishmania (Viannia) braziliensis promastigotes. The purification strategy consisted of an association of Triton X-114 extractionmethod with chromatography in Concanavalin A-Sepharose column, followed by chromatography in DEAE-Sephacell column. In the assay pf chromatographic fractions, we observed a peak of enzymatic activity against the pEFLpNan substrate (165 x 10-32 amp;#956;M of pNan/minute) in over 1010 parasites, coincident with the major protein peak eluted from thecolumn. SDS-PAGE analysis of the material eluted from the ionic exchange column showed four main protein bands with relative molecular mass of 63 , 43, 30 and 27 kDa. Gelatin-SDS-PAGEassays indicated that the 43kDa and the 63kDa bands can hydrolyze gelatin at neutral pH and are sensitive to E-64. Also, both enzymes can hydrolyze pEFLpNan substrate: 63 kDa (2,2 ± 0,3 amp;#956;M of pNan/minute) and 43 kDa (0,05 ± 0,2 amp;#956;M of pNan/minute) being both inhibited by E-64 (47 %and 36 % inhibition, respectively). Immunological recognition assays, with a specific polyclonal antibody against CPB from L. (L.) mexicana, showed that bands of 63 kDa and 43 kDa arerecognized in the fractions of the ionic exchange column. Agglutination, flow cytometry and immunocytochemistry assays performed with anti-CPB antiserum revealed that homologous ofCPB are located on the promastigote membrane surface. Moreover, the incubation of promastigotes with phospholipase C reduced the number of CPBs homologues-positive cells.Both anti-cross-reacting determinant (CRD) and anti-CPB antisera recognized 63kDa and 43kDa bands in the supernatant of phospholipase C-treated cells, suggesting that isoforms of these proteins are attached to the plasma membrane by glycosylphosphatidylinositol (GPI)-anchors. Also, our data suggest that GPI-anchored CPBs are present in the detergent-resistant lipid rafts. We observed that the CPB homologues do not remain stable on the membrane surface when theparasites are maintained under successive cultures; also, the total enzymatic activity over the substrate pEFLpNan is altered. We additionally showed by real-time RT-PCR thatL. (V). braziliensis cpb genes are active and their relative expression is increased throughout the successive cultures. Thus, the presented data support the hypothesis of that studied parasite presents CPs of membrane active, beyond homologous of intracellular CPB.
Advisor:Carlos Roberto Alves
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Leishmania braziliensis Cisteína Endopeptidases Cysteine Polymerase Chain Reaction Enzymes
Date of Publication:04/10/2008