Characterization and cDNA Cloning of a Promastigote-Specific Membrane Protein of Leishmania tropica (original) (raw)
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Journal of Biological Chemistry, 1985
A major integral membrane glycoprotein of 63 kDa (p63), present at 500,000 copies/ceIl, was found on the surface of Leishmania major LEM 513 promastigotes. This protein was labeled either by surface iodination of the cells or by metabolic incorporation of f36S]methionine. Peptide maps of the proteins labeled by the two procedures were identical. Protein p63 was purified in three steps: extraction and phase separation in the nonionic detergent Triton X-114, chromatography on DEAE-cellulose, and finally chromatography on a Mono-& column. The carbohydrate content as well as the concanavalin A receptor activity were characterized. A hydrophilic form of p63 was generated during the purification of the protein. This form was not derived by proteolysis from the amphiphilic protein found in the membrane, but may have been generated by the hydroIysis of a lipid containing myristyl residue(s) anchoring the protein in the membrane.
Vaccine, 2005
The integral membrane proteins (IMP's) of promastigotes of two virulent strain of Leishmania (L.) donovani Dd8 and Leishmania (L.) infantum LV9 and one avirulent viscerotropic strain Leishmania tropica UR6 were extracted by phase separation technique using a non-ionic detergent "Triton X-114". This detergent is homogeneous at 0 • C but divides in an aqueous phase and a detergent phase at above 20 • C. The phase partition resulted in solubilisation of hydrophilic proteins in aqueous phase, and IMP's with an amphiphilic nature were recovered in the detergent phase. The strain wise quantitative recovery rates of IMP's were estimated. These proteins were purified by chill methanol centrifugation and used as vaccinogen, alone or in combination with adjuvant against L. donovani challenge in hamster model. Among all the combinations, hamsters immunised with IMP of L. donovani (Dd8) in combination with CFA resulted in 75% parasite inhibition in spleen (P < 0.001), however, 61.14% (P < 0.001) and 77.60% (P < 0.001) parasitic inhibition was achieved in liver and bone marrow respectively as compared to their unvaccinated counter part. Similar combinations with UR6 and LV9 strain inhibited the parasite establishment up to 65.12% (P < 0.001) and 66.87% (P < 0.001), respectively on splenic site. The specific IgG level against (Dd8 strain) soluble leishmania promastigote antigen was monitored at different stages by enzyme linked immunosorbent assay (ELISA) corresponds to the level of parasitic establishment. Similar observations were made in cases of LV9 and UR6 strains. However, significant lymphoproliferative response to IMPs of Dd8 strain (SI 3.5-4.9, P < 0.001) was noticed in all IMP + CFA vaccinated animals. Thus, this study will provide a lead for more manipulative trials to develop a subunit vaccine against the fatal disease.
Evidence that the major surface proteins of three Leishmania species are structurally related
Molecular and Biochemical Parasitology, 1985
were surface radioiodinated. The proteins of the parasites were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and labeled molecules were revealed by fluorography. A single major iodinated protein of M 63 000 (p63) was identified in each of the three species. These proteins were partially purified by phase separation in Triton X-114 solution, demonstrating that the p63 of each of the three species is the most abundant integral membrane protein in the promastigote. Peptide maps were obtained by partial proteolysis with N-chlorosuccinimide or Staphylococcus V8 protease followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maps of L. major and L. donovani were identical, but only partially homologous to the maps ofL. tropica p63. Finally, immunological crossreactivity among the three p63s was demonstrated with the serum of a mouse immunized with purified L. major p63, and the serum of a dog naturally infected with L. donovani. The data show that the major surface proteins found on promastigotes of three Old World Leishmania species are structurally related.
The Brazilian journal of infectious diseases : an official publication of the Brazilian Society of Infectious Diseases, 2006
The outcome of Leishmania infections is determined by both the parasite species and the host genetic makeup. While much has been learned regarding immune responses to this parasite, our knowledge on parasite-derived factors is limited. The recent completion of the L. major and L. infantum genome sequence projects and concurrent advancement in proteomics technology would greatly accelerate the search for novel Leishmania proteins. Using a proteomics-based approach to study species-specific Leishmania proteins, we developed high-resolution, broad pH (3-10) two-dimensional gel electrophoresis (2-DE) separations to determine protein-expression profiles between highly infectious forms of the parasitic species L. amazonensis (New World) and L. major (Old World). Approximately 1,650 and 1,530 distinct protein spots were detected in the L. amazonensis and L. major gels, respectively. While a vast majority of the spots had similar distribution and intensity, a few were computationally define...
Molecular and Biochemical Parasitology, 1989
The expression, processing and localization of the promastigote surface glycoprotein, gp63, in the amastigote form of Leishmania mexicana was examined. Metabolically labeled protein was immunoprecipitated from promastigotes and amastigotes. The isolated proteins were subjected to deglycosylation and partial peptide mapping. The cleavage products generated migrated similarly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the proteins were closely related. The majority of gp63 in amastigotes was inaccessible to surface-labeling procedures, and lacked the phosphatidylinositol membrane anchor. Immunolocalization of this subpopulation of gp63 revealed it to be present within the parasite's flagellar pocket. Despite the relative paucity of ‘membrane-form’ gp63, isolation and analysis of surface proteins from lesion amastigotes indicated that gp63 was the most abundant protein on the amastigote surface.
Memorias Do Instituto Oswaldo Cruz, 1998
The kinetoplastid membrane protein 11 has been recently described in Leishmania (Leishmania) donovani as a major component of the promastigote membrane. Two oligonucleotide primers were synthesized to PCR-amplify the entire coding region of New World Leishmania species. The Leishmania (Viannia) panamensis amplification product was cloned, sequenced and the putative amino acid sequence determined. A remarkably high degree of sequence homology was observed with the corresponding molecule of L. (L) donovani and L. (L) infantum (97% and 96%, respectively). Southern blot analysis showed that the KMP-11 locus is conformed by three copies of the gene. The L. (V) panamensis ORF was subsequently cloned in a high expression vector and the recombinant protein was induced and purified from Escherichia coli cultures. Immunoblot analysis showed that 80%, 77% and 100% sera from cutaneous, mucocutaneous and visceral leishmaniasis patients, respectively, recognized the recombinant KMP-11 protein. In a similar assay, 86% of asymptomatic Leishmania-infected individuals showed IgG antibodies against the rKMP-11. We propose that KMP-11 could be used as a serologic marker for infection and disease caused by Leishmania in America.
BackgroundVisceral leishmaniasis (VL), a parasitic disease causes serious medical consequences if treatment is delayed. Despite a decline in the number of VL cases in the Indian Subcontinent, commencement of the disease in newer areas continues to be a major concern. Although serological diagnosis mainly by immunochromatographic tests has been found to be effective, test for cure in different phases of treatment is still desired. Even though good prophylactic response has been obtained in murine models by a number of vaccine candidates, few have been proposed for human use. MethodsIn this study, nine antigenic components (31, 34, 36, 45, 51, 63, 72, 91 and 97 kDa) of Leishmania promastigote membrane antigens, LAg, were electroeluted and evaluated through ELISA to diagnose and distinguish active VL from one month cured and six month past infection. Further, to investigate the immunogenicity of electroeluted proteins, humans PBMCs of cured VL patients were stimulated with 31, 34, 51, ...