Immunoproteomic analysis of mycobacterium leprae derived cell membrane antigen
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The aim of the study was serological characterization of antigens of cell membrane proteins (MLMA) of armadillo derived Mycobacterium leprae, a causative organism of leprosy. Cell membrane proteins of Mycobacterium leprae were separated using two dimensional gel electrophoresis (2-DGE) and then immunoblotted with sera from leprosy patients, tuberculosis patients, healthy individuals and anti-human IgG/IgA/IgM antibodies. The immunoblots thus obtained were compared and proteins corresponding to spots detected only in leprosy immunblots were finally analyzed for their characterization by MALDI-TOF/TOF. Employing this approach, 14 (6 with anti-IgA, 5 with anti-IgM and 3 with IgG) immuno-reactive proteins with anti-human IgA/IgM/IgG were recorded. Out of these 14 proteins only 8 proteins (encoded as: MAL 1, MAL 4, MAL 5, MAL 6, MML 5, MML 6, MML 7 and MML 8) were identified to be M. leprae specific by MALDI-TOF/TOF and MASCOT search. Of these 8 proteins, 1 protein was identified as bacterioferritin (MMP-II) and remaining 7 proteins appeared to be as 5 isoforms of major membrane protein-1 (MMP-1), a 35kDa protein. To our knowledge this is the first report regarding existence of isoforms of MMP-1/35kDa protein. On preliminary examination for serological potential of these antigens, MAL5 was found to be the best (sensitivity= 82.6 specificity = 54.6%, efficiency= 68.9%) among IgA reactive proteins. On the other hand, antigen encoded as MML7 was best (sensitivity 90.9% with a specificity of 33.3%, efficiency of 70.6) among IgM reactive proteins. Though seroreactive, as such both of these antigens do not seem to be very promising serodiagnostic reagents due to their low specificities, information obtained out of this study has opened paths towards dissection of these proteins at the peptide level with the view to look for their efficient serological potential.Keywords:
Mycobacterium leprae
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Proteins secreted by strains of Mycobacterium tuberculosis during short-term, zinc-sufficient batch culture were identified in order to define antigens likely to be relevant to the pathogenesis of human disease. [35S]Methionine-labelled proteins in supernatants of 4-7 d cultures were separated by PAGE under both denaturing and non-denaturing conditions, and the position of labelled material was determined. Secreted protein patterns of M. tuberculosis were quite similar to those of Bacillus Calmette-Guérin (BCG) but differed by the absence of the 46 kDa dimeric protein specific to BCG and by the presence in large amounts of a 23 kDa protein which, when denatured, gave 13 kDa subunits. This 13 kDa subunit protein constituted up to 20% of secreted proteins in classical strains of M. tuberculosis of phage type B but was not detected in phage type I strains from South India. This may be relevant to the different pathogenicity of these strains. Western blot analysis showed that antigens defined in supernatants of short-term (3 d) cultures of M. tuberculosis constituted a small subset of those seen in supernatants of organisms cultured for longer periods. One of the secreted proteins has the interesting property of binding to fibronectin. The available monoclonal antibodies and antisera have been used to identify lines on immunoblots corresponding to the secreted/released antigens of M. tuberculosis. The present findings suggest that there are major secreted antigens to which antibodies do not yet appear to have been produced experimentally.
Secretory protein
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Monoclonal antibodies (MAbs) previously shown to recognize distinct epitopes selectively expressed on the surface of some Mycoplasma hyorhinis strains were used to define two discrete sets of lipid-modified membrane surface proteins showing marked size variation within this species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of Triton X-114 phase-fractionated proteins from six isolates of M. hyorhinis defined a set of amphiphilic integral membrane proteins of 23, 50, and 55 kilodaltons (kDa) recognized on respective isolates by one MAb and a second set of integral proteins of 88, 120, and 100 to 150 kDa recognized by another MAb. The first group of proteins all contained a common, amphiphilic 18-kDa limit tryptic polypeptide bearing the epitope. The size- and strain-variant surface antigens identified by the MAbs were shown to be lipid-modified proteins. Phase fractionation of [3H]palmitate-labeled organisms revealed numerous 3H-labeled proteins in all isolates, which partitioned exclusively into the hydrophobic phase. These proteins generally showed pronounced size variation among isolates and included the antigen variants recognized by the two MAbs, as demonstrated directly by immunoprecipitation of correspondingly sized 3H-labeled proteins from each isolate. A third MAb recognized an invariant, lipid-associated surface protein of 70 kDa on all M. hyorhinis isolates. Covalent modification of lipid-associated proteins was confirmed by identifying 3H-labeled methyl palmitate after acid methanolysis of Triton X-114 phase proteins derived from [3H]palmitate-labeled organisms. However, removal of covalently bound lipid from chloroform-methanol-extracted proteins by alkaline hydroxylamine was selective; complete removal was observed with only a few proteins, possibly including the 120-kDa form of one antigen variant. This suggested potential differences in the nature of covalent linkage among lipid-modified M. hyorhinis surface antigens. Intraspecies antigen variants described here in M. hyorhinis share some characteristics with size-variant antigens reported in phylogenetically related gram-positive eubacteria and may contribute to phenotypic diversification and differences in pathogenicity of mycoplasmas.
Immunoprecipitation
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Mitochondrial inner membrane proteins extracted from beef heart tissue were examined for reactivity to antimitochondrial antibody (AMA) present in sera of patients with primary biliary cirrhosis (PBC) by an immunoblotting technique. Four proteins, which reacted with AMA, had molecular masses of 70 kDa, 50 kDa, 47 kDa and 40 kDa, as defined by their relative mobility (Rf) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All sera of 114 PBC patients were positive with at least one and as many as four of the mitochondrial proteins. The major antigenic proteins of mitochondrial inner membrane to which AMA reacts were the 70 kDa and 47 kDa proteins. All PBC sera containing antibodies to the 50 kDa and/or 40 kDa proteins reacted with 70 kDa as well. The isolation of antigen reacting with AMA of PBC is important to warrant further study of AMA and the cause of the disease. The isolation of responsible antigens had been difficult because the four antigens were insoluble. However, the antigen newly found by us, the 36 kDa fragment, obtained by partial trypsin digestion, is soluble. Using several procedures, the antigenic protein target of AMA was purified from mitochondria for the first time. We determined the N-terminal sequence of the soluble 36 kDa fragment, 25 residues in length. Until now the N-terminal sequence of the 36 kDa protein has not shown significant homology with any known protein. The present results of antigen purification would contribute to the elucidation of the epitopes of AMA antigen.
Primary Biliary Cirrhosis
Molecular mass
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The characterization of membrane proteins having no identified function in Mycobacterium tuberculosis is important for a better understanding of the biology of this pathogen. In this work, the biological activity of the Rv2560 protein was characterized and evaluated. Primers used in PCR and RT‐PCR assays revealed that the gene encoding protein Rv2560 is present in M. tuberculosis complex strains, but transcribed in only some of them. Sera obtained from rabbits inoculated with polymer peptides from this protein recognized a 33 kDa band in the M. tuberculosis lysate and a membrane fraction corresponding to the predicted molecular mass (33.1 kDa) of this protein. Immunoelectron microscopy analysis found this protein on the mycobacterial membrane. Sixteen peptides covering its entire length were chemically synthesized and tested for their ability to bind to A549 and U937 cells. Peptide 11024 (121 VV ALS D RATTAYTNTSG VS S140) showed high specific binding to both cell types (dissociation constants of 380 and 800 n m , respectively, and positive receptor–ligand interaction cooperativity), whereas peptide 11033 (284LIGIPVAALIHVYTYRKLSGG304) displayed high binding activity to A549 cells only. Cross‐linking assays showed the specific binding of peptide 11024 to a 54 kDa membrane protein on U937. Invasion inhibition assays, in the presence of shared high‐activity binding peptide identified for U937 and A549 cells, presented maximum inhibition percentages of 50.53% and 58.27%, respectively. Our work highlights the relevance of the Rv2560 protein in the M. tuberculosis invasion process of monocytes and epithelial cells, and represents a fundamental step in the rational selection of new antigens to be included as components in a multiepitope, subunit‐based, chemically synthesized, antituberculosis vaccine.
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U937 cell
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The 18-kDa protein of Mycobacterium leprae was purified from recombinant plasmids pUL108 and pML-3 grown in Saccharomyces cerevisiae and Escherichia coli, respectively. Significant lymphoproliferative responses were observed when T cells from immunized mice were challenged in culture with purified 18-kDa protein. Synthetic peptides have been prepared that span most of the 148 amino acid residues that constitute the sequence of the 18-kDa protein and used to map epitopes recognized by T cells. When mice were immunized with 18-kDa protein and lymph node cells subsequently prepared and challenged in microculture proliferative assays by using synthetic peptides, only one region of the intact protein appeared stimulatory. This T cell epitope was located between residues 116 and 121, adjacent to an epitope between residues 110 and 115 which we have previously shown to bind the L5 mAb. Immunization of mice with peptides, and subsequent challenge of lymph node cells in assays by using the 18-kDa protein as Ag revealed that residues 111-125 were the most effective in priming responses. Furthermore, the ability of 18-kDa primed lymph node cells to recognize determinants on both M. leprae and Mycobacterium tuberculosis indicates that in addition to possessing an M. leprae-specific B cell determinant, the 18-kDa protein contains a cross-reactive T cell epitope(s).
Mycobacterium leprae
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Cellular immune responses are influential for protection against intracellular bacteria such as brucellae. Therefore, identification of Brucella abortus antigens that activate primed bovine lymphocytes is fundamental for discerning the breadth of cellular response in bovine brucellosis. Potentially antigenic components of B. abortus S19 were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by nitrocellulose blotting. Specific one-dimensional blot segments induced proliferation of peripheral blood lymphocytes from all 25 of the vaccinated cattle tested and were defined as immunodominant. Individual proteins that stimulated lymphocyte proliferation were further characterized by two-dimensional cellular immunoblotting by two different approaches. Individual one-dimensional stimulatory blot segments were eluted, concentrated, and then subjected to two-dimensional cellular immunoblotting. Alternatively, entire two-dimensional gels containing all of the B. abortus components were blotted and nitrocellulose sections containing individual proteins were assayed for lymphocyte activation. Thirty-eight Brucella proteins that induced lymphocyte proliferation were resolved by both procedures. Phenotypic analysis of the proliferating cell population demonstrated the presence of CD4+, CD8+, and immunoglobulin M+ lymphocytes. Two immunogenic proteins, 12 and 31 kDa, identified by two-dimensional cellular immunoblotting, were subjected to partial N-terminal amino acid analysis. The 12-kDa protein was within the area of greatest lymphocyte proliferation, while the 31-kDa protein was chosen for comparison with a 31-kDa protein previously reported by others. A search of the National Biomedical Research Foundation protein data bank showed that the sequences were not homologous with other known proteins. Identification of Brucella proteins immunogenic for bovine lymphocytes provides an important step in distinguishing the various proteins involved in pathogenicity and/or disease resistance.
Cellular immunity
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Creatine kinase
Epitope mapping
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Mycobacterium leprae
Molecular mimicry
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We present evidence that a highly purified pepsin extract of type 5 streptococcal M protein (pep M5) contains at least three epitopes that are cross-reactive with sarcolemmal membrane proteins of human myocardium. The tissue-cross-reactive determinants of pep M5 are also partially shared with pep M6 and pep M19. Three rabbits immunized with a single 300 micrograms dose of pep M5 developed significant levels of heart-cross-reactive antibodies, as determined by indirect immunofluorescence tests. All three sera also contained antibodies that cross-reacted with pep M6 and pep M19. The heart tissue--specific antibodies that were eluted from sarcolemmal membranes opsonized types 5, 6, and 19 streptococci, indicating that they were directed against protective M protein epitopes on the surface of virulent organisms. Immunofluorescence inhibition tests, using purified M proteins as soluble inhibitors of heart-cross-reactive antibodies, revealed the number and M protein serotype distribution of the tissue-cross-reactive epitopes. Immunoblot analyses demonstrated the sarcolemmal membrane proteins containing the various cross-reactive antigenic determinants.
Immunofluorescence
Streptococcus Pyogenes
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