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Molecular and immunological characterization of the highly conserved antigen 84 from Mycobacterium tuberculosis and Mycobacterium leprae.

ABSTRACT: Crossed immunoelectrophoresis (CIE) has been used to develop a reference system for classifying mycobacterial antigens. The subsequent use of specific antibodies allowed further determination of antigens by molecular weight. The monoclonal antibody F126-2, originally raised against a 34-kDa antigen of Mycobacterium kansasii, reacted with antigen 84 (Ag84) in the CIE reference system for Mycobacterium bovis BCG and Mycobacterium tuberculosis. To characterize Ag84, we screened a lambda gt11 gene library from M. tuberculosis with antibody F126-2 and identified the encoding gene. The corresponding Mycobacterium leprae Ag84 gene was subsequently selected from a cosmid library, using the M. tuberculosis gene as a probe. Both genes were expressed as 34-kDa proteins in Escherichia coli, and the recombinant proteins indeed corresponded to Ag84 in the CIE reference system. The derived amino acid sequences of the M. tuberculosis and M. leprae proteins showed 85% identity, which indicates that Ag84 constitutes a group of highly conserved mycobacterial antigens. Antibodies of almost 60% of lepromatous leprosy patients responded to Ag84, indicating that the protein is highly immunogenic following infection in multibacillary leprosy.

SUBMITTER: Hermans PW

PROVIDER: S-EPMC173095 | biostudies-other | 1995 Mar

REPOSITORIES: biostudies-other

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Molecular and immunological characterization of the highly conserved antigen 84 from Mycobacterium tuberculosis and Mycobacterium leprae.

Hermans P W PW Abebe F F Kuteyi V I VI Kolk A H AH Thole J E JE Harboe M M

Infection and immunity 19950301 3

Crossed immunoelectrophoresis (CIE) has been used to develop a reference system for classifying mycobacterial antigens. The subsequent use of specific antibodies allowed further determination of antigens by molecular weight. The monoclonal antibody F126-2, originally raised against a 34-kDa antigen of Mycobacterium kansasii, reacted with antigen 84 (Ag84) in the CIE reference system for Mycobacterium bovis BCG and Mycobacterium tuberculosis. To characterize Ag84, we screened a lambda gt11 gene l ...[more]

PMID: 7868268

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Project description:A novel immunogenic antigen, the 6-kDa early secretory antigenic target (ESAT-6), from short-term culture filtrates of Mycobacterium tuberculosis was purified by hydrophobic interaction chromatography and anion-exchange chromatography by use of fast protein liquid chromatography. The antigen focused at two different pIs of 4.0 and 4.5 during isoelectric focusing, and each of these components separated into three spots ranging from 4 to 6 kDa during two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent differences in molecular masses or pIs of these isoforms were not due to posttranslational glycosylation. The molecular weight of the purified native protein was determined by applying gel filtration and nondenaturing polyacrylamide gel electrophoresis and found to be 24 kDa. ESAT-6 is recognized by the murine monoclonal antibody HYB 76-8, which was used to screen a recombinant lambda gt11 M. tuberculosis DNA library. A phage expressing a gene product recognized by HYB 76-8 was isolated, and a 1.7-kbp fragment of the mycobacterial DNA insert was sequenced. The structural gene of ESAT-6 was identified as the sequence encoding a polypeptide of 95 amino acids. The N terminus of the deduced sequence could be aligned with the 10 amino-terminal amino acids derived from sequence analyses of the native protein. N-terminal sequence analysis showed that the purified antigen was essentially free from contaminants, and the amino acid analysis of the antigen was in good agreement with the DNA sequence-deduced amino acid composition. Thus, the heterogeneities observed in the pI and molecular weight of the purified antigen do not derive from contaminating proteins but are most likely due to heterogeneity of the antigen itself. Native and recombinant ESAT-6 are immunologically active in that both elicited a high release of gamma interferon from T cells isolated from memory-immune mice challenged with M. tuberculosis. Analyses of subcellular fractions of M. tuberculosis showed the presence of ESAT-6 in cytosol- and cell wall-containing fractions. Interspecies analyses showed the presence of ESAT-6 in filtrates from M. tuberculosis complex species. Among filtrates from mycobacteria not belonging to the M. tuberculosis complex, reactivity was observed in Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum.

Dataset Information

Molecular and immunological characterization of the highly conserved antigen 84 from Mycobacterium tuberculosis and Mycobacterium leprae.

Publications

Molecular and immunological characterization of the highly conserved antigen 84 from Mycobacterium tuberculosis and Mycobacterium leprae.

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