title: Homology of the 70-kilodalton antigens from Mycobacterium leprae and Mycobacterium bovis with the Mycobacterium tuberculosis 71-kilodalton antigen and with the conserved heat shock protein 70 of eucaryotes.
abstract: Two lambda gt11 recombinant clones, JKL2 and JKL15, each containing an insert coding for part of the highly immunogenic 70-kilodalton (kDa) protein antigen, were isolated from a Mycobacterium leprae genomic library by immunoscreening with the monoclonal antibody L7. Clone JKL2 contained the largest insert, 2.3 kilobase pairs. Nonoverlapping fragments of this insert were used as probes and showed strong hybridization to a number of Mycobacterium tuberculosis-lambda gt11 recombinants producing proteins recognized by an anti-M. tuberculosis 71-kDa monoclonal antibody, IT11. One clone from a recombinant Mycobacterium bovis library was also characterized by using L7, and the insert from this clone, B5bt, hybridized strongly to the M. leprae probes as well. The nucleotide sequence of the 1,037-base-pair coding region of the JKL2 M. leprae clone which encodes the carboxy-terminal half of the 70-kDa protein had extensive homology with genes from a number of species. In all cases, these genes, including the recently described Ag63 and Ag361 of Plasmodium falciparum, were found to be members of the heat shock protein 70 (hsp 70) family of genes. At the amino acid level, homology was maximal between amino acids 83 through 107 and 159 through 184, which showed extreme conservation (92 and 85% identity) with Escherichia coli DnaK amino acids 386 through 409 and 460 through 485, respectively, and was 51% homologous over the entire coding region (amino acids 1 through 344 of JKL2). In contrast, amino acids 129 through 158 had maximal homology with the phylogenetically more distant Xenopus laevis hsp70. Homology declined substantially in the carboxy-terminal 34 amino acids. The predicted ATP-binding functional activity of the 70-kDa antigen from M. bovis was confirmed with affinity purification of the antigen by binding to ATP-agarose and elution with ATP. In view of the conservation of sequences between these mycobacterial antigens and mammalian endogenous cellular enzymes, further evaluation of these molecules in vivo may aid in understanding tolerance to self-antigens as well as provide potentially useful immunodiagnostic reagents.
authors.affiliation: Clinical Immunology Research Centre, University of Sydney, New South Wales, Australia.
authors.forename: R J
authors.initials: RJ
authors.lastname: Garsia
authors.forename: L
authors.initials: L
authors.lastname: Hellqvist
authors.forename: R J
authors.initials: RJ
authors.lastname: Booth
authors.forename: A J
authors.initials: AJ
authors.lastname: Radford
authors.forename: W J
authors.initials: WJ
authors.lastname: Britton
authors.forename: L
authors.initials: L
authors.lastname: Astbury
authors.forename: R J
authors.initials: RJ
authors.lastname: Trent
authors.forename: A
authors.initials: A
authors.lastname: Basten
mesh_terms: D000255:Adenosine Triphosphate; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D002846:Chromatography, Affinity; D004269:DNA, Bacterial / Q000302:isolation & purification; D004274:DNA, Recombinant; D005057:Eukaryotic Cells; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009163:Mycobacterium bovis / Q000235:genetics / Q000276:immunology*; D009166:Mycobacterium leprae / Q000235:genetics / Q000276:immunology*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology*; D012689:Sequence Homology, Nucleic Acid
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D006360:Heat-Shock Proteins; D000255:Adenosine Triphosphate