title: Identification and Characterization of Lipase Activity and Immunogenicity of LipL from Mycobacterium tuberculosis.
abstract: Lipids and lipid-metabolizing esterases/lipases are highly important for the mycobacterial life cycle and, possibly, for mycobacterial virulence. In this study, we expressed 10 members of the Lip family of Mycobacterium tuberculosis. Among the 10 proteins, LipL displayed a significantly high enzymatic activity for the hydrolysis of long-chain lipids. The optimal temperature for the lipase activity of LipL was demonstrated to be 37°C, and the optimal pH was 8.0. The lipase active center was not the conserved motif G-x-S-x-G, but rather the S-x-x-K and GGG motifs, and the key catalytic amino acid residues were identified as G50, S88, and K91, as demonstrated through site-directed mutagenesis experiments. A three-dimensional modeling structure of LipL was constructed, which showed that the GGG motif was located in the surface of a pocket structure. Furthermore, the subcellular localization of LipL was demonstrated to be on the mycobacterial surface by Western blot analysis. Our results revealed that the LipL protein could induce a strong humoral immune response in humans and activate a CD8+ T cell-mediated response in mice. Overall, our study identified and characterized a novel lipase denoted LipL from M. tuberculosis, and demonstrated that LipL functions as an immunogen that activates both humoral and cell-mediated responses.
authors.affiliation: Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Cao
authors.affiliation: Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, PR China.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Dang
authors.affiliation: Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, PR China.
authors.forename: Huafang
authors.initials: H
authors.lastname: Li
authors.affiliation: Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, PR China.
authors.forename: Tiantian
authors.initials: T
authors.lastname: Li
authors.affiliation: Heilongjiang Provincial Hospital for Prevention and Treatment of Tuberculosis, Harbin, PR China.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Yue
authors.affiliation: Heilongjiang Provincial Hospital for Prevention and Treatment of Tuberculosis, Harbin, PR China.
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.affiliation: Heilongjiang Provincial Hospital for Prevention and Treatment of Tuberculosis, Harbin, PR China.
authors.forename: Yajun
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, PR China.
authors.forename: Siguo
authors.initials: S
authors.lastname: Liu
authors.affiliation: Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, PR China.
authors.forename: Liping
authors.initials: L
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D005798:Genes, Bacterial; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000276:immunology*; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D040681:Structural Homology, Protein; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
chemical_list: D003902:Detergents; D050505:Mutant Proteins; D008049:Lipase