Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica
Cathepsin L proteases are a major class of endopeptidases expressed at a high level in Fasciola parasites. Several isoforms of cathepsin L were detected and they may perform different functions during the parasite development. In this study, a complete cDNA encoding a cathepsin L protease was cloned...
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th-mahidol.318582018-10-19T12:14:43Z Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica Veerawat Sansri Narin Changklungmoa Pannigan Chaichanasak Prasert Sobhon Krai Meemon Mahidol University Immunology and Microbiology Medicine Cathepsin L proteases are a major class of endopeptidases expressed at a high level in Fasciola parasites. Several isoforms of cathepsin L were detected and they may perform different functions during the parasite development. In this study, a complete cDNA encoding a cathepsin L protease was cloned from a newly excysted juvenile (NEJ) cDNA library of Fasciola gigantica and named FgCatL1H. It encoded a 326 amino acid preproenzyme which shared 62.8-83.1% and 39.5-42.9% identity to Fasciola spp. and mammalian cathepsins L, respectively. All functionally important residues previously described for cathepsin L were conserved in FgCatL1H. Phylogenetic analysis demonstrated that FgCatL1H belonged to a distinct group, clade 4, with respect to adult and other juvenile Fasciola cathepsin L genes. FgCatL1H expression was detected by RT-PCR, using gene specific primers, in metacercariae and NEJ, and the expression gradually decreased in advanced developmental stages. A recombinant proFgCatL1H (rproFgCatL1H) was expressed in the yeast Pichia pastoris, affinity purified, and found to migrate in SDS-PAGE at approximately 47.6 and 38.3. kDa in glycosylated and deglycosylated forms, respectively. The molecular mass of the activated mature rFgCatL1H in glycosylated form was approximately 40.7. kDa. Immunoblotting and immunohistochemistry using rabbit antibodies against rproFgCatL1H showed that FgCatL1H was predominantly expressed in epithelial cells of the digestive tract of metacercariae, NEJs and juveniles of F. gigantica. FgCatL1H could cleave the synthetic fluorogenic substrate Z-Phe-Arg-MCA preferentially over Z-Gly-Pro-Arg-MCA at an optimum pH of 6.5. It also showed hydrolytic activity against native substrates, including type I collagen, laminin, and immunoglobulin G (IgG) in vitro, suggesting possible roles in host tissue migration and immune evasion. Therefore, the FgCatL1H is a possible target for vaccine and chemotherapy for controlling F. gigantica infection. © 2013 Elsevier B.V. 2018-10-19T05:00:56Z 2018-10-19T05:00:56Z 2013-10-01 Article Acta Tropica. Vol.128, No.1 (2013), 76-84 10.1016/j.actatropica.2013.06.013 18736254 0001706X 2-s2.0-84883741456 https://repository.li.mahidol.ac.th/handle/123456789/31858 Mahidol University SCOPUS https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84883741456&origin=inward |
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Immunology and Microbiology Medicine Veerawat Sansri Narin Changklungmoa Pannigan Chaichanasak Prasert Sobhon Krai Meemon Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica |
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Cathepsin L proteases are a major class of endopeptidases expressed at a high level in Fasciola parasites. Several isoforms of cathepsin L were detected and they may perform different functions during the parasite development. In this study, a complete cDNA encoding a cathepsin L protease was cloned from a newly excysted juvenile (NEJ) cDNA library of Fasciola gigantica and named FgCatL1H. It encoded a 326 amino acid preproenzyme which shared 62.8-83.1% and 39.5-42.9% identity to Fasciola spp. and mammalian cathepsins L, respectively. All functionally important residues previously described for cathepsin L were conserved in FgCatL1H. Phylogenetic analysis demonstrated that FgCatL1H belonged to a distinct group, clade 4, with respect to adult and other juvenile Fasciola cathepsin L genes. FgCatL1H expression was detected by RT-PCR, using gene specific primers, in metacercariae and NEJ, and the expression gradually decreased in advanced developmental stages. A recombinant proFgCatL1H (rproFgCatL1H) was expressed in the yeast Pichia pastoris, affinity purified, and found to migrate in SDS-PAGE at approximately 47.6 and 38.3. kDa in glycosylated and deglycosylated forms, respectively. The molecular mass of the activated mature rFgCatL1H in glycosylated form was approximately 40.7. kDa. Immunoblotting and immunohistochemistry using rabbit antibodies against rproFgCatL1H showed that FgCatL1H was predominantly expressed in epithelial cells of the digestive tract of metacercariae, NEJs and juveniles of F. gigantica. FgCatL1H could cleave the synthetic fluorogenic substrate Z-Phe-Arg-MCA preferentially over Z-Gly-Pro-Arg-MCA at an optimum pH of 6.5. It also showed hydrolytic activity against native substrates, including type I collagen, laminin, and immunoglobulin G (IgG) in vitro, suggesting possible roles in host tissue migration and immune evasion. Therefore, the FgCatL1H is a possible target for vaccine and chemotherapy for controlling F. gigantica infection. © 2013 Elsevier B.V. |
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Mahidol University |
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Mahidol University Veerawat Sansri Narin Changklungmoa Pannigan Chaichanasak Prasert Sobhon Krai Meemon |
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Article |
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Veerawat Sansri Narin Changklungmoa Pannigan Chaichanasak Prasert Sobhon Krai Meemon |
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Veerawat Sansri |
title |
Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica |
title_short |
Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica |
title_full |
Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica |
title_fullStr |
Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica |
title_full_unstemmed |
Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica |
title_sort |
molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin l of fasciola gigantica |
publishDate |
2018 |
url |
https://repository.li.mahidol.ac.th/handle/123456789/31858 |
_version_ |
1763495500785385472 |