Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis
Abstract Cathepsin L is an important cysteine protease, but its function in T. spiralis remains unclear. The aim of this research was to explore the biological characteristics of T. spiralis cathepsin L (TsCatL) and its role in T. spiralis-host interactions. Bioinformatic analysis revealed the prese...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2022-06-01
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| Series: | Veterinary Research |
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| Online Access: | https://doi.org/10.1186/s13567-022-01065-6 |
| _version_ | 1818257055926976512 |
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| author | Ruo Dan Liu Xiang Yu Meng Chen Le Li Shao Rong Long Jing Cui Zhong Quan Wang |
| author_facet | Ruo Dan Liu Xiang Yu Meng Chen Le Li Shao Rong Long Jing Cui Zhong Quan Wang |
| author_sort | Ruo Dan Liu |
| collection | DOAJ |
| description | Abstract Cathepsin L is an important cysteine protease, but its function in T. spiralis remains unclear. The aim of this research was to explore the biological characteristics of T. spiralis cathepsin L (TsCatL) and its role in T. spiralis-host interactions. Bioinformatic analysis revealed the presence of the cysteine protease active site residues Gln, Cys, His and Asn in mature TsCatL, as well as specific motifs of cathepsin L similar to ERFNIN and GYLND in the prepeptide of TsCatL. Molecular docking of mature TsCatL and E64 revealed hydrophobic effects and hydrogen bonding interactions. Two domains of TsCatL (TsCatL2) were cloned and expressed, and recombinant TsCatL2 (rTsCatL2) was autocatalytically cleaved under acidic conditions to form mature TsCatL. TsCatL was transcribed and expressed in larvae and adults and located in the stichosome, gut and embryo. Enzyme kinetic tests showed that rTsCatL2 degraded the substrate Z-Phe-Arg-AMC under acidic conditions, which was inhibited by E64 and PMSF and enhanced by EDTA, L-cysteine and DTT. The kinetic parameters of rTsCatL2 were a Km value of 48.82 μM and Vmax of 374.4 nM/min at pH 4.5, 37 °C and 5 mM DTT. In addition, it was shown that rTsCatL2 degraded haemoglobin, serum albumin, immunoglobulins (mouse IgG, human IgG and IgM) and extracellular matrix components (fibronectin, collagen I and laminin). The proteolytic activity of rTsCatL2 was host specific and significantly inhibited by E64. rTsCatL2 possesses the natural activity of a sulfhydryl-containing cysteine protease, and TsCatL is an important digestive enzyme that seems to be important for the nutrient acquisition, immune evasion and invasion of Trichinella in the host. |
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| institution | Directory Open Access Journal |
| issn | 1297-9716 |
| language | English |
| last_indexed | 2024-12-12T17:37:34Z |
| publishDate | 2022-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Veterinary Research |
| spelling | doaj.art-8e48cbf575d74c169c3b1a4778aae1692022-12-22T00:17:10ZengBMCVeterinary Research1297-97162022-06-0153111910.1186/s13567-022-01065-6Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralisRuo Dan Liu0Xiang Yu Meng1Chen Le Li2Shao Rong Long3Jing Cui4Zhong Quan Wang5Department of Parasitology, Medical College, Zhengzhou UniversityDepartment of Parasitology, Medical College, Zhengzhou UniversityDepartment of Parasitology, Medical College, Zhengzhou UniversityDepartment of Parasitology, Medical College, Zhengzhou UniversityDepartment of Parasitology, Medical College, Zhengzhou UniversityDepartment of Parasitology, Medical College, Zhengzhou UniversityAbstract Cathepsin L is an important cysteine protease, but its function in T. spiralis remains unclear. The aim of this research was to explore the biological characteristics of T. spiralis cathepsin L (TsCatL) and its role in T. spiralis-host interactions. Bioinformatic analysis revealed the presence of the cysteine protease active site residues Gln, Cys, His and Asn in mature TsCatL, as well as specific motifs of cathepsin L similar to ERFNIN and GYLND in the prepeptide of TsCatL. Molecular docking of mature TsCatL and E64 revealed hydrophobic effects and hydrogen bonding interactions. Two domains of TsCatL (TsCatL2) were cloned and expressed, and recombinant TsCatL2 (rTsCatL2) was autocatalytically cleaved under acidic conditions to form mature TsCatL. TsCatL was transcribed and expressed in larvae and adults and located in the stichosome, gut and embryo. Enzyme kinetic tests showed that rTsCatL2 degraded the substrate Z-Phe-Arg-AMC under acidic conditions, which was inhibited by E64 and PMSF and enhanced by EDTA, L-cysteine and DTT. The kinetic parameters of rTsCatL2 were a Km value of 48.82 μM and Vmax of 374.4 nM/min at pH 4.5, 37 °C and 5 mM DTT. In addition, it was shown that rTsCatL2 degraded haemoglobin, serum albumin, immunoglobulins (mouse IgG, human IgG and IgM) and extracellular matrix components (fibronectin, collagen I and laminin). The proteolytic activity of rTsCatL2 was host specific and significantly inhibited by E64. rTsCatL2 possesses the natural activity of a sulfhydryl-containing cysteine protease, and TsCatL is an important digestive enzyme that seems to be important for the nutrient acquisition, immune evasion and invasion of Trichinella in the host.https://doi.org/10.1186/s13567-022-01065-6Trichinella spiraliscathepsin Lcysteine proteaseenzymatic characterizationinhibitor |
| spellingShingle | Ruo Dan Liu Xiang Yu Meng Chen Le Li Shao Rong Long Jing Cui Zhong Quan Wang Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis Veterinary Research Trichinella spiralis cathepsin L cysteine protease enzymatic characterization inhibitor |
| title | Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis |
| title_full | Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis |
| title_fullStr | Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis |
| title_full_unstemmed | Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis |
| title_short | Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis |
| title_sort | molecular characterization and determination of the biochemical properties of cathepsin l of trichinella spiralis |
| topic | Trichinella spiralis cathepsin L cysteine protease enzymatic characterization inhibitor |
| url | https://doi.org/10.1186/s13567-022-01065-6 |
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