Abstract |
Cathepsin S is a critical protease for the regulation of MHC class II immune responses, and thus is a potential target for developing immunosuppressive drugs in the pathogenesis of degenerative and autoimmune diseases. In this study, we cloned a cDNA encoding for cathepsin S (PoCtS) from the olive flounder, Paralichthys olivaceus. The 1170 bp PoCtS cDNA contained an open reading frame of 1014 bp, which consisted of a 25-residue putative signal peptide, a 96-residue propeptide and the 216-residue mature enzyme. The tissue-specific expression pattern of PoCtS, determined via RT-PCR and real-time PCR analysis, revealed ubiquitous expression throughout the entirety of healthy flounder tissues; however IL-1beta, IL-6, IL-8 and PoCtS expression increased significantly in muscle 6h post-injection of bacterial lipopolysaccharide (LPS). The cDNA encoding proenzyme of PoCtS was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in a pGEX-4T-1 vector. Also, the recombinant proPoCtS protein was overexpressed in E. coli BL21(DE3) as a 60 kDa fusion protein. Cathepsin S activity was detected through the cleavage of synthetic fluorogenic peptide substrates, such as Z- Val-Val- Arg-AMC and Z-Phe-Arg-AMC. The optimum pH for the protease activity was determined to be 8. This is the first report that characterized the enzymatic properties and analyzed the expression of piscine cathepsin S.
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Authors | Na Young Kim, Sang Jung Ahn, A Ram Lee, Jung Soo Seo, Moo-Sang Kim, Joong Kyun Kim, Joon Ki Chung, Hyung Ho Lee |
Journal | Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology
(Comp Biochem Physiol B Biochem Mol Biol)
Vol. 157
Issue 3
Pg. 238-47
(Nov 2010)
ISSN: 1879-1107 [Electronic] England |
PMID | 20601061
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Copyright | Copyright 2010 Elsevier Inc. All rights reserved. |
Chemical References |
- Fish Proteins
- Recombinant Proteins
- Cathepsins
- cathepsin S
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Topics |
- Amino Acid Sequence
- Animals
- Base Sequence
- Cathepsins
(genetics, metabolism)
- Cloning, Molecular
- Fish Proteins
(genetics, metabolism)
- Flounder
(genetics, metabolism)
- Gene Expression
- Molecular Sequence Data
- Phylogeny
- Recombinant Proteins
(metabolism)
- Tissue Distribution
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