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Cloning, expression analysis and enzymatic characterization of cathepsin S from olive flounder (Paralichthys olivaceus).

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.
AuthorsNa Young Kim, Sang Jung Ahn, A Ram Lee, Jung Soo Seo, Moo-Sang Kim, Joong Kyun Kim, Joon Ki Chung, Hyung Ho Lee
JournalComparative 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
PMID20601061 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright 2010 Elsevier Inc. All rights reserved.
Chemical References
  • Fish Proteins
  • Recombinant Proteins
  • Cathepsins
  • cathepsin S
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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