title: Molecular cloning, mRNA expression and enzymatic characterization of cathepsin F from olive flounder (Paralichthys olivaceus).
abstract: Cathepsin F is a recently described papain-like cysteine protease of unknown function, and unique among cathepsins due to an elongated N-terminal pro-region, which contains a cystatin domain. In the present study, the cDNA of olive flounder (Paralichthys olivaceus) cathepsin F (PoCtF) was cloned by the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) approaches. The PoCtF gene was determined to consist of the 1844 bp nucleotide sequence which encodes for a 475-amino acid polypeptide. The results of RT-PCR analysis revealed ubiquitous expression throughout the entirety of healthy flounder tissues; however the PoCtF expressions increased significantly in gill at 3h post-injection with lipopolysaccharide (LPS). Also, immunostaining using anti-PoCtF antibody was strongest on the epidermal mucus in the fin. The cDNA encoding mature enzyme of PoCtF was expressed in Escherichia coli using the pGEX-4T-1 expression vector system. Its activity was quantified by cleaving the synthetic peptide Z-Phe-Arg-AMC, a substrate commonly used for functional characterization of cysteine proteinases, and the optimal pH for the protease activity was 7.5. The findings of the present study suggest that PoCtF has a higher optimum pH than mammalian cathepsin F, and PoCtF is an interesting target for future investigations of the role of cathepsin F in the epidermal mucus and fish innate immune system.
authors: Department of Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea.
authors: Sang Jung
authors: SJ
authors: Ahn
authors: Na Young
authors: NY
authors: Kim
authors: Jung Soo
authors: JS
authors: Seo
authors: Ju Eun
authors: JE
authors: Je
authors: Ji Hea
authors: JH
authors: Sung
authors: Sang Hwan
authors: SH
authors: Lee
authors: Moo-Sang
authors: MS
authors: Kim
authors: Joong Kyun
authors: JK
authors: Kim
authors: Joon Ki
authors: JK
authors: Chung
authors: Hyung Ho
authors: HH
authors: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D056646:Cathepsin F / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005432:Flounder; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D056646:Cathepsin F