Molecular cloning, mRNA expression and enzymatic characterization of cathepsin F from olive flounder (Paralichthys olivaceus).
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.
Ahn
Sang Jung
SJ
Department of Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea.
Kim
Na Young
NY
Seo
Jung Soo
JS
Je
Ju Eun
JE
Sung
Ji Hea
JH
Lee
Sang Hwan
SH
Kim
Moo-Sang
MS
Kim
Joong Kyun
JK
Chung
Joon Ki
JK
Lee
Hyung Ho
HH
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
D018076:DNA, Complementary; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D056646:Cathepsin F