title: Olive flounder (Paralichthys olivaceus) cystatin B: cloning, tissue distribution, expression and inhibitory profile of piscine cystatin B.
abstract: Among the cystatin superfamily, cystatin B, also known as stefin B, is an intracellular inhibitor that regulates the activities of cysteine proteases, such as papain and cathepsins. In this study, the 536 bp cystatin B cDNA (referred to hereafter as PoCystatin B) was cloned from olive flounder (Paralichthys olivaceus) using a combination of the rapid amplification of cDNA ends (RACE) approach and olive flounder cDNA library screening. To determine the tissue distribution of PoCystatin B mRNA, the expression of PoCystatin B in normal and lipopolysaccharide (LPS)-stimulated flounder tissues were compared with that of the inflammatory cytokines interleukin (IL)-1β, IL-6, and IL-8 by reverse transcription (RT)-polymerase chain reaction (PCR). The results of the RT-PCR analysis revealed ubiquitous PoCystatin B expression in normal and LPS-stimulated tissues. To characterize the enzymatic activity of PoCystatin B protein, recombinant PoCystatin B protein was overexpressed in Escherichia coli BL21(DE3) cells in the pCold™ TF DNA expression vector as a soluble fusion protein of 67-kDa. PoCystatin B inhibited papain cysteine protease, bovine cathepsin B, and fish cathepsins F and X to a greater extent, whereas fish cathepsins L, S, and K were inhibited to a lesser extent. These results indicate that the enzymatic characteristics of the olive flounder cystatin B are similar to those of mammalian cystatin B proteins, and provide a better understanding of the mechanisms of regulation of cathepsins and cystatins in marine organisms.
authors: Department of Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea.
authors: Sang Jung
authors: SJ
authors: Ahn
authors: Hye Jin
authors: HJ
authors: Bak
authors: Ju Hyeon
authors: JH
authors: Park
authors: Seon Ah
authors: SA
authors: Kim
authors: Na Young
authors: NY
authors: Kim
authors: Jin Young
authors: JY
authors: Lee
authors: Ji Hea
authors: JH
authors: Sung
authors: Soo Jin
authors: SJ
authors: Jeon
authors: Joon Ki
authors: JK
authors: Chung
authors: Hyung Ho
authors: HH
authors: Lee
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D055313:Cystatin B / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D018076:DNA, Complementary / Q000235:genetics; D005432:Flounder / Q000235:genetics*; D020869:Gene Expression Profiling; D009928:Organ Specificity; D010206:Papain / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D012333:RNA, Messenger; D055313:Cystatin B; D010206:Papain