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.

Introduction

The cystatins are a family of cysteine protease inhibitors that belong to family I25 in the MEROPS peptidase database classification (Rawlings et al., 2004), and they can interact with and neutralize cathepsin cysteine proteases, which play important roles in many physiological processes, including protein degradation, arthritis, tumor invasion, metastasis, antigen presentation, bone resorption, and muscular dystrophy (Turk and Bode, 1991, Chapman et al., 1997, Otto and Schirmeister, 1997, Sol-Church et al., 2002, Rudenskaya and Pupov, 2008, Turk et al., 2008).

The cystatins are further divided into three families, the type 1 (stefin family), type 2 (cystatin family), and type 3 (kininogen family) families (Rawlings and Barrett, 1990, Turk and Bode, 1991, Abrahamson et al., 2003). The type 1 cystatin family is composed of stefin A and B (also known as cystatin A and B), which are intracellular cystatins that are present in the cytosol. Type 1 cystatins are single-chain polypeptides of about 100 residues and about 11 kDa in size, with neither disulphide bonds nor carbohydrate side chains (Barrett, 1986, Turk and Bode, 1991).

Cystatin B (stefin B) is an endogenous inhibitor of cysteine protease localized in the lysosomes, cytoplasm and the nucleus (Riccio et al., 2001). Mutations in the stefin B gene cause progressive myoclonus epilepsy of type 1 (EPM1), also known as Unverricht–Lundborg disease (Pennacchio et al., 1996). Cystatin B-deficient mice are likely to undergo anti-apoptotic processes in the cerebellum (Pennacchio et al., 1998, Di Giaimo et al., 2002, Lehesjoki, 2003).

Piscine cystatin B has been identified in several species, including zebrafish (Padhi et al., 2004), turbot (Scophthalmus maximus; Xiao et al., 2010), Atlantic cod (Gadus morhua, Rajan et al., 2011), and rock bream (Oplegnathus fasciatus, Premachandra et al., 2012) with molecular and typical inhibitory assays. However, there is a paucity of reports focused on the biochemical characterization and enzymatic properties of cystatin B in teleosts. Olive flounder, Paralichthys olivaceus, is an economically-important mariculture fish species in Asian countries like China, Japan, and Korea, and is a well-studied teleost model species for enzymatic characterization of the cathepsin family members, including cathepsins S (Kim et al., 2010), K (Je et al., 2009), F (Ahn et al., 2009), and X (Ahn et al., 2008). For these reasons, we cloned the cystatin B cDNA from olive flounder and analyzed the expression of cystatin B in various tissues. We also expressed recombinant fish cystatin B from olive flounder in E. coli cells, and carried out the enzymatic characterization of the purified recombinant cystatin B protein, with piscine cathepsins as substrates, to explore the potential roles of cystatin B in the regulation of cathepsins.