Molecular aspects and functional characterization of cystatin B and Glutaredoxin-1 from two commercially important teleostean species interpreting the impact on host immunity.

Abstract

Aquaculture has become one of the leading world industries with its rapid growth arising from the demand from the vast consumption market. However, fueled by rising domestic income, consumers in emerging economies are experiencing a diversification of the types of available fish through an increase in fishery imports. This significant growth in fish consumption has enhanced people's diets around the world through diversified and nutritious food. Fish is usually high in unsaturated fats and provides health benefits in protection against cardiovascular diseases. It also aids fetal and infant development of the brain and nervous system. With its valuable nutritional properties, it can also play a major role in correcting unbalanced diets and, through substitution, in countering obesity. In 2013, fish accounted for about 17 percent of the global population's intake of animal protein and 6.7 percent of all protein consumed. With the aforementioned concept on mind, the current research was conducted to provide an insight into the immune system of two commercially important fish species. Cystatins are a large superfamily of proteins involved in the competitive reversible inhibition of C1 class cysteine proteases. Plant derived papain proteases and cysteine cathepsins are the major cysteine proteases that interact with cystatins. Cystatins superfamily can be further clustered into three groups as Stefins, Cystatins and Kininogens. Among them cystatin B is placed under Stefins. The cystatin B lacks a signal sequence, disulfide bonds or carbohydrate groups. However, it contains the conserved cystatin family signature including single cystatin like domain, cysteine protease inhibitory signature concealing pantapeptide (QXVXG) consensus sequence and N-terminal two conserved neighboring glycine (8GG9) residues. In the current study, a member of cystatin B was identified from Korean black rockfish (Sebastes schlegeli) using a cDNA database and designated as RfCytB. The full length cDNA of RfCytB consisted of 573 bp, with a coding region of 294 bp. The 5´-untranslated region (UTR) comprised of 55 bp, and 3´-UTR of 263 bp consisting a polyadenylation signal sequence and a polyA tail. The coding sequence encodes a polypeptide consisting of 97 amino acids with a predicted molecular weight of 11 kDa and theoretical isoelectric point of 6.3. The RfCytB shared homology features with other teleost and vertebrate species and was clustered with the Cystatin family 1 in our phylogenetic reconstruction. RfCytB was ubiquitously expressed in all tissue types of healthy animals with gill and spleen being the highest. Temporal expression of RfCytB displayed significant up-regulation upon infection with Aeromonas salmonicida. Recombinantly expressed RbCytB showed concentration dependent papain inhibitory activity with high thermal stability and transient expression of RfCytB in LPS activated murine macrophages induced the expression of genes related to pro-inflammatory conditions, such as iNOS and TNF α, providing evidence for its protease inhibitory and immunity relevant roles in host, respectively. Reactive oxygen species (ROS) can be generated as by-products in all oxygenic organisms during aerobic metabolism. External or internal oxidants such as superoxide, hydrogen peroxide, and hydroxyl radicals, referred to as ROS, can result in oxidative damage to proteins, lipids, and nucleic acids. Cells possess many antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (Gpx), thioredoxin peroxidase (Tpx), glutaredoxin (Grx), and thioredoxin (Trx) systems, to resist oxidative damage. Grxs and Trxs play important roles in maintaining intracellular thiolredox homeostasis by scavenging reactive oxygen species. However, only a few Grxs have been functionally characterized in teleostean species. In this study, we identified Glutaredoxin 1 (GLRX1) from Rock bream and investigated their connection to antioxidant defense. In the current study, a member of Glutaredoxin 1 was identified from Korean black rockfish (Sebastes schlegeli) using a cDNA database and designated as RbGLRX1. The full length cDNA of RbGLRX1 consisted of 833 bp, with a coding region of 318 bp. The 5´- untranslated region (UTR) comprised of 24 bp, and 3´-UTR of 491 bp. The coding sequence encodes a polypeptide consisting of 105 amino acids with a predicted molecular weight of 11.5 kDa and theoretical isoelectric point of 7.65. The RbGLRX1 shared homology features with other teleost and vertebrate species and was clustered mainly with other teleostean species in our phylogenetic reconstruction. RbGLRX1 was ubiquitously expressed in all tissue types of healthy animals with blood and liver being the highest. Temporal expression of RbGLRX1 displayed significant up-regulation upon infection with Edwardsiella tarda, Streptococcus iniae and by the rock bream iridovirus (RBIV). Recombinantly expressed RbGLRX1 exhibited concentration dependent alkyl and DPPH radical scavenging activities. Functional studies of RbGLRX1 gave positive results for insulin disulfide assay and classical HED assay thus proving its functional characteristics. Overall, the research provided valuable information on the role of RbGLRX1 on host immune defense against ROS.