Serine Proteases, Production And Clearance Of Reactive Oxygen Species And Two Cystein Proteases Function In Innate Immunity In Chinese White Shrimp (Fenneropenaeus Chinensis)

In China, Chinese white shrimp(Fenneropenaeus chinensis) was an economically important cultivation and was mainly distributed in Yellow and Bo sea. Recently, the cultivation of F. chinensis has been beset with serious problems linked to the viral outbreak such as white spot syndrome virus (WSSV), yellow head virus (YHV), and Taura syndrome virus (TSV), which have been regarded as the most serious shrimp viruses. Like most arthropods, shrimp relies on innate immunity. Innate immunity includes cellular and humoral responses. Cellular responses includes encapsulation, phagocytosis, and nodule formation while humoral responses include clotting, synthesis of antimicrobial peptides, and activation of the prophenoloxidase (proPO) system. Activation of proPO can induce the production of melanin. During the process of melanin synthesis, reactive oxygen species (ROS) is also produced as byproducts. Phagocytosis, as one of the cellular immunity, primarily by hemocytes, have important role in combating infection. Hemocytes kill invading bacteria through the production of large amounts of ROS, such as superoxide anion (02·-), hydroxyl radical (OH·) and hydrogen peroxide (H2O2), which become abundant in inflamed tissues following respiratory bursts in response to bacterial challenge. Because high levels of ROS can cause cytotoxicity to host cells, organisms have evolved different strategies for coping with the negative reactions of ROS. In this paper, we mainly focused on the shrimp serine proteases related to the proPO activation and we are also interesting in shrimp thioredoxin, GPx, GST and FABP, which function in the clearance of ROS. In addition, we study another two cystein protease, which may participation of shrimp innate immunity.1. Clip domain serine protease and its homolog respond to Vibrio challenge in Chinese white shrimp, Fenneropenaeus chinensisClip domain serine proteases and their homologs are involved in invertebrate innate immunity, including hemolymph coagulation, antimicrobial peptide synthesis, cell adhesion, and melanization. Recognition of pathogens by pattern recognition receptors can trigger activation of a serine protease cascade. We report here the cDNA cloning of a serine protease (FcSP) and a serine protease homolog (FcSPH) from Chinese white shrimp, Fenneropenaeus chinensis. Both FcSP and FcSPH possess a clip domain at the N-terminal and an SP or SP-like domain at the C-terminal. In contrast to FcSP, FcSPH lacks a catalytic residue and is catalytically inactive. Tissue distribution and time course qRT-PCR analysis indicates that FcSP and FcSPH can respond to Vibrio anguillarum challenge in hemocytes, hepatopancreas and intestine. In situ hybridization analysis shows that FcSP is distributed in hemocytes and gills, and originated mainly from the hemocytes. FcSPH protein is expressed in gills and stomach of non-challenged shrimp. Its expression in gill mainly originates from the hemocytes in it. Two immunoreactive bands of FcSP can be detected in gills and stomach of non-challenged shrimp. FcSP protein is partially cleaved in non-challenged shrimp, while FcSPH protein is unprocessed in unchallenged shrimp and is partially cleaved after V. anguillarum challenge. Our results suggest that this Clip domain serine protease and its homolog may be involved in the serine protease cascade and play an important role in innate immunity of the shrimp.2. A selenium-dependent glutathione peroxidase (Se-GPx) and two glutathione S-transferases (GSTs) from Chinese shrimp(Fenneropenaeus chinensis)Glutathione S-transferases (GSTs) and glutathione peroxidases (GPxs) are essential components of cellular detoxification systems that defend cells against reactive oxygen species (ROSs). Two GSTgenes have recently been cloned from Fenneropenaeus chinensis and BLAST P analysis shows that one GST, designated FcMuGST, is similar to members of MuGST while the other has similarities to ThetaGST (FcThetaGST). A selenium dependent glutathione peroxidase (Se-GPx) has also been cloned from F. chinensis. The alignment of the deduced GST and GPx amino acid sequences with those from other species showed that the residues essential for enzymatic function of these three proteins are highly conserved. Tissue distribution and response to pathogens for the three genes was investigated by RT-PCR analysis, which showed that the transcript of FcMuGST and FcGPx increased in response to Vibrio anguillarum infection, while FcThetaGST showed little change at the transcript level. GPx activity in gill tissues quickly increased at 6 h after V. anguillarum challenge and maintained at a relatively high level from 6 h to 24 h. Total GST activity in hepatopancreas and intestines of the bacterial challenged shrimp was increased at 6 h, and gradually recovered from 12 and 24 h to the normal level. These three genes were all predicted to play an important role in detoxification defense reactions. FcMuGST primarily scavenges excess ROS produced after bacterial infection, while clearance of endogenous hydrophobic electrophile molecules was mainly dependent on activities of FcThetaGST.3. A thioredoxin response to the WSSV challenge on the Chinese white shrimp, Fenneropenaeus chinensisThioredoxin (TRX) is involved in cell redox homeostasis. In addition, it is responsible for maintaining proteins in their reduced state. In our study, a Fenneropenaeus chinensis thioredoxin (FcTRX) gene was identified from the Chinese white shrimp. The full length of FcTRX was 777 bp, including a 60 bp 5'untranslated region (UTR), a 318 bp open reading frame (ORF) encoding a 105amino acids protein, and a 399 bp 3'UTR. FcTRX contained a TRX domain with a conserved motif of Cys-Gly-Pro-Cys (CGPC). No signal peptide was predicted by SMART analysis. The molecular mass and pI of FcTRX were 12 kDa and 4.62, respectively. FcTRX is awidely distributed gene, and its mRNA is detected in hemocytes, hearts, hepatopancreas, gills, stomach, and intestine from an unchallenged shrimp. The expression level of FcTRX was the highest in hepatopancreas, where it was down-regulated to the lowest level at 12 h white spot syndrome virus (WSSV) challenge. In the gills, itwent up to the highest level at 6 h. Western blot showed that FcTRX protein in hepatopancreas challengedwithWSSV was down-regulated from2 h to 12 h and then restored to the level similar to that of unchallenged shrimp at 24 h. In the gills challenged with WSSV, the FcTRX protein was up-regulated from 6 h to 24 h. Our research indicated its possible role in the anti-WSSV innate immunity of shrimps.4. A fatty acid binding protein (FcFABP) respond to microbial infection in Chinese white shrimp, Fenneropenaeus chinensisFatty acid-binding protein (FABP) are involved in lipid metabolism. FABPs, members of the lipid-binding protein superfamily, play an important role in lipid metabolism and also participate in vertebrate innate immunity. A cDNA of FABP (FcFABP) cloned from the hepatopancreas of the shrimp was 715 bp in size and encoded a 14 kDa protein. FcFABP appeared to be a basic fatty acid binding protein with a predicted isoelectric point of 9.16. It showed sequence similarity to both vertebrate and invertebrate FABPs. Phylogenetic analysis showed that FcFABP, together with LvFABP, were clustered into one group. FcFABP was detected mainly in the hepatopancreas and expression level increased after a challenge with WSSV. FcFABP was down-regulated by V. anguillarum challenge. The protein also had bacterial binding activity. This lipid metabolism related proteins may play important roles in shrimp innate immunity.5. Two Cysteine Proteinases respond to Bacterial and WSSV Challenge in Chinese white shrimp Fenneropenaeus chinensisCathepsin L (CathL) and legumain/asparaginyl endopeptidase (AEP) are two kinds of cysteine proteases (CPs). The cDNAs encoding CathL and legumain from Chinese white shrimp Fenneropenaeus chinensis (FcCathL, FcLegu) were obtained. FcCathL protein includes a signal peptide, an Inhibitor_I29 domain and a Pept_C1 domain. FcLegu protein has a signal peptide and a Peptidase_C13 domain. Both FcCathL and FcLegu mRNA were expressed mainly in the hepatopancreas of unchallenged shrimp. FcCathL was also detected in the stomach and intestine post 24 h Vibrio anguillarum challenge. Time-course analysis of FcCathL showed that FcCathL was up-regulated in the hepatopancreas of shrimp challenged with white spot syndrome virus (WSSV) at 12 h. There was no significant difference in the statistical analysis post Vibrio challenge. FcLegu mRNA in hepatopancreas was down-regulated by Vibrio. FcLegu transcript first declined from 2 h to 6 h and then recovered from 12 h to 24 h in hepatopancreas challenged with WSSV. FcCathL protein was detected in the hemocytes, hepatopancreas, gill, stomach, and intestine of unchallenged shrimp. Three bands of FcCathL protein detected in some tissues may represent preproenzyme, single chain and mature double chain form respectively. An additional band could be detected only in gills. Mature double chain of FcCathL could be detected in stomach only in shrimp challenged with WSSV for 24 h and in intestine post 24 challenge with V. anguillarum or WSSV. In hemocytes, only preproenzyme of FcCathL could be detected and it was up-regulated by Vibrio or WSSV. In intestine and gills, single chain of FcCathL was upregulated by Vibrio or WSSV. In stomach, FcCathL single chain was upregulated only by WSSV. FcLegu protein was detected in all detected tissues. In hepatopancreas, FcLegu was detected in the proenzyme form. In other tissues, only active form could be detected. The protein of FcLegu was down-regulated by Vibrio or WSSV challenge in the stomach and gills. FcCathL and FcLegu were proposed to play a role in shrimp innate immunity for the first time.