Fish and Shellfish Immunology 86 (2019) 695–701 Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Short communication Molecular characterization, expression and functional analysis of cystatin C in Japanese flounder (Paralichthys olivaceus) T ∗ Haiyang Yu, Xin Xu, Quanqi Zhang, Xubo Wang Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 266003, Qingdao, Shandong, China ARTICLE INFO ABSTRACT Keywords: Cystatins are natural tight-binding reversible inhibitors of cysteine proteases found in a wide arrange of or- Paralichthys olivaceus ganisms. Studies have shown that cystatins play important roles under both physiological and pathological Cystatin C conditions in mammals. However, much less is known about fish cystatins. In this study, we described the Immune response identification and analysis of the gene encoding cystatin C in Japanese flounder (Paralichthys olivaceus). This Inhibit bacteria gene had a high homology with the sequence of cystatin C in many fish species and had a signal peptide and three conserved functional sites. The results of qRT-PCR showed that the gene was highly expressed in the liver. Lipopolysaccharide, peptidoglycan and polyinosinic-polycytidylic acid all increased its expression after stimu- lation. Functional analysis showed that the recombinant P. olivaceus cystatin C purified from Escherichia coli had cysteine protease inhibitory activity and could inhibit bacterial growth by binding to bacteria. Meanwhile, rPocystatin C could up-regulate the expression of cytokines tumor necrosis factor α and interleukin 10. These results indicated that cystatin C of P. olivaceus might be considered to have the similar immunomodulatory function to mammalian cystatin. 1. Introduction revealing valuable results: Some of these cystatin-like proteins may respond to bacterial challenge or are involved in other immune pro- Protease inhibitors have inhibitory effects on proteolytic enzymes cesses [12–14]. Despite of all these efforts, the expression patterns and and play crucial roles in the innate immune response of vertebrates. potential functions of teleost cystatins remain limited. Cystatins are a family of cysteine protease inhibitors that contain at Japanese flounder (Paralichthys olivaceus) is an important cultured least one cystatin domain. They interact with and neutralize cathepsin fish in China. Along with the deterioration of the culture environment, cysteine proteases, hence possessing pivotal functions in various phy- the cultured population is susceptible to several kinds of virus, bacteria siological processes, such as protein degradation, arthritis, tumor in- and pathogens, which leads to high mortalities and poor benefits [16]. vasion, metastasis and antigen presentation [1–3]. Based on structural All these disadvantages let researchers to develop stocks with higher differences, cystatins are classified into several major types. Type 1 resistance to diseases. In this paper, we aimed to analyze the expression cystatins (cystatin A and B) are cytoplasmic proteins composed of and function of P. olivaceus cystatin C (Pocystatin C). LPS, peptidoglycan ∼100 amino acid residues that are ∼11 kDa in size. They lack signal (PGN) and polyinosinic-polycytidylic acid (poly I:C) stimulation was peptide, disulfide bonds and carbohydrates. Type 2 cystatins, including performed to detect the expression changes of Pocystatin C mRNA. The cystatins C, D, E/M, F, G, S, SA and SN, are extracellular polypeptides of antibacterial effect of recombinant P. olivaceus cystatin C protein ∼120 amino acid residues. They contain two conserved disulfide bonds (rPocystatin C) was tested. Furthermore, we examined the effects of and localize mainly to the extracellular matrix [4,5]. One of the features rPo-cystatin C on cytokines tumor necrosis factor α (TNF-α) and in- shared by these two types of cystatins is the tertiary structure of a terleukin 10 (IL-10). By these experiments, we could acquire a deeper “cystatin fold” formed by a five-stranded β-sheet wrapped around a understanding about the role of cystatin C in teleost immune system. central α-helix [6,7]. Up to now, many studies on fish cystatins have been performed in Oncorhynchus keta [8], Cyprinus carpio [9], Oncorhynchus mykiss [10], Acipenser sinensis [11], Pseudosciaena crocea [12], Scophthalmus max- imus [13], Oplegnathus fasciatus [14] and Clarias batrachus [15], ∗ Corresponding author. E-mail address: [email protected] (X. Wang). https://doi.org/10.1016/j.fsi.2018.12.015 Received 29 September 2018; Received in revised form 3 December 2018; Accepted 9 December 2018 Available online 10 December 2018 1050-4648/ © 2018 Elsevier Ltd. All rights reserved. H. Yu et al. Fish and Shellfish Immunology 86 (2019) 695–701 2. Materials and methods 2.4. Quantitative real-time PCR (qRT-PCR) 2.1. Fish and cell line qRT-PCR was conducted on LightCycler 480 (Roche, Forrentrasse, Switzerland). The SYBR Green Master Mix (Roche, Basel, Switzerland) P. olivaceus individuals of one-year-old were obtained from a fish was used in the PCR detection system. Thermocycling consisted of an farm in Yantai, Shandong Province, China, and kept at 17 °C. Three initial polymerase activation of 2 min at 95 °C, followed by 40 cycles at male and three female individuals were anesthetized by MS-222 95 °C for 15 s and 60 °C for 45 s. A pair of specific primers PocystatinC- (30 mg/mL) and then killed by severing the spinal cord. Brain, heart, RT-Fw/Rv (Table 1) was used for qRT-PCR. The β-actin gene was se- intestine, head kidney, liver, spleen, gill and muscle of specimens were lected to normalize relative gene expression for its convincing and collected from each individual, immediately frozen in liquid nitrogen statistically repeatable results. All qRT-PCR experiments were per- −ΔΔ and stored at −80 °C for further use. formed in triplicate. Data were analyzed through the 2 Ct method. P. olivaceus gill cell line (FG-9307) was derived from gill tissue of P. The expression level of genes (Pocystatin C, TNF-α and IL-10) relative to olivaceus in 1997 by Tong et al. [17]. Cells was cultured in Eagle's that of β-actin was calculated. Fold changes represented the ratio of minimal essential medium (EMEM, BI, Israel) supplemented with 10% relative expression levels between experimental and control groups. (V/V) fetal bovine serum (BI, Israel), 1% (V/V) non-essential amino acids (NEAA, Gibco, US), and 100 U/mL penicillin at 24 °C. 2.5. Immune challenge assay Monolayer-cultured gill cells with good growth state were trans- 2.2. RNA extraction and cDNA synthesis ferred to 12-well plates and cultured for 24 h. When the rate of cell coverage reached 90%–95%, LPS, PGN and poly I:C were added to each Total RNA was extracted using Trizol Reagent (Invitrogen, Carlsbad, well of the experimental group to a final concentration of 50 μg/mL (the CA, USA) in accordance with the manufacturer's protocol, treated with concentration was determined based on pre-experiments). The control RNase-free DNase I (TaKaRa, Dalian, China) to degrade genomic DNA, group was added with the same concentration of PBS. Four wells of and then frozen at −80 °C. Reverse transcription and cDNA synthesis each group were sampled at every time point (0, 1, 2, 4, 8 and 12 h after were performed with 1 μg of total RNA and random primers using the stimulation). After washed 3 times in PBS, cells were sampled and Reverse Transcriptase M-MLV Kit (TaKaRa, Dalian, China) in ac- stored in TRIzol at −80 °C until further use. cordance with the manufacturer's instructions. Quality and quantity of the cDNA were evaluated via 1.5% agarose gel electrophoresis and 2.6. Construction of expression vector spectrophotometry using NanoPhotometer Pearl (Implen, Germany). The cDNA sequence (the sequence encoding the signal peptide and the transmembrane region was not included) of Pocystatin C was am- fi 2.3. Identi cation of Pocystatin C cDNA and bioinformatic analysis plified by PCR with the specific primers PocystatinC-Fw2/Rv2 (Table 1). The reaction was performed under the following conditions: an initial To acquire the Pocystatin C gene, the available sequences of teleosts denaturation step at 94 °C for 5 min, a step containing 35 cycles of and mammals cystatin C genes were obtained from NCBI and Ensembl. denaturation at 94 °C for 30 s, annealing for 30 s at 68 °C and extension Then, the retrieved sequences were used as query sequences in BLAST at 72 °C for 1 min, and an additional extension step at 72 °C for 7 min. fi searches. The mRNA sequence of Pocystatin C gene was identi ed using The PCR product was digested with BamHI (TaKaRa, Dalian, China) and tBLASTn analysis from P. olivaceus transcriptome previously sequenced hindIII (TaKaRa, Dalian, China), and sub-cloned into the expression fi by our laboratory [18]. A pair of speci c primers (PocystatinC-Fw1/ vector pET-32a (Novagen, USA) treated with the same restriction en- Rv1, Table 1) was designed to amplify the gene. The PCR products were zymes. The correct recombinant plasmid was screened by sequencing fi puri ed, sub-cloned and sequenced. and named pET32a-Pocystatin C. Multiple alignment analysis of the protein sequences was performed using MUSCLE3.8.31. The domains in Pocystatin C protein structure 2.7. Expression and purification of rPocystatin C were predicted by SMART software, and SignalP 4.1 server was used to search canonical signal peptides. The phylogenetic tree was constructed The constructed plasmid pET32a-Pocystatin C was transformed into based on the deduced amino acid sequences by using MEGA 7.0. The E. coli BL21 (DE3) and cultured overnight in Luria-Bertani (LB) broth reliability of each node was estimated by bootstrapping with 1000 re- containing kanamycin (100 mg/mL). The culture was then diluted plications. 1:100 with LB and incubated at 37 °C for 4 h.