173 ISJ 16: 173-183, 2019 ISSN 1824-307X RESEARCH REPORT HC-SPH: A conserved serine protease homolog of S1 superfamily in the triangle-shell pearl mussel (Hyriopsis cumingii) Q Liu1,2, B Xu1,2*, T Xiao1,2* 1Hunan Engineering Technology Research Center of Featured Aquatic Resources Utilization, Hunan Agricultural University, Changsha 410128, China 2Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan Province, Changde 415000, China Accepted September 12, 2019 Abstract Serine proteases play central roles in immune defense in invertebrates through innate immunity, and are particularly important complement system in molluscs because their susceptibility to infection due to lack of an adaptive immune ability. A gene encoding the serine protease homolog from the triangle - shell pearl mussel (Hyriopsis cumingii) was identified and designated as HC-SPH in this study. Protein sequence analysis revealed that HC-SPH consists of a typical Tryp_SPc functional domain of serine protease of S1 family lead by a signal peptide, and the molecule shares a highly conserved sequence and structural organization with other members, including a cleavage site, 3 enzymatic active sites and 3 substrate binding sites, so that it was clustered into a trypsin-like serine protease subfamily of the S1 superfamily. Semi - quantitative analysis of the amplicons separated on agarose gel by comparing to the β-actin products revealed that the digestive gland had a strong expression while the gonads were seen as weak expression sites. Infected by Aeromonas hydrophila, the gene expression was significantly up - regulated in the kidney at the 6 hours post challenge (hpc), stomach at 12 hpc and gills at 24 hpc while the expression maintained steadily unchanged in the digestive gland. However, up to 48 hpc, the expression levels in all four tissues reached significantly high, and also joined by a high level of expression in intestine that was down - regulated before 24 hpc, to build up an enhanced immune defense. The complementary up - regulation of the gene expression in these tissues suggested a temporal and spatial reinforce model for HC-SPH in immune response. Key Words: Tryp_SPc domain; Serine protease homolog; S1 superfamily; immune response; mollusk Introduction Serine proteases (SPs), commonly recognized by their serine activity, are hydrolyse protein family of enzymes with wide distribution and important biological actions (Ross et al., 2003; Liu et al., 2019; Wei et al., 2019). SPs are molecularly characterized by a conserved Tryp_SPc domain together with the catalytic triad histidine (His, H), aspartic acid (Asp, D) and the serine (Ser, S) (Li et al., 2012b; Wang et al., 2014). Inactive SPs with key residue(s) of catalytic triads replaced by other amino acid are defined as SP homologs (SPHs), which are similar to SPs in amino acid sequence (Liu et al., 2010) and classified ___________________________________________________________________________ Corresponding authors: Baohong Xu Tiaoyi Xiao Hunan Engineering Technology Research Center of Featured Aquatic Resources Utilization Hunan Agricultural University Changsha 410128, China E-mail: xbht568@126.com; tyxiao1128@163.com as protease precursors. SPHs assist SPs as cofactors to exert their functions (Wang and Jiang, 2004; Lee et al., 2018) by activating/inhibiting the original protease activities in variety of biological processes such as digestion, cell differentiation, tissue remodeling, angiogenesis and embryonic development as well as their involvement in cellular and humoral immune responses (Rawlings and Barrett, 1993; Krem and Di Cera, 2001, 2002). Up to date, SPH was identified in many invertebrates and some of which were shown to play roles in different biological processes, including antimicrobial activity from horseshoe crab Tachypleus tridentatus (Kawabata et al., 1996), defense of pathogen challenge in Chinese white shrimp Fenneropenaeus chinensis (Ren et al., 2009,2011), cell adhesion in crayfish Pacifastacus leniusculus (Huang et al., 2000) and the tiger shrimp Penaeus monodon (Liu et al., 2006), and activation of the prophenoloxidase (proPO) system (Kwon et al., 2000; Charoensapsri et al., 2009; Cui et al., mailto:xbht568@126.com 174 2010), which represent an important host natural immune system that recognizes foreign invasive substances by molecular pattern of proteins. The molecular characterization, gene cloning and expression profile of the SPH were also abundantly studied in insect and other arthropods (Jitvaropas et al., 2009; Ren et al., 2011) while little is known in mollusks. The freshwater triangle - shell pearl mussel Hyriopsis cumingii is a mollusk species widely cultured in China for pearl production (Ren et al., 2012). However, mussel aquaculture has been declining in the recent years because of the diseases by bacterial and viral infection. Like other mollusks, the mussel lacks acquired immune system and mainly relies on innate immunity to defense pathogens invasion (Wang et al., 2013). In this study, a gene (termed HC-SPH) coding for SPH (termed HC-SPH for encoded proteins) was cloned and characterized from the H. cumingii. The tissue - specific gene expression profiles of the gene before and after infection with the common infectious bacteria Aeromonas hydrophila were analyzed to provide evidence of its potential role in innate immune responses as suggested from other species. The results reported here summarized the molecular identification and characterization of HC-SPH, which contribute to general knowledge of SPH from a mollusk and particularly provide important molecular data for further investigation of HC-SPH towards a better understanding for its role in immune defense against infections. Materials and methods Experimental animal and immune challenge Mussels (Hyriopsis cumingii), 285.33 ± 55.18 g and 14.37 ± 1.28cm, were obtained from a commercial farm in Changde, Hunan Province, China and cultured in freshwater at 24 - 28 °C. They were acclimatized for a week before being used for experiments. The healthy mussels were injected in axe feet with 109 cfu/mL (≈ 0.5 mL) of Aeromonas hydrophila isolated and purified from dying mussels for challenging, and with an equal volume of phosphate buffer saline (PBS) as controls. Three challenged and three control mussels were chosen for sample collection from the digestive gland, stomach, intestine, gill, heart, mantle, axe foot, adductor muscle, kidney and the gonad at the time points of 0 h, 3 h, 6 h, 12 h, 24 h, 48 h after injection. All samples were snap - frozen in liquid nitrogen and immediately stored at - 80 °C under RNase - free conditions. RNA extraction and cDNA synthesis Total RNA samples were isolated using the RNAprep Pure tissue kit (Tiangen Biotech, China) according to the manufacturer’s instruction, and treated with DNase I. RNA samples were kept in DEPC - treated water, qualified by gel electrophoresis and quantified using a biophotometer (Eppendorf, Germany). About 5 μg total RNA from the digestive gland of healthy mussel was converted into 5′-RACE - Ready cDNA and 3’-RACE-Ready cDNA by reverse transcription using the SMARTerTM RACE (Rapid amplification of cDNA ends) cDNA Amplification Kit (Clontech, USA) for full - length cDNA cloning. About 2 μg total RNA from each tissue sample was reverse transcribed by RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Canada) for gene expression study. RACE and sequencing analysis The designing of gene specific primers used for this study was based on the expressed sequence tag (EST, GenBank accession number: FE968619) from a cDNA library generated by suppression subtractive hybridization (Xiao et al., 2009). The 5′-RACE - Ready cDNA was synthesized using the SMART II™ A Oligonucleotide and the 5′-RACE CDS Primer A (Table 1); The 5′-RACE was performed using gene specific primer pairs UPM/ HC-SPH - GSPR - out and UPM/ HC-SPH - GSPR - in (Table1) in nested PCRs. 3’-RACE-Ready cDNA was synthesized using the 3′ - RACE CDS Primer A (Table 1). The 3′-RACE was performed using gene specific primer pairs UPM/HC-SPH-GSPF - out and UPM/ HC-SPH - GSPF - in (Table 1) in nested PCRs. The PCR reactions were catalyzed by Ex Taq polymerase (Takara, Japan) and the amplified fragments were cloned into the pUCm - T vector (Biobasic, Canada) for sequencing analysis (Sangon, China). The full - length cDNA coding for the SP homolog of H. cumingii was amplified and identified by its overlapping sequence to the EST. DNA and amino acid sequence analysis The cDNA sequence of HC-SPH was submitted and translated into amino acid sequence (http://www.vivo.colostate.edu/molkit/translate/), termed as HC-SPH, which was further characterized for its molecular property and homology prediction. The protein sequence was submitted to BLASTP searching against various databases through UniProtKB/SwissProt (http://www.uniprot.org/blast/) for a primary homology result and collected close related amino acid sequence for alignment. The sequence was used for a prediction of its functional domains and motifs by the online service of the Eukaryotic Linear Motif (ELM) resource for functional sites in proteins. The SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) was used to define the signal peptide and the cleavage site. The amino acid sequences related to HC-SPH found by UniProtKB were retrieved for annotating the place of HC-SPH, sequences clustered directly with HC-SPH and the sequences with well-defined Tryp_SPc domains used for functional motif alignment were listed in Table S1. The sequences were aligned and edited by CLUSTALX 1.83 (Thompson et al., 1997). Phylogenetic analysis was performed with MEGA 6.0 using maximum likehood method with a bootstrap valve of 1000. The HC-SPH gene expression by RT-PCR and RT-qPCR The expression profiling of HC-SPH transcripts in the digestive gland, stomach, intestine, gill, heart, mantle, axe foot, adductor muscle, kidney and gonad from healthy groups were measured using RT-PCR. The quantity of HC-SPH transcripts in the 175 Table 1 Primers designed for cloning and expression analysis of HC-SPH Primer name Sequence SMART II™ A Oligonucleotide 3′-RACE CDS Primer A(3′-CDS) 5′-RACE CDS Primer A (5′-CDS) 10×Universal Primer A Mix (UPM) Long Primer Short PrimerRandom Primer Oligo (dT)20 HCSHP-GSPR-out HCSPH-GSPR-in HCSPH-GSPF-out HCSPH-GSPF-in HC-SPH-pf HC-SPH-pr β-actin -pf β-actin -pr 5'-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3' 5'-AAGCAGTGGTATCAACGCAGAGTAC(T)30V N-3' * 5'-(T)25V N-3' * 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3′ 5'-CTAATACGACTCACTATAGGGC-3' 5'-(dN)9-3' * 5'-(dT)20-3' 5'-GGCGATGTCGTCCTTGAGACTGGTG-3' 5'-CGCTGAATCCTGGACCCTCAAAATG-3' 5'-TTGACCTTGACCTCAGACATTGGCAG-3' 5'-ATGCCTCTCATCACTACCGTGTTGGA-3' 5'-TGAGGGTCCAGGATTCAGCGTAT-3' 5'-CCCCAACCAGGGAGGTAGCAAAC-3' 5'-ACTCACACCGTCCCCATCTAT-3' 5'-CGATTTCTCTTTCAGCAGTGG-3' * N = A, C, G, or T; V = A, G, or C in primer sequences digestive gland, stomach, intestine, gill and kidney from both challenged and control groups were measured using RT-qPCR. The HC-SPH gene - specific primer pairs, HC-SPH - pf and HC-SPH - pr (Table 1), was designated to amplify a fragment of 279 bp located between the position 326 and 603 of the gene. The house - keeping gene β-actin was used as an internal control amplified by the gene specific primer pair β-actinpf/β-actin-pr (Table 1) for an expected fragment of 148 bp. The RT-qPCR reaction was conducted using the TransStartTM Top Green qPCR SuperMix (TransGen Biotech, China) on an ABI 7300 Real-Time Detection System (Applied Biosystems, USA) as instructed by the manufacturer. Briefly, the final PCR reaction mixture (25 μL) in H2O consisted of 12.5 μL of 2 x TransStartTM Top Green qPCR SuperMix, 0.5 μL of 50 × Passive Reference Dye1, 1 μL cDNA and 0.5 μL of each primer, was incubated at the denaturing temperature of 94 °C for 30 s first followed by 40 cycles of 5 s at 94 °C, 31 s at 61 °C. All PCR products were dissociated and confirmed a single amplicon from each sample by the system. A series of 10 - fold diluted mixture of first - strand cDNAs from all selected tissues were used to construct two standard curves (Chang et al., 2007) for HC-SPH and β-actin respectively against the no cDNA negative controls. Each sample was run in triplicates. Data collected were analyzed using the Sequence Detection System (SDS version1.4, Applied Biosystems, USA). The relative expression of HC-SPH was determined using the comparative threshold cycle (Ct) method (2-(△△CT) method) as described previously (Livak and Schmittgen, 2001; Jiang et al., 2019). The arithmetic mean ± standard deviation was calculated and used for determining the significance of differences by t - test using the Statistics Package for Social Science (SPSS) 17.0. p < 0.05 means significant differences and p < 0.01 for most significant differences. Results Isolation of HC-SPH, a full - length cDNA sequence coding for HC-SPH A cDNA fragment of 478 bp (EST tag of SP from the triangle shell mussel, Genbank accession number: FE 968644) was retrieved from the database, based on which a pair of gene specific primers were designed (GSPF and GSPR listed in Table 1) for amplifying the full - length cDNA sequence (Fig. 1A). A 344 bps 5′-RACE fragment and a 495 bps 3′-RACE fragment (Fig. 1B) overlapped to the EST fragment to give the rise of a full - length cDNA sequence of 1046 bp (Fig. 1C). The HC-SPH contains a 5′ - terminal untranslated region (UTR) of 73 bps, an 864 bps open reading frame (ORF) encoding 287 amino acid residues and a 3′-UTR of 109 bps with a canonical polyadenylation signal sequence AATAAA and a labile motif ATTTA (both underlined) followed by a poly (A) tail (Fig. 1C). Bioinformatic analysis of amino acid sequence of HC-SPH protein Full-length HC-SPH cDNA and its encoded SP in H. cumingii (HC-SPH) were submitted to the GenBank Accession number: GU222695. The amino acid sequence from ORF (303 aa) of the HC-SPH was analyzed using UniProtKB and ELM in conjunction with biological databases and found a typical Tryp_SPc (Trypsin-like SP) domain of the SP family between R52 and I290 (Fig. 2A). The N - terminus (K1 - I70) was recognized as a signal peptide (Fig. 2A) for proteolytic cleavage around R52. Highly conserved motifs important for proteolytic cleavage, typical enzymatic active sites, and substrate binding sites for the enzyme are shown along the molecule (Fig. 2A). Multiple sequence alignment of HC-SPH with six other well-defined SPs showed those conserved motifs (Fig. 2B) around the 176 Fig 1 The full - length cDNA cloning of HC-SPH gene and its encoded amino acid sequence (HC-SPH). The HC-SPH EST - tag sequence from GenBank was used for designing of the gene specific primers for RACE PCRs (A), which amplified specific products as single bands (sizes of molecular weight maker and the products were given as indicated by arrows) for 5’ - and 3’ - RCAE respectively (B). The determined nucleotide sequence, contained a canonical polyadenylation signal sequence AATAAA and a labile motif ATTTA (both underlined) followed by a poly (A) tail, and the deduced amino acid sequence were given in single letter format (C). The translation initiation methionine (M) is labelled by +1 on top enzymatic important sites with the ranges numbered at the bottom. The numbers of non - conserved amino acid residues before, between and after the motifs are given for each species respectively in brackets (Fig. 2B). The UniProtKB BLAST search against databases revealed a high similarity of HC-SPH to the Tryp_SPc superfamily, amongst which protein sequences with defined biological properties were retrieved for similarity analysis (Fig. 3). A phylogenetic tree was generated to show the position and similarity relationship of HC-SPH in the unrooted tree (Fig. 3). HC-SPH was clearly clustered with a group of proteins that have strong trypsin activities. The HC-SPH expression in tissues and regulation in response to bacterial infection The presence of HC-SPH gene transcripts in different tissues of healthy H. cumingii was detected by RT-PCR as strategized (Fig. 4A) using a pair of gene - specific primers, HC-SPH-pf and HC-SPH-pr (Table 1). A fragment with the expected size (279 bp) was amplified from the tissues listed in all replicates in comparison with the relative amount of cDNA template reflected by PCR amplification of the house 177 Fig. 2 Characterization of the molecular properties of HC-SPH. A signal peptide sequence was identified at the N - terminus with a proteolytic cleavage site, presented by SignalP 4.1 (A). Using UniProtKB BLAST conjunct with ELM searches, a typical Tryp_SPc functional domain was detected (A), including a cleavage site (indicated by blue ▲), 3 enzymatic active sites (indicated by red ▲) and 3 substrate binding sites (indicated by black ▲). The conserved motifs containing the sites were aligned with the corresponding residues numbered at the bottom and the numbers of non - conserved residues were given in brackets (B) keeping gene β-actin (Fig. 4B). RT-PCR amplification result showed HC-SPH gene expression in tissues of 2 years old mussels examined, including the digestive gland, stomach, intestine, gills, heart, mantle, axe foot, adductor muscle, kidney and gonads, by the gene-specific primers (Fig. 4B). Semi-quantitative analysis of the amplicons separated on agarose gel by comparing to the β-actin products revealed that the digestive gland had a strong expression while the gonads were seen as weak expression sites. In the time course RT-qPCR analysis of gene expression in five selected tissues at hours post challenge (hpc), the HC-SPH expression in digestive gland was slightly increased at 6 hpc followed by a decrease at 12 hpc detected, then the expression kept rising to a highest (p < 0.01) level till 48 hpc (Fig. 5A). The HC-SPH gene expression in kidney (Fig. 5E), stomach (Fig. 5B) and the gills (Fig. 5D) rather fluctuated with a highest expression detected at 6 hpc for kidney, 12 hpc for stomach and 24 hpc for the gills. While kidney expressed basal level of HC-SPH after a surge at 6 hpc, the expression in the stomach and gills showed complementary expression pattern (Fig. 5). The intestine showed a constant low levels of HC-SPH expression (although expression was significantly high at 3 hpc (p < 0.01) than that at 0 hpc), particularly between 6 hpc and 24 hpc at a significantly lower level (p < 0.01) than 0 hpc, before an expression surge starting from 24 hpc and reach a peak (p < 0.01) at 48 hpc (Fig. 5C). Discussion HC-SPH, a secreted trypsin-like SP in H. cumingii SPs are enzymes widely conserved across species not only in their molecular structures but also in their biological activities (Ross et al., 2003). A cDNA library was established using suppression subtraction hybridization achieves an EST tag for SP gene of the H. cumingii in our previous study (Xiao et al., 2009), which is similar with the genes encoding the factors responded to immunogenic stimulation (Lund and Olafsen, 1999; Gerwick et al., 2000,2002). In the present study, the information of the EST tag was used for identify and clone the full-length cDNA, which contends an ORF coding for HC-SPH. The UniProtKB BLAST analysis against databases 178 Fig. 3 The phylogenetic analysis of HC-SPH. The phylogenetic analysis showing the position of HC-SPH as a member of serine proteases of S1 superfamily (brunch was highlighted with bold) and protein was clustered into the group of with trypsin-like proteins revealed HC-SPH exhibited a typical molecular characteristic of a SP. A well-conserved Tryp_SPc domain of the SP family proteins was found in HC-SPH by functional search, including enzymatic active sites for its processing and activities along the molecule (Fig. 2A). The molecule was recognized as an inactive trypsin-like SP precursor. A cleavage site around I37 (Fig. 2B) was found in the precursor that are the characteristics of enzymatic activities and also known as catalytic triad for SPs (Perona and Craik, 1995), and 3 substrate binding sites at around E226, S252 and 3 S254 (Fig. 2B) of the molecule, which bind to the substrates for the function. These conserved motifs are ensuring the actions (Li et al., 2012a). However, key residues of catalytic triads were replaced by other amino acids (Q82 for H82, L232 for S232), similar to SPs in amino acid sequence, are classified as protease precursors (Liu et al., 2010). Furthermore, the numbers of the amino acid residuals in non - conserved regions were very close between species (Fig. 2B), which were significant in ensuring similar molecular conformations essential for their function. SPHs assist SPs as cofactors to exert their functions (Wang and Jiang, 2004) by activating/inhibiting the original protease activities, including cell differentiation, tissue remodeling, embryonic development and humoral immune responses in the cell (Rawlings and Barrett, 1993; Krem and Di Cera, 2001, 2002). 179 Fig. 4 The HC-SPH gene expression in tissues of healthy Hyriopsis cumingii. A pair of gene specific primers were designed to amplify a fragment of 279 bp between the nucleotide position 326 and 603 (A). The RT-PCR products were confirmed by agarose gel electrophoresis showing the right size (top, B) and the relative template concentrations were judged by the house - keeping gene β-actin (bottom, B) Signal peptide at the amino terminus of a nascent protein, mediating protein targeting to the membrane of the endoplasmic reticulum (Blobel, 1980, 2000), directs the protein transportation and the secretory pathway, which is recognized by the signal recognition particle (SRP) and cleaved by the signal peptidase (Duffaud et al., 1985). These residuals are comprising a characteristic tripartite structure of a hydrophilic, usually positively charged n - region, a central hydrophobic h - region and a cleavage site (von Heijne, 1985), which are represented by C-score (raw cleavage site score), S-score (signal peptide score) and Y-score (combined cleavage site score) in the SignalP prediction (Petersen et al., 2011). The HC-SPH protein sequence was analyzed by software and a clear signal peptide region of 31 amino acids (the translation initiation starts at the M17, see Fig. 1C) with a cleavage site (D valve = 0.829 by setting - up D-cutoff 0.450) between C31 and Q32 of the short stretch of VLC-QL recognized (Fig. 2A). The high raw C-score recognized this region as a signal peptide and enhanced by the Y-score of combined cleavage site, which compares the C-score peak(s) against the steep slope of the signal peptide score (S-score). Signal peptides are rather heterogeneous and interchangeable between different species, but the signal peptide specifies the protein secretion (von Heijne, 1985; Kober et al., 2013). HC-SPH was therefore regarded as a secreted trypsin-like SP. In addition, the HC-SPH was very similar to the complement factor D (CFAD_HUMAN, P00746), which is also single - domain SP. The factor D is a component of the alternative complement pathway. Recent studies show that an ancient proto - complement complex (C3, SP factor B and complement receptor CR) exists in bivalves (Gorbushin, 2018, 2019). A complement -like activity is shown in bivalves Mytilus edulis, Mya Arenaria, and Sinonovacula constricta (Klimovich and Gorbushin, 2017; Niu et al., 2018). However, homolog of the factor D cleaving the factor B to Bb and Ba is not described until now (Gerdol et al., 2018). Our results implied that the HC-SPH was a potential candidate for this role, although it was still need to further confirmation. HC-SPH is a member of the peptidases S1 family Hundreds of sequences producing significant alignments with low expect values (E values) were found by both BLASTP (Altschul et al., 2005) and UniProtKB (Apweiler et al., 2012) searches. They share similar enzymatic functions and classified as proteins of the peptidases S1 family (Goldman et al., 2006), the largest of all of the peptidase families, by both the number of sequenced proteins and the 180 Fig 5 The HC-SPH gene expression profiles in tissues of mussels challenged by A. hydrophila infection. The transcripts were quantified using RT-qPCR in time-course manner and indicated by hours post challenge (hpc). The amplicons detected by qPCR in digestive gland (A), stomach (B), intestine (C), gill (D) and kidney (E) were normalized by control value (0 h) and adjusted to the scale for summarizing the result. The relative abundancy of the transcripts in a tissue at a time of hpc was compared with the control (0 h) to rule out the difference statistically. The up- and down-regulated expression levels of significant difference (p < 0.05, N=3) were indicated by single asterisk (*), and of highly significance (p < 0.01, N=3) by double asterisks (**) number of distinct peptidase activities (serine endopeptidases) (Rawlings and Barrett, 1993) fell into three main activity types: trypsin-like, chymotrypsin-like and elastase-like SPs (Cooley et al., 2001; Ovaere et al., 2009; Madala et al., 2010). The peptidases of the S1 family proteins are secretory precursors led by an N - terminal signal peptide that is cleaved to form the active enzyme. The cleavage initiates a structural rearrangement to produce a “new” N - terminus for binding. The phylogenetic tree of the S1 peptidase (Fig. 3) clearly showed that the HC-SPH was clustered to a group of trypsin-like type SPs roughly flanked by a group of elastase-like SPs at the bottom part and a large group of chymotrypsin-like proteases on top. Gene expression in tissues regulated by bacterial infection suggested temporal and spatial active roles of HC-SPH in immune - response Immune responses to infections are a series of cellular and molecular actions in invertebrate species (Peteiro et al., 2007). Understanding of the reaction process is particularly important in molluscs because their susceptibility to microbial infection and transmitting diseases (Barcia and Ramos-Martinez, 2008) due to the lack of an adaptive immune system (Wang et al., 2013). The mollusk defenses through a chemicophysical barriers preventing host invasion and the circulating hemocytes and secreted bio - factors to initiate immune responses (Hine, 1999; Garcia-Garcia et al., 2008), including the activation of phenoloxidase system (Little et al., 2005) and the complement system capable of recognizing and eliminating invading pathogens (Kardos et al., 2008). The SPs are the central components of the complement system, specifically bind to and cleave the substrates in forms of super - molecular complexes with other SPs and non - enzymatic proteins (Harmat et al., 2004). The expression patterns of SP in tissues would be altered to activate immune response stimulated by infection. The gene expression in physiological conditions examined by RT-PCR in this study showed the presence of HC-SPH gene transcripts in all detected tissues (Fig. 4B). In the time course RT-qPCR analysis of gene expression in five selected tissues of A. hydrophila challenged mussels showed that the trends of the HC-SPH expression were similar to the CfCUBSP in Chlamys farreri which indicated CfCUBSP might be involved in immune response (Yang et al., 2017). These results implied that a temporal and spatial reinforce model for HC-SPH to response to foreign invaders that yet to be proved experimentally. The HC-SPH gene expressed at high levels in all secreting tissues at 48 hpc onwards when an immune acting system recruited and built up. The relatively low level of expression after the challenging and mechanisms of down - regulation before 24 hpc in the intestine remain to be further investigated. In summary, the full - length cDNA coding for the SPH of the S1 family protein was cloned from H. cumingii. HC-SPH possessed a typical functional domain of a Tryp_SPc with characteristics of signal 181 peptide cleavage, active and substrate binding sites of the protease, which was further classified as a member of trypsin-like SP of S1 superfamily. Under the physiological conditions, the gene expression remained high in the digestive gland, but the expression showed a temporal complementary pattern of high levels in the digestive gland, kidney, stomach and low levels in the gills upon the regulation of bacterial infection. 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