Trypsin-like Serine Protease Research Articles

Coevolved Positions Represent Key Functional Properties in the Trypsin-Like Serine Proteases Protein Family

Trypsin-like serine proteases are a group of homologous enzymes which exert multiple roles in both vertebrate and invertebrate organisms. Key properties of these enzymes include their activation from an inactive zymogen form to their active form by cleavage of residues in their N-terminus, the presence of a conserved catalytic triad of residues, and the existence of different patterns of substrate selectivity for residue cleavage between the various members of this protein family. In this article, we apply the decomposition of residue coevolution networks computational method to find sets of residues related to some of these key properties, especially to zymogen activation. Positive selection detection, normal modes analysis, and the calculation of thermal couplings between the bovine trypsinogen and bovine trypsin structures residues yielded further information for understanding the zymogen activation process and highlighted the importance of some of the coevolved set residues during these transitions.

Kallikrein-related peptidases protein expression in lymphoid tissues suggests potential implications in immune response

ObjectivesKallikrein-related peptidases (KLKs) are a subgroup of 15 secreted chymotrypsin- and trypsin-like serine proteases that have been reported to possess novel functions in innate immunity and inflammation. Since the potential role of KLKs in immunity has not been studied in detail at the protein level, we examined the expression pattern of 12 members of the KLK family in immune-related tissues. Design & MethodsProtein expression in tissue extracts was evaluated using immunoassays (ELISA). Immunohistochemistry (IHC) was performed on representative sections of tonsil and lymph nodes to determine the cellular localization of the KLK family members. ResultsELISA profiling of KLK3-KLK15 (except KLK12) revealed higher protein levels in the tonsil, compared to the lymph nodes and spleen. Relatively high protein levels in the tonsil were observed for KLK7, KLK9, KLK10 and KLK13. Expression of these KLKs was significantly lower in lymph nodes and spleen. IHC analysis in tonsil unveiled that KLK9 and KLK10 were differentially expressed in lymphoid cells. KLK9 was strongly expressed in the germinal center of lymphoid follicles where activated B-cells reside, whereas KLK10 was expressed in the follicular dendritic cells (FDCs) that are vital for maintaining the cycle of B cell maturation. ConclusionOverall, our study revealed the possible implications of KLK expression and regulation in the immune cells of lymphoid tissues.

Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Spike Protein Proteolytic Processing.

The coronavirus spike envelope glycoprotein is an essential viral component that mediates virus entry events. Biochemical assessment of the spike protein is critical for understanding structure–function relationships and the roles of the protein in the viral life cycle. Coronavirus spike proteins are typically proteolytically processed and activated by host cell enzymes such as trypsin-like proteases, cathepsins, or proprotein-convertases. Analysis of coronavirus spike proteins by western blot allows the visualization and assessment of proteolytic processing by endogenous or exogenous proteases. Here, we present a method based on western blot analysis to investigate spike protein proteolytic cleavage by transient transfection of HEK-293 T cells allowing expression of the spike protein of the highly pathogenic Middle East respiratory syndrome coronavirus in the presence or absence of a cellular trypsin-like transmembrane serine protease, matriptase. Such analysis enables the characterization of cleavage patterns produced by a host protease on a coronavirus spike glycoprotein.

The trypsin-like serine protease domain of Paralichthys olivaceus complement factor I regulates complement activation and inhibits bacterial growth

In mammals, complement factor I (CFI) is a serine protease in serum and plays a pivotal role in the regulation of complement activation. In the presence of cofactor, CFI cleaves C3b to iC3b, and further degrades iC3b to C3c and C3d. In teleost, the function of CFI is poorly understood. In this study, we examined the immunological property of CFI from Japanese flounder (Paralichthys olivaceus) (PoCFI), a teleost species with important economic value. PoCFI is composed of 597 amino acid residues and possesses a trypsin-like serine protease (Tryp) domain. We found that PoCFI expressions occurred in nine different tissues and were upregulated by bacterial challenge. Recombinant PoCFI-Tryp (rPoCFI-Tryp) inhibited complement activation and degraded C3b in serum. rPoCFI-Tryp exhibited apparent binding capacities to a board-spectrum of bacteria and inhibited bacterial growth. These results provide the first evidence to indicate that CFI in teleost negatively regulates complement activation via degradation C3b, and probably plays a role in host immune defense against bacterial infection.

A novel complement factor I involving in the complement system immune response from Lampetra morii

Complement factor I (CFI) is a serine protease which plays a key role in the modulation of complement system and the induced-fit factor responsible for controlling the complement-mediated processes. In this study, a CFI gene was cloned and characterized from Lampetra morii (designated as L-CFI) at molecular and cellular levels. The L-CFI protein included a factor I membrane attack complex domain (FIMAC), a scavenger receptor cysteine-rich domain (SRCR), a trypsin-like serine protease domain (Tryp_SPc) and 2 low-density lipoprotein receptor class A domains (LDLa) which would exhibit functional similarities to CFI superfamily proteins. Tissue expression profile analysis showed that L-CFI mRNA constitutively expressed in all tested tissues except erythrocytes, with the predominant expression in liver. The mRNA expression level of L-CFI increased significantly after Vibrio anguillarum and Staphylocccus aureus stimulation. It is demonstrated that L-CFI interacted with L-C3 protein and affected the deposition of L-C3 on the cell surface. Furthermore, lamprey serum after deplete L-CFI and L-C3 reduced the cytotoxic activity against HeLa cells. These findings suggest that L-CFI plays an important role in lamprey immunity and involved in the lamprey complement system.

Is the hormone a protease? Proteolytic properties of human recombinant anti-mullerian hormone

Anti-mullerian hormone (AMH) is a glycoprotein of the TGFβ cytokine superfamily that regulates the development of the mammalian reproductive system, as well as the functioning of mature gonads. Recombinant AMH (rAMH) is also able to induce apoptosis of malignant cells bearing AMH type II receptors (MISRII) on the surface. Development of rAMH-based anticancer drugs is hampered by the lack of accurate information about the tissues where the AMH active form is generated and about the enzyme that activates the hormone by specific proteolysis. According to one hypothesis, the proteolytic processing of the hormone is autocatalytic. The goal of this work was to investigate the proteolytic activity of rAMH and its biologically active form — C-terminal AMH fragment (C-AMH). We showed that two forms of the hormone possess both autoproteolytic activity and the ability to influence the structure of other proteins. A full-length molecule of the hormone, as well as C-AMH, also forms complexes with aprotinin, an inhibitor of trypsin-like serine proteases. We determined that aprotinin competes for binding to C-AMH with antibodies blocking C-AMH interaction with MISRII. The obtained data suggest that AMH has protease properties and that the site of specific AMH autoproteolysis is involved in the interaction of the hormone with a specific receptor.

A Clip Domain Serine Protease Involved in Egg Production in Nilaparvata lugens: Expression Patterns and RNA Interference.

Clip domain serine proteases play vital roles in various innate immune functions and in embryonic development. Nilaparvata lugens proclotting enzymes (NlPCEs) belong to this protease family. NlPCE1 was reported to be involved in innate immunity, whereas the role of other NlPCEs is unclear. In the present study, N. lugens proclotting enzyme-3 (NlPCE3) was cloned and characterized. NlPCE3 contains a signal peptide, a clip domain, and a trypsin-like serine protease domain. NlPCE3 was expressed in all tissues examined (gut, fat body, and ovary), and at all developmental stages. Immunofluorescence staining showed that NlPCE3 was mainly expressed in the cytoplasm and cytomembrane of follicular cells. Double stranded NlPCE3 RNA interference clearly inhibited the expression of NlPCE3, resulting in abnormal egg formation and obstruction of ovulation. These results indicate that NlPCE3 plays an important role in egg production in N. lugens.

6-Substituted amiloride derivatives as inhibitors of the urokinase-type plasminogen activator for use in metastatic disease

The oral K+-sparing diuretic amiloride shows anti-cancer side-activities in multiple rodent models. These effects appear to arise, at least in part, through moderate inhibition of the urokinase-type plasminogen activator (uPA, Ki = 2.4 µM), a pro-metastatic trypsin-like serine protease that is upregulated in many aggressive solid malignancies. In applying the selective optimization of side-activity (SOSA) approach, a focused library of twenty two 6-substituted amiloride derivatives were prepared, with multiple examples displaying uPA inhibitory potencies in the nM range. X-ray co-crystal structures revealed that the potency increases relative to amiloride arise from increased occupancy of uPA’s S1β subsite by the appended 6-substituents. Leading compounds were shown to have high selectivity over related trypsin-like serine proteases and no diuretic or anti-kaliuretic effects in rats. Compound 15 showed anti-metastatic effects in a xenografted mouse model of late-stage lung metastasis.

HC-SPH: A conserved serine protease homolog of S1 superfamily in the triangle-shell pearl mussel (Hyriopsis cumingii)

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.

Identification and enzymatic characterization of clip domain serine protease in the digestive fluid of the sea hare, Aplysia kurodai.

Clip domain serine proteases (CDSPs) participate in the extracellular signaling cascades of various biological processes such as innate immune responses in invertebrates. CDSP genes have been isolated from numerous invertebrates. Nevertheless, the enzymatic properties of mollusk CDSPs are poorly understood. In the present study, we demonstrated that the amino acid sequences of the trypsin-like serine protease purified from the digestive fluid of the sea hare, Aplysia kurodai resemble those of the unidentified CDSP-type protein (TPS3) of Aplysia californica predicted by genome analysis. The purified enzyme produced single 34 and 26.5 kDa bands on SDS-PAGE under non-reducing and reducing conditions, respectively. The 34-kDa band generated two amino-terminal sequences that were similar to the deduced sequences of the clip and catalytic domains of TPS3. The single amino-terminal sequence of the 26.5 kDa band showed a single sequence homologous to the catalytic domain. Thus, the purified enzyme consists of clip and catalytic domains bridged by disulfide linkage(s). The subsite specificity and inhibitor sensitivity of the purified enzyme were clearly distinct from those of horseshoe crab and silkworm CDSPs. A good substrate for the sea hare enzyme was pyroglutamyl-Arg-Thr-Lys-Arg-4-methyl-7-coumarylamide. The enzyme activity was strongly inhibited by aprotinin but not leupeptin. The physiological function of the enzyme in the digestive fluid remains to be determined.

Metacaspase-3 of Plasmodium falciparum: An atypical trypsin-like serine protease

Metacaspases are clan CD cysteine peptidases found in plants, fungi and protozoa that possess a conserved Peptidase_C14 domain, homologous to the human caspases and a catalytic His/Cys dyad. Earlier reports have indicated the role of metacaspases in cell death; however, metacaspases of human malaria parasite remains poorly understood. In this study, we aimed to functionally characterize a novel malarial protease, P. falciparum metacaspase-3 (PfMCA3). Unlike other clan CD peptidases, PfMCA3 has an atypical active site serine (Ser1865) residue in place of canonical cysteine and it phylogenetically forms a distinct branch across the species. To investigate whether this domain retains catalytic activity, we expressed, purified and refolded the Peptidase_C14 domain of PfMCA3 which was found to express in all asexual stages. PfMCA3 exhibited trypsin-like serine protease activity with ser1865 acting as catalytic residue to cleave trypsin oligopeptide substrate. PfMCA3 is inhibited by trypsin-like serine protease inhibitors. Our study found that PfMCA3 enzymatic activity was abrogated when catalytic serine1865 (S1865A) was mutated. Moreover, PfMCA3 was found to be inactive against caspase substrate. Overall, our study characterizes a novel metacaspase of P. falciparum, different from human caspases and not responsible for the caspase-like activity, therefore, could be considered as a potential chemotherapeutic target.

Aptamer-peptide conjugates as a new strategy to modulate human α-thrombin binding affinity

Aptamers are single-stranded RNA or DNA molecules that specifically recognize their targets and have proven valuable for functionalizing sensitive biosensors. α-thrombin is a trypsin-like serine proteinase which plays a crucial role in haemostasis and thrombosis. An abnormal activity or overexpression of this protein is associated with a variety of diseases. A great deal of attention was devoted to the construction of high-throughput biosensors for accurately detect thrombin for the early diagnosis and treatment of related diseases. Herein, we propose a new approach to modulate the interaction between α-thrombin and the aptamer TBA15. To this end, TBA15 was chemically conjugated to two peptide sequences (TBA-G3FIE-Ac and TBA-G3EIF-Ac) corresponding to a short fragment of the acidic region of the human factor V, which is known to interact directly with exosite I. Surface Plasmon Resonance (SPR) results showed enhanced analytical performances of thrombin with TBA-G3EIF-Ac than with TBA wild-type, reaching a limit of detection as low as 44.9 pM. Electrophoresis mobility shift assay (EMSA) corroborated the SPR results. Molecular dynamics (MD) simulations support experimental evidences and provided further insight into thrombin/TBA-peptide interaction. Our findings demonstrate that the combination of TBA15 with key interacting peptides offers good opportunities to produce sensitive devices for thrombin detection and potential candidates to block thrombin activity.

A Study of the Structure of Trypsin-Like Serine Proteinases. 2. A Study of Tryptophan Fluorescence in Variants of Miniplasminogen with an Altered Primary Structure

Abstract—Variants of miniplasminogen with an altered primary structure have been designed to study previously described changes in tryptophan fluorescence during plasminogen activation by urokinase. Miniplasminogens that contain the Trp141, Trp215, and Leu217 substitutions (the position of the amino-acid residue is specified by the Chymotrypsin nomenclature) have been tested. The activation of mutants containing a single tryptophan substitution induced changes in the fluorescence spectrum. No changes in the fluorescence spectrum were observed in the case of miniplasminogen containing a double tryptophan substitution. This indicates that both Trp141 and 215 contribute to the fluorescence spectrum shift; however, as suggested previously, the contribution of changes in the Trp215 microenvironment is greater. The Trp141, Trp215, and Leu217 amino-acid substitutions in miniplasminogen have no effect on the rate of plasminogen activation by urokinase. The replacement of the conserved Trp215 residue in serine proteases led to a significant reduction but not loss of the amidolytic activity of plasmin.

SUN-471 Corresponding Primary Transcript and mRNA Levels for Pomc and Prss56 Suggest Oscillating Transcriptions in Adult Rat Tanycytes

We recently reported mRNA and protein expression patterns in adult rat hypothalamic tanycytes that suggest oscillatory gene expression, demonstrating highly variable pro-opiomelanocortin (Pomc) mRNA and protein expression in these cells with 40% of rats expressing high, 40% low, and another 20% intermediate levels (Wittmann et al., J Comp Neurol, 2017). In addition, we demonstrated that mRNA expression for Prss56 , a trypsin-like serine protease, is comparably variable but inversely proportional to that of Pomc in tanycytes (Wittmann and Lechan, J Comp Neurol, 2018), proposing that the most likely explanation for this variability is that the two genes are expressed in an out-of-phase, oscillatory manner. Since the amount of mRNA depends on both the rate of transcription and mRNA degradation, we investigated whether increased transcription drives the accumulation of Pomc and Prss56 mRNAs. Fluorescent in situ hybridization was used to detect Pomc and Prss56 primary transcripts (hnRNA) using probes against intronic sequences. As expected, intronic hybridization signal was localized in nuclei rather than the cytoplasm; while Prss56 primary transcript was represented by only 1 or 2 intensely fluorescent nuclear dots, the hybridization signal for Pomc primary transcript ranged from a single dot to filling the entire nucleus. A high correlation was found between the expression levels of primary transcripts and the respective mRNAs. In general, high Pomc or Prss56 mRNA levels were accompanied by high Pomc or Prss56 hnRNA levels in tanycytes, whereas low hnRNA levels corresponded to low mRNA levels. These data indicate that variable Pomc and Prss56 mRNA levels highly correlate with the transcriptional activity of these genes, raising the possibility of oscillatory expression and/or activity of certain transcription factors in adult rat tanycytes. We hypothesize that this phenomenon may be related to the stem/progenitor cell function of tanycytes (Prevot et al., Endocr Rev, 2018) and reminiscent of the oscillatory expression of transcription factors in neural progenitor cells during development (Kageyama et al., Neurosci Res, 2018).

Cloning and Analysis of the Multiple Transcriptomes of Serine Protease Homologs in Crayfish (Procambarus clarkii)

ABSTRACTFive different serine protease homologs (SPH) transcripts presumably or possibly resulting from alternative splicing were cloned from the hemocytes of crayfish (Procambarus clarkii) in this paper. Although different deletions of cDNA of SPH-2 and SPH-4 were found in the 5ʹ untranslated regions, they shared the same open reading frame and encoded a 424 amino acids protein with a calculated molecular weight of 45.84 kDa compared with SPH-5. The predicted cutting site of the signal peptide was located between Ala22 and Glu23; a clip domain and a trypsin-like serine protease domain were located in the N-terminal and the C-terminal, respectively. Large deletions were found in the SPH-1 and SPH-3. Both of them lacked the clip domain. The 22 amino acids signal peptide existed in the SPH-1 coding protein, and a low complexity region (LCR) was formed in the N-terminal of it. The deduced protein of SPH-1 contained 358 amino acids with a molecular weight of 38.80 kDa. There was only one trypsin-like serine protease domain found in the C-terminal of the SPH-3 coding protein. The deduced protein of SPH-3 contained 250 amino acids with a molecular weight of 26.90 kDa. The amino acid Ser (S) of the catalytic triad in trypsin-like serine protease domain of the proteins analyzed in this paper was replaced by Gly (G), suggesting that the SPH-1, SPH-2, SPH-3, SPH-4, and SPH-5 were serine protease homologs.

The catalytic, stem, and transmembrane portions of matriptase-2 are required for suppressing the expression of the iron-regulatory hormone hepcidin

Matriptase-2 (MT2) is a type-II transmembrane, trypsin-like serine protease that is predominantly expressed in the liver. It is a key suppressor for the expression of hepatic hepcidin, an iron-regulatory hormone that is induced via the bone morphogenetic protein signaling pathway. A current model predicts that MT2 suppresses hepcidin expression by cleaving multiple components of the induction pathway. MT2 is synthesized as a zymogen that undergoes autocleavage for activation and shedding. However, the biologically active form of MT2 and, importantly, the contributions of different MT2 domains to its function are largely unknown. Here we examined the activities of truncated MT2 that were generated by site-directed mutagenesis or Gibson assembly master mix, and found that the stem region of MT2 determines the specificity and efficacy for substrate cleavage. The transmembrane domain allowed MT2 activation after reaching the plasma membrane, and the cytoplasmic domain facilitated these processes. Further in vivo rescue studies indicated that the entire extracellular and transmembrane domains of MT2 are required to correct the low-hemoglobin, low-serum iron, and high-hepcidin status in MT2-/- mice. Unlike in cell lines, no autocleavage of MT2 was detected in vivo in the liver, implying that MT2 may also function independently of its proteolytic activity. In conjunction with our previous studies implicating the cytoplasmic domain as an intracellular iron sensor, these observations reveal the importance of each MT2 domain for MT2-mediated substrate cleavage and for its biological function.

Kallikrein 5 inhibitors identified through structure based drug design in search for a treatment for Netherton Syndrome

Netherton syndrome (NS) is a rare and debilitating severe autosomal recessive genetic skin disease with high mortality rates particularly in neonates. NS is caused by loss-of-function SPINK5 mutations leading to unregulated kallikrein 5 (KLK5) and kallikrein 7 (KLK7) activity. Furthermore, KLK5 inhibition has been proposed as a potential therapeutic treatment for NS. Identification of potent and selective KLK5 inhibitors would enable further exploration of the disease biology and could ultimately lead to a treatment for NS. This publication describes how fragmentation of known trypsin-like serine protease (TLSP) inhibitors resulted in the identification of a series of phenolic amidine-based KLK5 inhibitors 1. X-ray crystallography was used to find alternatives to the phenol interaction leading to identification of carbonyl analogues such as lactam 13 and benzimidazole 15. These reversible inhibitors, with selectivity over KLK1 (10–100 fold), provided novel starting points for the guided growth towards suitable tool molecules for the exploration of KLK5 biology.

Purification and characterization of a sarcoplasmic serine proteinase from threadfin bream Nemipterus virgatus muscle

A sarcoplasmic serine proteinase (SSP) was purified from threadfin bream (Nemipterus virgatus) belly muscle by ammonium sulfate precipitation and a series of chromatographies including Q-Sepharose, Phenyl Sepharose and Superdex 200. The SSP was purified 1967 folds with a yield of 4.8%. The molecular weight of the SSP was estimated to be 43.5 kDa and 22.5 kDa on SDS-PAGE under non-reducing and reducing conditions, respectively. The N-terminal amino acid sequence of the two protein bands were determined as IVGGYEXQPYSQAHQVSLNSGY and corresponded. It is suggested that the SSP exists as a homodimer. Optimum pH and temperature were 9.5 and 50 °C, using Boc-Val-Pro-Arg-MCA as a substrate. Substrate specificity and effects of inhibitors indicated that the SSP was a trypsin-like serine proteinase. The SSP was responsible for hydrolyzing myosin heavy chain (MHC) and inducing modori phenomenon in the threadfin bream surimi gel. Thus, the SSP was considered as a modori-inducing proteinase.

Membrane-Anchored Serine Proteases and Protease-Activated Receptor-2-Mediated Signaling: Co-Conspirators in Cancer Progression.

Pericellular proteolysis provides a significant advantage to developing tumors through the ability to remodel the extracellular matrix, promote cell invasion and migration, and facilitate angiogenesis. Recent advances demonstrate that pericellular proteases can also communicate directly to cells by activation of a unique group of transmembrane G-protein-coupled receptors (GPCR) known as protease-activated receptors (PAR). In this review, we discuss the specific roles of one of four mammalian PARs, namely PAR-2, which is overexpressed in advanced stage tumors and is activated by trypsin-like serine proteases that are highly expressed or otherwise dysregulated in many cancers. We highlight recent insights into the ability of different protease agonists to bias PAR-2 signaling and the newly emerging evidence for an interplay between PAR-2 and membrane-anchored serine proteases, which may co-conspire to promote tumor progression and metastasis. Interfering with these pathways might provide unique opportunities for the development of new mechanism-based strategies for the treatment of advanced and metastatic cancers.

The function of two trypsin-like serine proteases from Eriocheir sinensis involved in Spiroplasma eriocheiris infection

Trypsin-like serine proteases (TLSPs), are members of the serine protease family and play important roles in innate immune responses. There are no previous studies regarding the role of TLSPs in immune responses to Spiroplasma eriocheiris in Eriocheir sinensis. In this paper, two TLSPs were identified from E. sinensis, namely EsTLSP-I and EsTLSP-II. Phylogenetic analysis showed that EsTLSP-I and EsTLSP-II clustered into two different groups that the crustaceans within each group were specifically. Tissue distribution analysis indicated that both EsTLSP-I and EsTLSP-II were mostly expressed in hemocytes, in the gill and in the intestine. The EsTLSP-I and EsTLSP-II transcription levels in hemocytes significantly increased after infection with S. eriocheiris. After dsRNA interference, the expression of both EsTLSP-I and EsTLSP-II distinctively declined from 48 h to 96 h. The expression of proPO and beta-1,3-glucan binding protein (LGBP) were also notably reduced after silencing. Compared to the control group, the copy number of S. eriocheiris increased significantly in interfering EsTLSP-I and EsTLSP-II groups during S. eriocheiris infection. Meanwhile, the mortality of crabs in the silenced EsTLSP-I and EsTLSP-II groups was higher than in the control group after the S. eriocheiris challenge. The results indicate that EsTLSP-I and EsTLSP-II play important roles in the innate immune system of Eriocheir sinensis, in combating infections of S. eriocheiris.