Ai-Qing Yu

A Double WAP Domain-Containing Protein Es-DWD1 from Eriocheir sinensis Exhibits Antimicrobial and Proteinase Inhibitory Activities

Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides, which are critical in the host immune response against microbial invasion. The common feature of these proteins is a single WAP domain maintained by at least one four-disulfide core (4-DSC) structure rich in cysteine residues. In this study, a double WAP domain (DWD)-containing protein, Es-DWD1, was first cloned from the Chinese mitten crab ( Eriocheir sinensis ). The full-length Es-DWD1cDNA was 1193 bp, including a 411 bp open reading frame (ORF) encoding 136 amino acids with a signal peptide of 22 amino acids in the N-terminus. A comparison with other reported invertebrate and vertebrate sequences revealed the presence of WAP domains characteristic of WAP superfamilies. As determined by quantitative real-time RT-PCR, Es-DWD1 transcripts were ubiquitously expressed in all tissues, but it was up-regulated in hemocytes post-challenge with pathogen-associated molecular patterns (PAMPs). The mature recombinant Es-DWD1 (rEs-DWD1) protein exhibited different binding activities to bacteria and fungus. Moreover, rEs-DWD1 could exert agglutination activities against Bacillus subtilis and Pichia pastoris and demonstrated inhibitory activities against the growth of Staphylococcus aureus, Aeromonas hydrophila and P . pastoris . Furthermore, rEs-DWD1 showed a specific protease inhibitory activity in B. subtilis. Coating of rEs-DWD1 onto agarose beads enhanced encapsulation of the beads by crab hemocytes. Collectively, the results suggest that Es-DWD1 is a double WAP domain containing protein with antimicrobial and proteinase inhibitory activities, which play significant roles in the immunity of crustaceans.

Immunoglobulin superfamily protein Dscam exhibited molecular diversity by alternative splicing in hemocytes of crustacean, Eriocheir sinensis

Be absent of adaptive immunity which have both specificity and memory, invertebrates seem to have evolved alternative adaptive immune strategies to resist various intruding pathogens. Whereas vertebrates could generate a wide range of immunological receptors with somatic rearrangement, invertebrates possibly depend on alternative splicing of pattern-recognition receptors (PRRs). Recently, it has been suggested that a member of the immunoglobulin superfamily (IgSF), Down syndrome cell adhesion molecule (Dscam), plays a crucial role in the alternative adaptive immune system of invertebrates. At present, we successfully isolated and characterized the first crab Dscam from Eriocheir sinensis. EsDscam has typical domain architecture compared with other Dscam orthologs, including one signal-peptide, 10 immunoglobulin (Ig) domains, 6 fibronectin type III domains (FNIII), one transmembrane domain (TM) and one cytoplasmic tail. We had detected four hypervariable regions of EsDscam in the N-terminal halves of Ig2 (25) and Ig3 domain (30), the complete Ig7 (18) and also the transmembrane domain (2), potentially generate 27,000 unique isoforms at least. Transcription of EsDscam were both a) detected in all tissues, especially in immune system, digestive system and nerve system; b) significantly induced in hemocytes post lipopolysaccharides (LPS), peptidoglycans (PG) and β-1, 3-glucans (Glu) injection. Importantly, we had detected membrane-bound and secreted Dscam isoforms in E. sinensis, and showed that secreted isoforms were extensively transcribed post different PAMPs challenge respectively. Results from immuno-localization assay revealed that EsDscam evenly distributed in the cell surface of hemocytes. These findings indicated that EsDscam is a hypervariable PRR in the innate immune system of the E. sinensis.

Two antibacterial C-type lectins from crustacean, Eriocheir sinensis, stimulated cellular encapsulation in vitro

The first step of host fighting against pathogens is that pattern recognition receptors recognized pathogen-associated molecular patterns. However, the specificity of recognition within the innate immune molecular of invertebrates remains largely unknown. In the present study, we investigated how invertebrate pattern recognition receptor (PRR) C-type lectins might be involved in the antimicrobial response in crustacean. Based on our previously obtained completed coding regions of EsLecA and EsLecG in Eriocheir sinensis, the recombinant EsLectin proteins were produced via prokaryotic expression system and affinity chromatography. Subsequently, both rEsLecA and rEsLecG were discovered to have wide spectrum binding activities towards microorganisms, and their microbial-binding was calcium-independent. Moreover, the binding activities of both rEsLecA and rEsLecG induced the aggregation against microbial pathogens. Both microorganism growth inhibitory activities assays and antibacterial activities assays revealed their capabilities of suppressing microorganisms growth and directly killing microorganisms respectively. Furthermore, the encapsulation assays signified that both rEsLecA and rEsLecG could stimulate the cellular encapsulation in vitro. Collectively, data presented here demonstrated the successful expression and purification of two C-type lectins proteins in the Chinese mitten crab, and their critical role in the innate immune system of an invertebrate.

Two novel short C-type lectin from Chinese mitten crab, Eriocheir sinensis, are induced in response to LPS challenged.

The basic mechanism of host fighting against pathogens is pattern recognition receptors recognized pathogen-associated molecular patterns. However, the specificity of recognition within the innate immune molecular of invertebrates remains largely unknown. For this reason, we investigated the immune functionality of two pattern recognition receptors, C-type lectin EsLecA and EsLecG, post lipopolysaccharides (LPS) challenge in Chinese mitten crab (Eriocheir sinensis), which is a commercially important and disease vulnerable aquaculture species. The cloning of full-length EsLecA and EsLecG cDNA were based on the initial expressed sequence tags (EST) isolated from a hepatopancreatic cDNA library via PCR. The EsLecA cDNA contained a 480-bp open reading frame that encoded a putative 159-amino-acid protein, while EsLecG cDNA contained a 465-bp open reading frame that encoded a putative 154-amino-acid protein. Comparison, with other reported invertebrate and vertebrate sequences, revealed the presence of carbohydrate recognition domains that were common among C-type lectin superfamilies. EsLecA and EsLecG mRNA expression in E. sinensis were (a) both detected in all tissues, including the hepatopancreas, gills, hemocytes, testis, accessory gland, ovary, muscle, stomach, intestine, heart, thoracic ganglia and brain, and (b) responsive in hepatopancreas, gill, hemocytes post-LPS immuno-challenge all appeared dramatically variation. Collectively, the data presented here demonstrate the successful isolation of two novel C-type lectins from the Chinese mitten crab, and their role in the innate immune system of an invertebrate.

Two novel Toll genes (EsToll1 and EsToll2) from Eriocheir sinensis are differentially induced by lipopolysaccharide, peptidoglycan and zymosan

Tolls/Toll-like receptors (TLRs) play an essential role in initiating innate immune responses against pathogens and are found throughout the insect kingdom but have not yet been reported in the crustacean, Eriocheir sinensis. For this purpose, we cloned two novel Toll genes from E. sinensis, EsToll1 and EsToll2. The full-length cDNA of EsToll1 was 3963 bp with a 3042-bp open reading frame (ORF) encoding a 1013-amino acid protein. The extracellular domain of this protein contains 17 leucine-rich repeats (LRRs) and a 139-residue cytoplasmic Toll/interleukin-1 receptor (TIR) domain. The cDNA full-length of EsToll2 was 4419 bp with a 2667-bp ORF encoding an 888-amino acid protein with an extracellular domain containing 10 LRRs and a 139-residue cytoplasmic TIR domain. By phylogenetic analysis, EsToll1 and EsToll2 clustered into one group together with Tolls from other crustaceans. Quantitative RT-PCR analysis demonstrated that a) both EsToll1 and EsToll2 were constitutively expressed in all tested crab tissues; b) EsToll1 and EsToll2 were differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsToll2 expression was significantly upregulated at almost all time intervals post-challenge with LPS, PG and GLU. Our study indicated that EsToll1 and EsToll2 are differentially inducibility in response to various PAMPs, suggesting their involvement in a specific innate immune recognition mechanism in E. sinensis.

A novel C-type lectin from Eriocheir sinensis functions as a pattern recognition receptor with antibacterial activity.

As pattern recognition receptors (PRRs), C-type lectins (CTLs) play significant roles in recognizing and eliminating pathogens in innate immunity. In this study, a novel CTL (EsLecD) was identified from the crustacean Eriocheir sinensis. The cloning of full-length EsLecD cDNA was based on the initial expressed sequence tags (ESTs) isolated from a hepatopancreatic cDNA library. The full-length EsLecD cDNA of 686 bp with an open reading frame of 468 bp encodes a putative protein of 155 aa residues, including an N-terminal signal peptide and a single carbohydrate-recognition domain (CRD). By quantitative RT-PCR analysis, the EsLecD transcript was mainly detected in the hepatopancreas but rarely in other tissues, and it was significantly upregulated in the hepatopancreas after immune challenge with lipopolysaccharides. The recombinant EsLecD protein (rEsLecD) exhibited the ability to bind to all tested microorganisms, including bacteria and yeast. Meanwhile, calcium significantly increased the binding affinity of rEsLecD toward microorganisms, but it was not essential. The binding of rEsLecD induced the aggregation of microbial pathogens. Moreover, rEsLecD was capable of inhibiting the growth of microorganisms and even directly killing bacteria. Interestingly, rEsLecD could stimulate cellular encapsulation in vitro. In conclusion, results of this study suggest that EsLecD acts as an antibacterial PRR participating in the innate immunity of invertebrates.

Association of a hepatopancreas-specific C-type lectin with the antibacterial response of Eriocheir sinensis.

Pattern recognition receptors (PPRs) are part of the initial step of a host defense against pathogens in detecting pathogen-associated molecular patterns. However, determinants of the specificity of this recognition by innate immune molecules of invertebrates remain largely unknown. In this study, we investigated the potential involvement of an invertebrate PRR C-type lectin in the antimicrobial response of the crustacean Eriocheir sinensis. Based on the initial expressed sequence tags (EST) of a hepatopancreatic cDNA library, the full-length EsLecF cDNA was cloned and determined to contain a 477-bp open reading frame encoding a putative 158-amino-acid protein. A comparison with other reported invertebrate and vertebrate C-type lectin superfamily sequences revealed the presence of a common carbohydrate recognition domain (CRD). EsLecF transcripts in E. sinensis were mainly detected in the hepatopancreas and were inducible by a lipopolysaccharide (LPS) injection. The recombinant EsLecF (rEsLecF) protein produced via a prokaryotic expression system and affinity chromatography was found to have a wide spectrum of binding activities towards various microorganisms, and its microbial-binding activity was calcium-independent. Moreover, the binding of rEsLecF induced the aggregation of microbial pathogens. Results of the microorganism growth inhibitory assay and antibacterial assay revealed capabilities of rEsLecF in suppressing microorganism growth and directly killing bacteria, respectively. Furthermore, rEsLecF could enhance cellular encapsulation in vitro. Collectively, the findings presented here demonstrated the successful isolation of a novel C-type lectin in a crustacean and highlighted its critical role in the innate immunity of an invertebrate.

Identification and characterization of Tube in the Chinese mitten crab Eriocheir sinensis

As a key component of the Toll signaling pathway, Tube plays central roles in many biological activities, such as survival, development and innate immunity. Tube has been found in shrimps, but has not yet been reported in the crustacean, Eriocheir sinensis. In this study, we cloned the full-length cDNA of the adaptor Tube for the first time from E. sinensis and designated the gene as EsTube. The full-length cDNA of EsTube was 2247-bp with a 1539-bp open reading frame (ORF) encoding a 512-amino acid protein. The protein contained a 116-residue death domain (DD) at its N-terminus and a 272-residue serine/threonine-protein kinase domain (S_TKc) at its C-terminus. Phylogenetic analysis clustered EsTube initially in one group with other invertebrate Tube and Tube-like proteins, and then with the vertebrate IRAK-4 proteins, finally with other invertebrate Pelle proteins. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that EsTube was highly expressed in the ovary and testis, and moderately expressed in the thoracic ganglia and stomach. EsTube was expressed at all selected stages and was highly expressed in the spermatid stage (October, testis) and the stage III-2 (November, ovary). EsTube was differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (β-1,3-glucan). Our study indicated that EsTube might possess multiple functions in immunity and development in E. sinensis.

Molecular cloning and expression analysis of a dorsal homologue from Eriocheir sinensis

Dorsal as a crucial component of Toll signaling pathway, played important roles in induction and regulation of innate immune responses. In this study, we cloned a NF-κB-like transcription factor Dorsal from Eriocheir sinensis and designated it as EsDorsal. The full-length cDNA of EsDorsal was 2493 bp with a 2022-bp open reading frame (ORF) encoding a 673-amino acid protein. This protein contained a 171-residue conserved Rel homology domain (RHD) and a 102-residue Ig-like, plexins and transcription factors domain (IPT). By phylogenetic analysis, EsDorsal was clustered into one group together with other invertebrate Dorsals or NF-κBs, and then clustered with vertebrate NF-κBs. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that (a) EsDorsal had higher expression level in immune organs; (b) EsDorsal differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsDorsal was more responsive to LPS than GLU and PG. Collectively, EsDorsal was differentially inducibility in response to various PAMPs, suggesting its involvement in a specific innate immune regulation in E. sinensis.

Molecular characterization and sub-cellular distribution of JNK and JIP4 protein kinases in spermatogenesis and acrosome reaction of the Chinese mitten crab Eriocheir sinensis H. Milne Edwards, 1853 (Crustacea: Brachyura: Varunidae)

The c-Jun N-terminal protein kinase mitogen-activated protein kinases (JNK MAPKs) are an evolutionarily conserved family of serine/threonine protein kinases involved in controlling cell growth, differentiation, and apoptosis in mammalian testis and germ cells. As a scaffold protein of the JNK pathway, JNK-interacting protein 4 (JIP4) is found in some mammalian spermatozoa and participates in certain male reproductive processes. Few have reported on the roles of JNK and JIP4 in the Chinese mitten crab Eriocheir sinensis H. Milne Edwards, 1853. Based on transcriptome sequences from the testis of E. sinensis , we successfully cloned full-length cDNAs of JNK and JIP4 (designated Es-JNK and Es-JIP4 , respectively). Western blotting showed relatively high expression of Es -JNK and Es -JIP4 proteins in the testis. Expression levels of total Es -JNK, phosphorylated Es -JNK ( Es -p-JNK), and Es -JIP4 proteins decreased gradually during spermatogenesis. Es-JNK and Es-JIP4 mRNAs were mainly localized in the nucleus from the spermatogonium stage to the secondary spermatocyte stage and were widely distributed in the nuclear cup, apical cap, acrosomal tubule, and acrosomal vesicle of spermatids. Es -JNK, Es -p-JNK, and Es -JIP4 proteins were nevertheless mainly dispersed in the cytoplasm and intercellular substance of spermatogonium and primary spermatocytes, in the nucleus of secondary spermatocytes, and in the apical cap, acrosomal tubule, acrosomal vesicle, and nuclear cup of spermatids. During the acrosome reaction induced by the calcium ionophore A23187, Es -JNK, Es -p-JNK, and Es -JIP4 proteins were found mainly expressed in the apical cap and acrosomal vesicle by immunofluorescence microscopy. The results implicate functions for Es -JNK and Es -JIP4 in spermatogenesis and the acrosome reaction in E. sinensis .