Structural and functional analysis of transforming growth factor beta regulator 1 (TBRG1) in the red swamp crayfish Procambarus clarkii: The initial insight into TBRG1's role in invertebrate immunity.

The transforming growth factor beta regulator 1 (TBRG1) is a growth inhibitory protein that acts as a tumor suppressor in human cancers, gaining its name for the transcriptional regulation by TGF-ß. While extensive research has been conducted on the tumor-related function of TBRG1 in mammals, its significance in invertebrates remains largely unexplored. In this study, a homolog of TBRG1 was first structurally and functionally analyzed in the red swamp crayfish Procambarus clarkii. The full-length cDNA sequence was 2143 base pairs (bp) with a 1305 bp open reading frame (ORF) encoding a deduced protein of 434 amino acids (aa). The changes of PcTBRG1 transcripts upon immune challenges indicated its involvement in innate immunity. After knocking down PcTBRG1, the decline of bacteria clearance capacity revealed the participation of PcTBRG1 in the immune response. Furthermore, the downregulation of AMPs' expression after the cotreatment of RNAi and bacteria challenge suggested that PcTBRG1 might participate in innate immunity through regulating AMPs' expression. These results provided initial insight into the immune-related function of TBRG1 in invertebrates.

Identification and functional analysis of cytosolic phospholipase A2 (cPLA2) from the red swamp crayfish Procambarus clarkii: The first evidence of cPLA2 involved in immunity in invertebrates.

Cytosolic phospholipase A2 (cPLA2) specifically liberates the arachidonic acids from the phospholipid substrates. In mammals, cPLA2 serves as a key control point in inflammatory responses due to its diverse downstream products. However, the role of cPLA2 in animals lower than mammals largely remains unknown. In the current research, a homolog of cPLA2 was first identified and characterized in the red swamp crayfish Procambarus clarkii. The full-length cDNA of PccPLA2 was 4432 bp in length with a 3036 bp-long open reading frame, encoding a putative protein of 1011 amino acids that contained a protein kinase C conserved region 2 and a catalytic subunit of cPLA2. PccPLA2 was ubiquitously expressed in all examined tissues with the highest expression in the hepatopancreas, and the expression in hemocytes as well as hepatopancreas was induced upon the immune challenges of WSSV and Aeromonas hydrophila. After the co-treatment of RNA interference and bacterial infection, the decline of bacteria clearance capability was observed in the hemolymph, and the expression of some antimicrobial peptides (AMPs) was significantly suppressed. Additionally, the phagocytosis of A. hydrophila by primary hemocytes decreased when treated with the specific inhibitor CAY10650 of cPLA2. These results indicated the participation of PccPLA2 in both cellular and humoral immune responses in the crayfish, which provided an insight into the role that cPLA2 played in the innate immunity of crustaceans, and even in invertebrates.

Identification and functional analysis of endoplasmic reticulum oxidoreductase 1 (ERO1) from the green mud crab Scylla paramamosain: The first evidence of ERO1 involved in invertebrate immune response.

Endoplasmic reticulum oxidoreductase 1 (ERO1) is an important mediator in regulating disulfide bond formation and maintaining endoplasmic reticulum homeostasis. Its activity is transcriptionally regulated by the unfolded protein response (UPR) in the endoplasmic reticulum, which is known to be essential in immunity. However, whether ERO1 is involved in innate immunity in invertebrates remains unclear. In the present study, two subtypes of ERO1 from Scylla paramamosain were first identified and characterized. Sequence analysis revealed the conserved ERO1 domain and the oxidative capacity assay verified the oxidative capacity of SpERO1 recombinant protein. Moreover, SpERO1s were found to be ubiquitously expressed in all the tested tissues, with the highest expression observed in hemocytes. Two SpERO1s exhibited distinct expression patterns in response to Vibrio alginolyticus and White Spot Syndrome Virus (WSSV). Importantly, the downregulation of the expression of immune factors upon bacterial challenge in SpERO1-silenced crabs was observed. These results provided an initial foundation for further investigations into the role of ERO1 in the innate immunity of invertebrates.

The first identification and functional analysis of two drosophila mothers against decapentaplegic protein genes (SpSmad1 and SpSmad2/3) and their involvement in the innate immune response in Scylla paramamosain.

Smad，a member of the TGF-ß superfamily，controls cell proliferation，growth and guiding cell differentiation, thus playing a crucial role in diseases. However, the presence as well as specific function of Smad in crabs is still unknown. In this study, two Smads (Smad1 and Smad2/3) were identified for the first time from the mud crab Scylla paramamosain. The complete open reading frames of SpSmad1 and SpSmad2/3 were 1,497bp and 1,338bp, encoding deduced proteins of 498 and 445 amino acids respectively. Moreover, under the administration of Vibrio alginolyticus and WSSV, the relative expression levels of SpSmad1 and SpSmad2/3 were significantly increased, indicating their involvement in the innate immune response of mud crabs. Knockdown of SpSmad1 and SpSmad2/3 in vivo not only led to the increasement of the expressions of NF-κB signaling genes and antimicrobial peptides genes, but also significantly affected the bacterial clearance process of mud crabs. Additionally, overexpression of SpSmad1 and SpSmad2/3 in HEK293T cells could markedly activate NF-κB signaling. These results indicated that Smad1 and Smad2/3 participated in the innate immunity of Scylla paramamosain, and might provide a better understanding of the presence and immune regulatory functions of Smad1 and Smad2/3 in crabs and even invertebrates.

Characterization of fibroblast growth factor receptor 4 (FGFR4) from the red swamp crayfish Procambarus clarkii and its role in antiviral and antimicrobial immune responses.

FGFRs involved multiple physiological processes, such as endocrine homeostasis, wound repair, and cellular behaviors including proliferation, differentiation and survival. In the present study, the homologs of fibroblast growth factor receptor 4 (FGFR4) were identified and characterized from the red swamp crayfish Procambarus clarkii for the first time. The full-length cDNAs of pcFGFR4 were 2878 bp with 2451 bp open reading frame (ORF), respectively. The deduced pcFGFR4 protein contained an immunoglobulin, two immunoglobulin C-2 Type, a transmembrane region and a catalytic domain. Real-time PCR analysis showed that pcFGFR4 were highly expressed in muscle and hemocyte. Moreover, the expression levels of pcFGFR4 in the hepatopancreas and hemocyte were positively stimulated after challenge with Aeromonas hydrophila and WSSV, implying the involvement of pcFGFR4 against bacterial and viral infections in innate immune responses. While pcFGFR4 were silenced in vivo, the expression levels of antimicrobial peptide (AMP) genes (pcALF1-5,8 and pcCrustin1-2) and NF-κB signaling components (pcDrosal and pcRelish) were significantly reduced. Additionally, NF-κB signaling could be markedly activated by overexpression of pcFGFR4 in HEK293T cells. Finally, our results indicated that pcFGFR4 regulated crayfish's innate immunity by modulating NF-κB signaling. These findings may provide new insights into pcFGFR4-mediated signaling cascades in crustaceans and provide a better understanding of crustacean innate immune system.

Identification and functional analysis of two drosophila mothers against decapentaplegic protein(Smad)genes and their involvement in immune responses in the red swamp crayfish Procambarus clarkii.

Drosophila mothers against decapentaplegic proteins (Smads), the crucial signal transducers in activating downstream gene transcription through transforming growth factor beta (TGF-ß) receptors, are the pleiotropic factors with important role in mediating cell proliferation, homeostasis, differentiation, apoptosis and immune response. However, whether Smads are involved in immune response in crustaceans remains unexplored. In the present study, the Smad3 and Smad4 were firstly identified and functionally characterized from the Red Swamp Crayfish Procambarus clarkii. The full-length cDNAs of pcSmad3 and pcSmad4 were 1, 670 bp and 3, 060 bp with 1, 326 bp and 1, 875 bp open reading frame (ORF), respectively. Real-time PCR analysis of the expression profiles demonstrated that pcSmad3 and pcSmad4 were predominantly expressed at in stomach, heart, and hemocytes. Notably, the expression levels of pcSmad3 and pcSmad4 both Aeromonas hydrophila and WSSV challenges were significantly altered, suggesting the involvement of pcSmad3 and pcSmad4 in innate immune responses. Knockdown of pcSmad3 and pcSmad4 in vivo dramatically activated the transcriptions of NF-κB signaling genes and anti-lipopolysaccharide factor genes. The overexpression of pcSmad3 and pcSmad4 could significantly activate NF-κB signaling in HEK293T cells. Meanwhile, the clearance of bacteria was significantly reduced with knockdown of pcSmad3 and pcSmad4 in vivo. Results indicated that pcSmad3 and pcSmad4 played an immune-regulatory role in crayfish's innate immunity, which might pave the for a better understanding of the TGF-ß superfamily members in crustacean.

Identification and functional analysis of drosophila mothers against decapentaplegic protein gene 1 (Smad1) from the red swamp crayfish Procambarus clarkii: The first evidence of Smad1 involved in immunity in invertebrates.

Smads, part of signaling cascades that represent downstream pathways of the TGF-ß super family proteins, are pleiotropic cytokines with important role in mediating cell proliferation, homeostasis, differentiation, apoptosis and immune response. However, the specific functions of Smads remain unknown in crustaceans. In the present study, the drosophila mothers against decapentaplegic protein gene 1 (Smad1) was firstly identified and characterized from the Red Swamp Crayfish Procambarus clarkii. The obtained cDNA sequence of pcSmad1was 2, 503 bp long with a 1, 488 bp open reading fame, which encoded a putative protein of 496 amino acids. Furthermore, pcSmad1 responded to both Aeromonas hydrophila and WSSV challenge, suggesting the involvement of pcSmad1 in innate immune responses. Knockdown of pcSmad1 in vivo dramatically increased the expressions of NF-κB signaling genes and anti-lipopolysaccharide factor genes. Additionally, overexpression of pcSmad1 in HEK293T cells could markedly activate NF-κB signaling. Taken together, these results indicated that pcSmad1 played an immune-regulatory role in crayfish's innate immunity, which may provide a better understanding of TGF-ß superfamily members in crustacean.

Identification and function analysis of two fibroblast growth factor receptor (FGFR) from Scylla paramamosain: The evidence of FGFR involved in innate immunity in crustacean.

The fibroblast growth factor receptor (FGFR) belongs to the tyrosine kinase family consisting of four members (FGFR1-4). This study involved identification and characterization of FGFR1 and FGFR3 from mud crab Scylla paramamosain for the first time. The obtained cDNAs of SpFGFR1 and SpFGFR3 were 2,380 bp and 2,982 bp in length with a 1,503 bp and 2,310 bp open reading frame, respectively. The predicted SpFGFR1 protein included three immunoglobulin domains and a transmembrane region, while SpFGFR3 protein possessed a typical TyrKc (Tyrosine kinase, catalytic) domain. Real-time PCR analysis showed that SpFGFR1 and SpFGFR3 were highly expressed in the hepatopancreas. Furthermore, the expression levels of SpFGFR1 and SpFGFR3 in the hepatopancreas were enhanced following challenges with Vibro alginolyticus, Staphylococcus aureus, Poly (I:C) and White spot syndrome virus, which shows the involvement of SpFGFR1 and SpFGFR3 in innate immune response to infections from bacteria and virus. There was significant suppression of six antimicrobial peptide genes (SpALF1-5 and SpCrustin) and three NF-κB members (SpDorsal, SpIKK and SpRelish) when SpFGFR1 and SpFGFR3 was interfered in vivo. Also, treatment of the hemocytes with specific inhibitor of SpFGFR for 24 h consistently down-regulated SpDorsal, SpRelish and AMPs. These results suggested that SpFGFR1 and SpFGFR3 played important roles in regulating the Toll signaling pathway and immune deficiency (IMD) pathway through NF-κB signaling pathway. These findings may provide new insights into the role of FGFRs in the innate immune function of crustaceans.

Identification and functional analysis of transforming growth factor-ß type III receptor (TßR3) from Scylla paramamosain: The first evidence of TßR3 involved in development and innate immunity in invertebrates.

Transforming growth factor-ß type III receptor (TßR3), as a co-receptor of TGF-ß superfamily, plays critical roles in development and growth as well as some disease pathogeneses by presenting ligands to other receptors in vertebrates. However, the identification and functional characterization of TßR3 had not been reported yet in invertebrates. In the present study, TßR3 was first identified and characterized in mud crab Scylla paramamosain. The obtained cDNA length of SpTßR3 was 2, 424 bp with a 1, 854 bp open reading frame, which encoded a putative peptide of 617 amino acids containing a typical transmembrane region and a Zona pellucida (ZP) domain. Real-time PCR results showed that SpTßR3 was predominantly expressed at early embryonic development stage and early postmolt stage, suggesting its participation in development and growth. We report, for the first time in invertebrates, the challenge of both Vibro alginolyticus and Poly (I:C) could alter the expression patterns of SpTßR3. Notably, the expression levels of SpIKK, two NF-κB members (SpRelish and SpDorsal), and five antimicrobial peptide genes (SpCrustin and SpALF1-4) were significantly suppressed when SpTßR3 was interfered in vivo. Secondly, the overexpression of SpTßR3 in vitro could activate NF-κB signaling through the dual-luciferase reporter assays. Furthermore, the bacterial clearance assay after SpTßR3 was silenced in vivo highlighted the potential of SpTßR3 in activating the innate immune responses. These results implied the involvement of SpTßR3 in the innate immune responses by regulating the NF-κB pathway. This study first indicated that TßR3 was present in invertebrate, and it participated in not only the development and growth but also the innate immunity of S. paramamosain. It also provided new insights into the origin or evolution of TGF-ß receptors in crustacean species and even in invertebrates.

Transforming growth factor-ß-activating kinase 1 and its binding protein 1 participate in the innate immune responses via modulating the IMDNFκB signaling in mud crab (Scylla paramamosain).

Transforming growth factor-ß-activating kinase 1 (TAK1) is essential for diverse important biological functions, such as innate immunity, development and cell survival. In the present study, the homologs of TAK1 and TAK1-binding protein 1 (TAB1) were identified and characterized from mud crab Scylla paramamosain for the first time. The full-length cDNAs of SpTAK1 and SpTAB1 were 2, 226 bp and 2, 433 bp with 1, 782 bp and 1, 533 bp open reading frame (ORF), respectively. The deduced SpTAK1 protein contained a conserved S_TKc (Serine/threonine protein kinases, catalytic) domain, and the putative SpTAB1 protein possessed a typical PP2Cc (Serine/threonine phosphatases, family 2C, catalytic) domain and a potential TAK1 docking motif. Real-time PCR analysis showed that SpTAK1 and SpTAB1 were highly expressed at early development stages, suggesting their participation in crab's development process. Moreover, the expression levels of SpTAK1 and SpTAB1 in hepatopancreas were positively stimulated after challenge with Vibro alginolyticus and Poly (I:C), implying the involvement of SpTAK1 and SpTAB1 in innate immune responses against both bacterial and viral infections. When SpTAK1 or SpTAB1 were silenced in vivo, the expression levels of two IMDNFκB signaling components (SpIKKß and SpRelish) and six antimicrobial peptide (AMP) genes (SpALF1-5 and SpCrustin) were significantly reduced, and the bacteria clearance capacity of crabs was also markedly impaired in SpTAK1 or SpTAB1 silenced crabs. Additionally, overexpression of SpTAK1 and SpTAB1 in HEK293T cells could markedly activate the mammalian NF-κB signaling. Collectively, our results suggested that TAK1 and TAB1 regulated crab's innate immunity via modulating the IMDNFκB signaling. These findings may provide new insights into the TAK1/TAB1-mediated signaling cascades in crustaceans and pave the way for a better understanding of crustacean innate immune system.