Studies On Structure And Immune Function Of The Complement Related Molecules In Portunus Trituberculatus

Complement system,as the important component and immune effector system of innate immunity,performs a crucial role in innate immune defense of invertebrates.Until now,studies on complement system remain deficient in crustaceans,especially in brachyuran crabs.In the current study,five complement like molecules,including the receptor for C1q(PtgC1qR),alpha-2 macroglobulin(PtA2M-1 and PtA2M-2),thioester-containing protein(PtTEP)and mannose-binding lectin(PtMBL),were isolated and functional studied from Portunus trituberculatus through gene cloning,quantitative real-time PCR,prokaryotic expression system and RNA interference techniques.We further investigated the relationship and immune function of these molecules in crab immune system.The receptor for the globular head of complement component C1 q,gC1qR,is a multifunctional and multiligand binding protein.In this study,a full-length cDNA sequence of a gC1 qR homolog(PtgC1qR)in P.trituberculatus was identified.PtgC1 qR was a 268-amino-acid polypeptide with a conserved MAM33 domain and a mitochondrial targeting sequence in the first 56 amino acids.The transcripts of PtgC1 qR were detected in all examined tissues with the highest level detected in hepatopancreas.Compared with other embryonic stages,PtgC1 qR was highly expressed in the fertilized eggs and embryos at the cleavage stage,which suggests PtgC1 qR may be a maternal gene.The transcripts of PtgC1 qR in hemocytes exhibited time-dependent response expression pattern after challenged with bacteria(Vibrio alginolyticus,Micrococcus luteus)and fungi(Pichia pastoris).Moreover,the recombinant PtgC1qR(rPtgC1qR)exhibited strong antibacterial activity and microbial-binding activity,suggesting its crucial role in immune defense and immune recognition.Further phenoloxidase(PO)assay showed that rPtgC1 qR could suppress the crab PO activity in vitro in a dose-dependent manner,and it could result in nearly 100% inhibition of PO activity under the concentration of 11.65 μM.Knockdown of PtgC1 qR could significantly enhance the expression of serine protease related genes(PtcSP1-3 and PtSPH),prophenoloxidase(proPO)associated genes(PtproPO and PtPPAF)and C3-like genes(PtA2M-1 and PtTEP).However,the phagocytosis related genes(PtMyosin,PtRab5 and PtArp)and PtA2M-2 were significantly down-regulated in the PtgC1qR-silenced crabs.Alpha-2 macroglobulin(A2M)is a ubiquitous protease inhibitor.Herein,two distinct A2 M genes(designated as PtA2M-1 and PtA2M-2,respectively)were isolated from P.trituberculatus.PtA2M-1 and PtA2M-2 encoded proteins with 1,541 or 1,516 amino acids,respectively,containing the typical functional domains of A2 M.Unlike highly expressed in hemocytes of most arthropods,PtA2M-1 and PtA2M-2 were predominantly detected in gill,eyestalk and digestive tracks.During the embryonic stages,PtA2 Ms were found to be expressed most highly in fertilized eggs,suggesting their maternal origin.After challenged with V.alginolyticus,the transcripts of PtA2 Ms showed similar time-dependent response expression pattern,while PtA2M-1 was more sensitive to M.luteus and P.pastoris infection than PtA2M-2.Knockdown of PtA2M-1 or PtA2M-2 could significantly enhance the expression of proPO-associated genes(PtproPO and PtPPAF)and serine protease related genes(PtcSP1-3 and PtSPH),however,PtLSZ and the phagocytosis-related genes(PtMyosin and PtRab5)were effectively inhibited.These results were further supported by the PO and lysozyme activities in hemolymph of the PtA2M-1-or PtA2M-2-silenced crabs.In addition,PtA2M-1 or PtA2M-2 could regulate the expression of antimicrobial peptide(AMP)genes(PtALF1-3,PtCrustin1 and PtCrustin3)through the Toll and NF-κB pathways.Thioester-containing proteins(TEPs)are an ancient superfamily of secreted effector proteins.In the present study,a TEP gene,designated as PtTEP,was identified from P.trituberculatus.The open reading frame(ORF)of PtTEP was 4,434 bp in length,encoded a polypeptide with 1,478 amino acids containing the conserved sequence features of insect TEP.PtTEP was highly expressed in most immune-related tissues,such as intestine,gill and hepatopancreas,and the expression of PtTEP was significantly upregulated in hemocytes after bacterial and fungal challenges.Compared to that in fertilized eggs,the transcripts of PtTEP showed up-regulated in the blastula,gastrula and heart beating stages.RNA interference-mediated suppression of PtTEP could significantly increase the expression of proPO-associated genes(PtproPO and PtPPAF)and serine protease related genes(PtcSP1-3 and PtSPH)but lead to a lower expression of PtLSZ and phagocytosis-related genes(PtMyosin and PtRab5).These results were further supported by the PO and lysozyme activities in hemolymph of the PtTEP-silenced crabs.Moreover,silencing of PtTEP reduced the expression of AMP genes and genes involved in activation of the Toll and NF-κB pathways and additionally increased the mortality of V.alginolyticus infected crabs.Mannose-binding lectin(MBL)is an essential pattern recognition receptor involved in activation of the complement lectin pathway.In this study,a novel MBL gene(PtMBL)was cloned from P.trituberculatus.The complete cDNA of PtMBL gene was 994 bp in length with an ORF of 777 bp.It encoded a protein with 259 amino acids containing the conserved carbohydrate-recognition domain(CRD)domain with QPD motif.PtMBL was highly expressed in most immune-related tissues,such as muscle,intestine,stomach and hepatopancreas,and the expression of PtMBL was significantly upregulated at early time after being challenged with bacteria and fungi.With the embryonic development,the transcripts of PtMBL were decreased significantly and reached to the lowest level at the heart-beating stage.Combined with high expression in ovary,it is suggested that PtMBL may be a maternal immune factor.Knockdown of PtMBL could significantly enhance the expression of PtTEP,proPO-associated genes(PtproPO and PtPPAF)and serine protease related genes(PtcSP1-3 and PtSPH),while the expression of PtA2 Ms and phagocytosis-related genes(PtArp,PtMyosin and PtRab5)was significantly suppressed.The PO activities were increased significantly in hemolymph of the PtMBL-silenced crabs.Furthermore,silencing of PtMBL gene enhanced the expression of AMP genes and genes involved in activation of the Toll and NF-κB pathways.The studies on complement-like molecules from the swimming crab indicated that they were as similar with vertebrate complement molecules in both structure and immune function.Research showed that PtgC1 qR and PtMBL as PRRs were participated in activation of the primitive complement system through regulating C3-like molecules(PtA2M-1,PtA2M-2 and PtTEP),and the primitive complement system in P.trituberculatus may perform its immune function via the serine protease cascades,proPO-activating system,phagocytosis,as well as the synthesis and secretion of AMP mediated by Toll and NF-κB immune pathways.Moreover,complement-like molecules may be maternally-derived immune factors to participate in the immune defense response to pathogens.Taken together,our results indicated that the complement-like molecules played an important role in the innate immune defense of the swimming crab.Meanwhile,it provided new clues and theoretical support for further understanding the evolution of complement system in invertebrates and systematically investigating the function of primitive complement system.