Identification And Function Studies Of Serpins In Litopenaeus Vannamei

Litopenaeus vannamei,also known as the Pacific white shrimp,is main shrimp species cultured in China.In recent years,due to the outbreak of diseases,shrimp farming industry have suffered huge losses in China and even the whole world.Therefore,efforts should be strengthened on the basic immunology research of shrimp while we enhanced the diseasecontrol programs,which may lay the molecular basis for shrimp resistance breeding.As low invertebrates,lacking of adaptive immunity compel shrimp to defense against the invasion of pathogens only depending on innate immune system.Pathogen invasion will activate the Toll/IMD pathways and the prophenoloxidase(proPO)activation system,by which the antimicrobial peptides and melanin were induced for killing microorganisms.Serine protease cascades were needed for the activation of these immune responses.However,the redundant proteases will cause damage for the host and further lead to the shrimp death.Thus activated serine proteases needed to be mediated strictly to function in specific time and location.Serine protease inhibitors(SPIs),typically known as serpins were the moderator and actively participated in the regulation process and irreversibly inhibited their specific target proteases.By homology-based cloning,shared network data and RACE methods,four serpin genes were isolated and identified from L.vannamei,and corresponding bioinformatic analysis were performand;the mRNA expression profiles of these four serpin genes were analyzed be the qRT-PCR;the gene-EGFP fusion expression vectors of serpins were constructed and transfected into Drosophila S2 cells for subcellular localization analysis;we use dual luciferase report system to detect the inhibitory effect of serpins on promoter activity of some antibacterial peptide;double stranded RNA mediated RNA interference was performed to study the regulating role of serpins;we constructed the prokaryotic expressional vector of serpins and induce the expression of serpins in vitro.The purified recombinant serpins protein(rLvserpins)was used to test the inhibitory activity;site-specific mutagenesis technology was used to investigate the functional role of P1P1’;the target protein interacted with serpinB3 were analyzed by GST-Pull down;western blot was used to study the inhibitory mechanism between serpin and proteases.The main research results obtained as follows:1.Four serpin genes were identified from L.vannamei,and named Lvserpin3,Lvserpin7,Lvserpin8,LvserpinB3 respectively.Putative signal peptide,characteristic serpin domain,RCL sequence and the canonical seven to eight α-helices and three β-sheets were identified from the four serpins.Moreover,a transmembrane helix(TMHs)was predicted to be located on the N-terminal of LvserpinB3.2.In the present study,we study the subcellular localization of Lvserpin3 and LvserpinB3.Confocal microscopy detected that Lvserpin3 distributes in the whole cell,LvserpinB3 locates mostly in mitochondria.Overexpression of Lvserpin3 in Drosophila S2 cells led to significant decreace of promoter activities of Drosophila Attacin A,while overexpression of LvserpinB3 led to significant decreace of promoter activities of L.vannamei Penaeidin4.Inhibition on the promoter activity might suggest that Lvserpin3 and LvserpinB3 can participate in the regulation of some antibacterial peptides and further to participate in the prawn antibacterial immunity.3.L.vannamei serpin3,Lvserpin8 and LvserpinB3 was highly expressed in hepatopancreas,Lvserpin7 was highly expressed in hemocytes;Being stimulated by V.anguillarum,the expression of Lvserpin3 was significantly increased in the early stage,while the transcripts of Lvserpin7,Lvserpin8 and LvserpinB3 were remarkably elevated at late stage compared to the PBS group.After challenged with M.lysodeikticus,the expression of Lvserpin3 and Lvserpin7 was significantly increased in the early stage,while the expression of Lvserpin8 and LvserpinB3 was significantly elevated at late stage.Moreover,the transcripts of these four serpin genes were significantly increased in the mid-to late stage of injection of WSSV.Silenced the expression of Lvserpin3 by dsRNA-mediate RNAi then challenged with V.anguillarum significantly increased the transcription of LvPPAF,LvproPO and PO activity and also led to increased mortality.Suppression of Lvserpin7 markedly increased the transcription of LvPPAF,LvPPAE and cumulative mortality.Silencing of Lvserpin8 remarkably increased the expression of LvPPAE2 and cumulative mortality.Suppression of LvserpinB3 significantly increased the mRNA expression of LvSP1,LvPPAE2 and cumulative mortality.4.The purified recombinant serpins protein(rLvserpins)was used to test the inhibitory activity in vitro.rLvserpin3 could solidly bind to B.subtilis and V.anguillarum,and weakly bind to M.lysodeikticus and E.coil.Moreover,rLvserpin3 could effectively restrain the growth of M.lysodeikticus and B.subtilis by inhibiting the secreted bacterial proteases.rLvserpin7 protein not only showed strong binding activity to B.subtilis and V.anguillarum,but also could inhibit the protease secreted by B.subtilis.rLvserpin8 exhibited stronger inhibitory activity against trypsin,while rLvserpinB3 displayed significant inhibition activities on trypsin,elastase,and papain.What’s more,rLvserpin3,rLvserpin7,rLvserpin8 and rLvserpin B3 could impede the extent of proPO cascade.5.The protease inhibitory activities of the R373 G mutants dramatically decreased compared to that of wild-type.The inhibitory activities of the I374 G mutants were decreased but no significance was found compared to that of wild-type.While mutations of RI to GG also led to a significant decrease of protease inhibitory activities.GST-Pull down analysis showed that serpinB3 could interact with LvSP1 protein which with the same P1P1’ residues.This suggests that serpin can identify the proteases with the same P1P1 ’site and further play the inhibition role.Change of the P1P1’ sites lead to the different effect on inhibition of protease,which might suggest that the P1 residue of serpin determines the primary selectivity,as well as determines the target specificity.There was no corresponding band of the serpinB3-protease complex was detected by western blot analysis.Instead,a peptide band with small molecular weight was detected and became stronger with increased of serpinB3 levels.In light of the above findings,this paper suggests that different serpin members have different effects on shrimp response to invaders.It can be developed through regulating synthesis of AMPs or even by inhibiting bacterial protease,and the protease involved in the proPO activating system.Studying of the immune regulation role of serpins,which being used as a selection index will provide theoretical basis for L.vannamei disease-resistant breeding.