Study On Gasdermin D Cleavage And Effect Mediated By Porcine Caspase-1

Pigs are an important livestock,a major source of protein and a potential donor for organ transplantation.Studying pig immune system and immune mechanism is of great significance to promote the development of pig industry and explore the feasibility of xenotransplantation.Pyroptosis is a newly discovered inflammatory type of programmed cell death,which is different from the apoptosis.In the pathway of pyroptosis,the activated caspases cleaves the "executor" Gasdermin(GSDM)family proteins and produces GSDM-N,which causes the cell membrane to rupture.At present,the research on pyroptosis is mainly focused on humans and mice,but there are few studies on the mechanism of porcine pyroptosis and related proteins.In this study,the prokaryotic expression system was used to express recombinant porcine caspase-1(rpcaspase-1)p20 protein.New Zealand rabbits and BALB/c mice were immunized with rpcaspase-1 p20 to prepare pcaspase-1 p20 specific polyclonal and monoclonal antibodies.Western blot results showed that the prepared polyclonal antibody can recognize rpcaspase-1 p20 and pcaspase-1 p20 overexpressed in human embryonic kidney cells(HEK293).A series of identifications were carried out on three hybridoma cells 4E7,3C2 and 2H5 that can secrete pcaspase-1 p20 monoclonal antibody.Western blot results showed that the monoclonal antibodies secreted by the three cell lines can specifically recognize rpcaspase-1 p20 and the natural pcaspase-1 expressed in porcine duodenum and jejunum tissues.Then,this study used the prepared pcaspase-1 antibody and the porcine Gasdermin D(pGSDMD)specific monoclonal antibody prepared and stored in our laboratory to detect some tissue proteins of piglets infected enterotoxigenic Escherichia coli(ETEC),Western blot results showed that the activation of pcaspase-1 and the cleavage of pGSDMD occurred in the duodenum and jejunum tissues of piglets.In view of the fact that pcaspase-1 activation and pGSDMD cleavage events are consistent in the tissue distribution characteristics,it is speculated that pcaspase-1 may be related to the cleavage of pGSDMD.Therefore,this study further studied the ability and mechanism of pGSDMD cleaved by pcaspase-1.On the one hand,this study used prokaryotic expression system to prepare recombinant pGSDMD(rpGSDMD),rpGSDMD mutant and rpcaspase-1 with enzymatic activity.The prepared recombinant protein proved that rpcaspase-1 has the ability to cleave rpGSDMD;the mutation of aspartic acid(D)at position 279 of rpGSDMD to alanine(A)can completely inhibit the cleavage of rpGSDMD by rpcaspase-1,indicating that pcaspase-1 cleaves pGSDMD after Asp279.On the other hand,the eukaryotic expression plasmids of pcaspase-1 and pGSDMD were used to cotransfect HEK293 cells,which proved that the eukaryotic expression of pcaspase-1 can cleave pGSDMD.In cell-free systems and eukaryotic cell overexpression systems,both Z-VAD-FMK(pan-caspase inhibitor)and VX-765(caspase-1 inhibitor)can inhibit the cleavage of pGSDMD by pcaspase-1,indicating that cleavage of pGSDMD depends on enzyme activity of pcaspase-1.Finally,this study demonstrated that overexpression of amino-terminal segment of pGSDMD(pGSDMD-N)lead to release of lactate dehydrogenase significantly increase in HEK293 cells,Intestinal porcine enterocytes(IPEC-J2),Intestinal mouse fibroblast cells(L929)and Madin-Darby bovine kidney cells(MDBK).Moreover,HEK293 cells died with apparent pyroptosis morphology and were sensitive to propidium iodide staining,indicating that pGSDMD-N can destroy the integrity of cell membranes.In ad dition,expressing rpGSDMD-N in E.coli can kill bacteria.To sum up,pcaspase-1 p20 polyclonal antibody and pcaspase-1 p20 monoclonal antibody were prepared in this study,then used as a tool to cooperate with the pGSDMD specific monoclonal antibody prepared earlier in our laboratory,proving that pcaspase-1 can cleave pGSDMD at Asp279,and the produced pGSDMD-N has the characteristics of destroying cell membrane and bacteriostasis.This study provides research tools and important basic information for comprehensively elucidating the basic mechanism of porcine pyroptosis and further exploring the relationship between porcine pyroptosis and disease.