Crystal Structure Analysis And Functional Study Of Type Ⅰ Interferon In Grass Carp

Cytokines are soluble extracellular proteins or glycoproteins that play a huge role in predisposing and adaptive immunity.Although cytokines are occasionally produced constitutively,they are usually produced by virtually every nucleated cell type in response to injurious stimuli.Cytokines have been assigned to various family groups based on the structural homologies and receptors.Interferons(IFNs)are widely expressed cytokines that have potent antiviral.This study focuses on the crystal structure,ligand receptor binding mode,signaling pathway,antiviral function and antiinflammatory function of grass carp type I interferon.To check the response of grass carp(Ctenopharyngodon idella,Ci)ifna to viruses,viral analogues,and viral proteins.We used different means to stimulate the EPC cell line,and used q PCR to detect the classical genes of the IFN pathway,and found that SVCV-N,SVCV-P,SVCV-M,SVCV-L and SVCV-G all reduced the expression of ifna and its downstream antiviral genes to varying degrees,but the results of different proteins were different,and then concluded that viral proteins are not classical pathogen-associated molecular patterns(PAMPs)that can be sensed by pattern recognition receptors(PRRs),but are specifically recognized by host antibodies.Viruses evade host immunity in different ways.In order to understand the synthesis,secretion and immune functions of type I interferons in grass carp,Ci IFNa and Ci IFNd were expressed in the Escherichia coli(E.coli)cells and purified.Hybridoma cells were obtained using the PEG method and injected into the abdominal cavity of mice to generate ascites.Two monoclonal antibodies of Ci IFNa and Ci IFNd were purified,respectively,and characterized by SDS-PAGE,ELISA,Western blot and immunofluorescence assay.It has been shown that the monoclonal antibodies produced had good specificity and high titers against the antigens and could specifically recognize recombinant proteins expressed in E.coli and eukaryotic cells.The Ci IFNa antibodies did not react with Ci IFNd and vice versa.Taken together,the Ci IFNa and Ci IFNd monoclonal antibodies prepared in this study laid the basis for further in-depth study of the cellular sources and protein expression profiles of IFNs in grass carp.Crystal is a solid with three-dimensional periodic atomic structure and polyhedral shape under certain growth conditions.It is an important means to study cytokines.The crystal structure of Ci IFNa was determined at 1.58 (?) resolution with space group C121 by molecular replacement.As expected,the structure reveals a typical type I IFN architecture of 6 structural elements,denoted A through F.The helices A,C,D and F form an anti-parallel four helix bundle,which are arranged in an up-up-down-down orientation.The helix F is straight,which is the hallmark of type I IFN.Unlike mammals,Ci IFNa lacks helix element in the AB loop.One disulfide bond formed by Cys3-Cys99 is present in the Ci IFNa,linking the N terminal region with helix D,and is analogous to the disulfides found in other vertebrates’ type I IFN.Type I IFN in teleost fish are divided into subgroups I and II,and previous studies have shown that they activate antiviral responses by binding to different receptors.However,the interaction between IFN ligands and receptors is not fully understood.We found that,unlike previous studies,IFNa can bind to CRFB1,CRFB2,and CRFB5,respectively,and the three receptors can bind to each other to form a paired receptor complex.We also identified four key residues Leu27,Glu103,Lys117 and His165 that interact with receptors in the grass carp IFN superposition model.Subsequently,four mutated plasmids L27 A,E103A,L117 A and H165 A of grass carp IFNa were constructed to verify its binding ability to receptors,and it was found that the mutation of a single site had little effect on its binding ability to receptors.Further,Mutation of Leu27,Glu103,Lys117 and His165 markedly decreased the phosphorylation of signal transducerand activator of transcription(STAT)1a induced by Ci IFNa in the EPC cells.Interestingly,wildtype and mutant Ci IFNa proteins did not alter the phosphorylation levels of STAT1 b.Fluorescence quantitative PCR and plaque assay showed that Glu103 and His165 were necessary for IFNa activation of cell antiviral activity in grass carp.Our results demonstrate that fish type I IFNs,although structurally conserved,interact with the receptors in a manner that may differ from mammalian homologs.In addition,we also conducted a preliminary study on the crystal structure and function of IFNd,another important member of grass carp type I interferon,and found that although the two have similar structure,the function of IFNd and IFNa of the same group is quite different,which lays a foundation for further study of the difference IFNs in the future.The above studies preliminally explored the function of type I interferon in grass carp immune system,analyzed its key sites from the perspective of crystal structure,and identified four key sites binding to receptors.Glu103 and His165 were identified as the key antiviral sites of IFNa in grass carp from multiple perspectives.It lays a foundation for further elaboration of the antiviral function and signal transduction pathway of grass carp,and provides a theoretical basis for fish immunity and disease prevention and control.