Study On The Crystal Structure And Function Of Chicken MHCI Presenting Viral Peptide

Cellular immunity is an important part of the chicken immune system,which plays a crucial role in resisting the invasion of pathogens,protecting the health of the body,especially Cytotoxic T lymphocytes(CTL)immune response mediated by CD8+ T cell.The activation of Cytotoxic T lymphocytes response depends on the process that T cell receptor recognizes the peptides which are degradated by intracellular proteasomeand presented by the major histocompatibility complex I(MHC I).The Chicken MHC I was also known as BF2,and there were several mainly distributed alleles in the flocks including BF2*0201,BF2*0401,BF2*1501,BF2*2101,BF2*1201 and so on.However,the structure and function of BF2*1201 had not been resolved so far,and its mechanism for presenting viral peptides was also unkown.Here,the BF2*1201 heavy chain and chicken β2m were expressed using Escherichia coli expression system respectively.These two proteins were co-refolded with antigenic peptide in vitro respectively,and some peptides with a higher affinityfor MHC I molecule such as A6(AVKGVGTMV)、P1、P2、A1 and vSRC(LPACVLEV),were screened out,Based on the above data,it was concluded that the anchoring motif of the BF2*1201 was X(V/A/P/E)XX(V/I)XX(V/M),and the high quality crystals of the complexs(BF2*1201-vSRCLV8-Chβ2m,BF2*1201-A6AV9-Chβ2m)were obtained by further purification and crystallization of the complexs.Then,the crystal structure of BF2*1201 complexed with peptide(vSRCLvs and A6Av9)were solved by X-ray diffraction.the amino acid composition of pockets of peptide binding groove(B12)and the interaction between peptide binding groove(B12)and presenting peptides were demonstrated.We found that the B,C and F pocket were important to MHCI molecules for presenting peptides.When the BF2*1201 present a nine amino acid of the peptide,the P8 residue replace the P9 residue,and its side chain inserted into the F pocket,and the P9 residue side chain extended out of the C-terminus of the binding groove,the side chain of R83 which located on BF2*1201 molecule swinged towards the outside of the groove to forming an "Open back door" conformation.This unique combination of C-terminal open structure was firstly found among those solved poultry MHCI class molecular structures.In a sense,the avian MHCI may have a symmetric peptide anchoring motif(-X-----X-)with anchored residue position PN +1 and PC-1.The bioinformatics method was used to analyze the fuction of the residue R83,and we found that R83 was conserved in MHCI class molecules of most low vertebrates including reptiles,amphibians,fish and poultry.In mammals,Position 83 Y(Tyr)was conserved;in other known MHC class Ⅱ molecules,the corresponding loci 83R(Arg)was also more conserved.The conservativeness of this MHC Ⅰ and Ⅱ interacting with the residue of peptide provided some evidences for the hypothesis that MHCI was originated from MHCII.Then,mutations of Gly(G)were replaced by the 2nd,5th and 8th residues of vSRCLV8 and A6Av9,respectively.The mutant peptides were standardized refolded with BF2*1201 and thermal stability expriment of the complex were further done.The results showed that the binding ability of the mutant peptide to the target molecule decreased significantly,the N-terminal 5th(P5)and the 8th(P8)residue are particularly important,with dominant anchoring characteristics.We extended the C-terminus of peptides eluted from the BF2*1201 molecule and demonstrated that the target molecule was able to present a peptide of C-terminal extension by refolding.Based on structural data and related reports,appropriate Avian Influenza T cell epitopes were designed and immunized SPF chickens with avian influenza inactivated Re-6 strain antigen together.Effective candidates for avian Influenza T cell epitopes were screened out by monitoring antibody levels and related cytokines(IFN-y and IL-4)expression levels of immunized animals.Finally,we found that the peptide PS0804 and PS0816 had a significant effect on the promotion of the antibody titers of chickens immunized by avian influenza inactivated antigens,and PS0804 also promoted the cellular immune response.This work provided an important data for the development of avian influenza epitope vaccine.In summary,we first solved the crystal structure of pBF2*1201and clarified its mechanism for presenting peptide.We found new characters of poultry peptide binding groove and novel feature of MHCI presenting peptides,we also screened out some effective avian Influenza T cell epitopes through animal experiments.This work provided valuable experience for the application of effective T cell epitope in Vaccine production and theoretical basis for the appropriate design of new poultry peptide epitope vaccine.