| Major Histocompatibility Complex I(MHC I)is expressed on the surface of nucleated cells,and presents endogenous antigens to CD8+ T lymphocytes to help them recognize and clear virus-infected cells or cancer cells.In the past 30 years,the processing and presention of human and murine MHC I have been extensively studied,but few studies have been found to focus on the molecular polymorphism of cat MHC I(feline leucocyte antigen,FLA I),especially in the field of structural biology.Feline immunodeficiency virus(FIV)is a significant pathogen that causes acquired immunodeficiency syndrome(AIDS)in cats,this pathogen belongs to the same lentivirus as human immunodeficiency virus(HIV)and has a similar infection process with it.Thus,FIV-infected cat has been become the smallest natural model for HIV infection.MHC I molecules are able to present viral polypeptides to cytotoxic T cell for their recognition,which are crucial for clearing the virus infection such as HIV.However,the structure and function of FLA I molecules are unclear,which strongly limit the further study on the anti-FIV infection.Firstly,in this study,24 FLA I E alleles sequences were successfully cloned from the peripheral blood lymphocytes in 12 domestic cats by PCR technology.The highly variable locus of the constituted MHC I peptide binding groove(PBG)mapped to the three dimensional structure of FLA-E*01801 to complete the detailed analysis.Results embodied the high variable locus that constituted PBG were able to affect the epitope presentation by FLA I.Secondly,in order to identify the binding motif used for presenting the immunodeficiency virus by FLA I in domestic cats,we finally selected one FIV nonapeptide(gag:40-48;RMANVSTGR)that could bind FLA-E*01801 in vitro,based on the prediction of the epitope peptide that combined with the allele FLA-E*01801 by bioinformatics.The structure of FLA-E*01801 complex was analyzed by in vitro refolding and X-ray.Glu63 and Trp167are key amino acids for FLA-E*01801 A pocket construction,they interact with the peptide P1-Arg through hydrogen bond and van der Waals force.But these two amino acids of MHC I in different spices have variable conservations.The results of structural analysis,mutagenesis of the polypeptide motif and circular dichroism found that all the positive peptides were not able to assist the mutations with the formation of stable complexes of FLA-E*01801-63e/N,but could assist FLA-E*01801.167w/s with the formation of stable complexes,the increased refolding efficiency,and the relieved restriction of Pi-Asp.Furthermore,the structure of FLA-E*01801-67w/s presented RMA9-P1D determined that the large side chain of Trp167 resulted in the N-terminal closure of the FLA-E*01801 A pocket,meanwhile,P1-Asp was repulsed by Glu63,the mutated N terminal of Ser167 was opened by following releasing the P1-Asp restriction.Hence,FLA-E*01801 complex A pocket could restrictedly bind with P1-Asp,which indicated that FLA-E*01801 complex A pocket had restriction.Thirdly,the nonapeptide binding motif of FLA-E*01801 was X(except D)-M/T/A/V/I/L/SXXXXXXR/K identified by the biochemical assay,and a total of 125 binding epitopes of FIV potential nonapeptides have been determined according to the peptide binding motif of FLA-E*01801.In addition,our study conceded the fCD8aa molecular existed as dimer via analyzing the crystal structure of fCD8aa.This study,for the first time,found that the MHC I A pocket was restricted and identified the binding motif of FLA-E*01801,which laid the foundation for further study of FIV and HIV polypeptide vaccines.This study firstly discovered the MHC IA pocket had the restriction,identified the binding modify of FLA-E*01801,and found the existence format of fCD8??,which lay a foundation to the further study on the FIV and HIV polypeptide vaccines. |
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