The Study Of Interaction Among HLA-B27,ERAP1 And CRT In The Pathogenesis Of Ankylosing Spondylitis

BackgroundThe human immune system recognizes damaged and infected cells by degrading peptides produced by intracellular and endocytic proteins,including abnormal and unnecessary proteins.These potentially immunogenic peptides can be bound to major histocompatibility complex(MHC)class I molecules,which expressed on all nucleated cells and platelets,and then be presented to CD8+T lymphocytes,thereby initiating an immune response.Endoplasmic reticulum aminopeptidase 1(ERAP1)is an aminopeptidase,which is a kind of proteolytic enzyme that selectively excises the N-terminal amino acids of peptides.The canonical function of ERAP1 is to splice peptides(antigen peptide precursors)degraded from proteasome in the endoplasmic reticulum,making the peptide precursors into antigen peptides that can be recognized,bound,and presented by MHC class Ⅰ molecules,thereby determining immunogenicity.Genome-wide association studies(GWAS)have demonstrated that ERAP1 single nucleotide polymorphism(SNP)s are associated with a variety of diseases,especially with ankylosing spondylitis(AS).AS is a complex disease characterized by inflammation,periarticular ossification,and systemic osteoporosis/osteopenia,mainly affecting the sacroiliac joint,lumbar spine,and peripheral joints.The pathogenesis of AS involves multifactorial interaction between host immune response and environmental factors.Human leukocyte antigen(HLA)-B*27 is tightly related to AS susceptibility,accounting for about 20%of AS genetic risk.ERAP1 is still the most strongly related gene to AS except HLA-B*2 7,and this correlation has been confirmed in a variety of populations.More noteworthy is that the strong association between ERAP1 and AS only exists in HLA-B*27 positive AS patients.The interaction between ERAP1 and HLA-B*27 at the genetic and functional level suggests that ERAP1 SNPs may cause changes in amino acid sequence,resulting in changes in ERAP1 protein expression level,cutting activity,and substrate specificity,which will directly cause changes in ERAP1 processing peptide repertoire.In AS disease-related HLA-B*27 positive individuals,such changes in peptide repertoire may induce abnormal immune responses and mediate the occurrence and development of AS.The implementation of protein function is closely related to its three-dimensional spatial structure,so the description of protein structure is generally considered to be an important capture of its working mechanism.Protein structure can explain how amino acid sequence mutations affect the occurrence and development of diseases,and how to design small molecule drugs targeting protein structure for treatment.X-ray crystallography studies have found that ERAP1 has two conformations:an active closed state and an inactive open state.However,the dynamic changes of ERAP1 under physiological conditions can not be fully reflected by these two static structures.ObjectiveThe purpose of this study is to investigate whether there is an interaction between HLA-B*27 and ERAP1 protein;If there is a direct interaction,in which conformation does ERAP1 bind to HLA-B*27;If it is an indirect interaction,is there any intermediate protein,especially the protein that plays a role in antigen processing and presentation,that mediates the binding of HLA-B*27 and ERAP1.MethodHLA-B*27 protein expression was induced in Escherichia coli,and the protein was purified using the inclusion body protein refolding method.The properties of the protein were analyzed through size exclusion chromatography,SDS-PAGE gel electrophoresis.ERAP1 protein was secreted and expressed in HEK293F,purified by metal chelation affinity chromatography,and characterized via size exclusion chromatography,SDS-PAGE gel electrophoresis,negative staining-120kV electron microscopy.The optimal protein concentration and sample preparation conditions were determined by using 200kV Talos Arctica.The protein structure was analyzed via single-particle cryo-EM.To identify protein interactions,transient transfection and immunocoprecipitation,in vitro co-migration analysis of proteins,and in vivo co-expression immunoprecipitation-mass spectrometry were performed in HEK293T cells.ResultsWe firstly verified the interaction between HLA-B*27:05 and ERAP1 protein through co-immunoprecipitation.Then,the recombinant protein in the monomer form of HLA-B*27:05 was obtained by the renaturation method of inclusion body protein,which expressed in the prokaryotic system E.coli.The properties of the protein sample were detected by size exclusion chromatography and SDS-PAGE gel electrophoresis.The recombinant protein in the monomer form of ERAP1 was expressed and purified via the secretory expression in HEK293F cell.Then,the recombinant protein ERAP1 was purified by metal chelation affinity chromatography.The recombinant protein ERAP1 was detected by size exclusion chromatography,SDS-PAGE gel electrophoresis,negative stain-120kV EM until the ideal protein was obtained.Then,we prepared the frozen electron microscopy samples using the ERAP1 protein,and screened the required protein concentration and sampling conditions by 200kV Talos Arctica.We try to analyze the structure of ERAP1 via single-particle cryo-EM,and found one conformation between closed and open states.Subsequently,co-migration experiments were conducted using the HLA-B*27:05 protein and ERAP1 protein obtained above,and the result was negative.It is considered that the interaction between HLA-B*27:05 and ERAP1 protein is indirect,indicating the presence of an intermediate protein.Then,His-HLA-B*27:05 and Flag ERAP1 were overexpressed in HEK293T cells,and an intermediate protein CRT mediating their interaction was identified through immunoprecipitation-mass spectrometry technology.It was confirmed through co-immunoprecipitation that CRT can bind to ERAP1 and HLA-B*27:05,and the binding can occur in the endoplasmic reticulum.We also constructed the expression vector of CRT in the eukaryotic system(mammalian cells)in vitro by molecular cloning technology.ERAP1 and CRT were co-expressed in 293F cells,and they were expressed and purified in vitro.A small amount of ERAP1-CRT complex protein was obtained,which established a foundation for subsequent related structure and function studies.ConclusionIn summary,our study found the intermediate protein CRT that mediates the interaction between HLA-B*27 and ERAP1;successfully expressed and purified the recombinant protein in HLA-B*27 monomer form;explored the dynamic conformation of ERAP1 via single-particle cryo-EM.These contents not only can help us better understand the functions of HLA-B*27 and ERAP1,but also provide theoretical supplement for the pathogenesis of AS.