Characterization of HLA-DQ2 and HLA-DM interaction

描述（由申请人提供）：人们普遍认为特定MHC II类分子与自身免疫性疾病之间存在强关联，但这种关联的机制尚不清楚。HLA-DQ2等位基因与乳糜泻和1型胰岛素依赖型糖尿病之间的遗传联系很久以前就已经被发现，然而，这种联系的解释仍然未知。MHC II分子是细胞表面蛋白，能够向CD4+ T细胞呈递抗原肽。多肽进入MHC II的装载是由两种称为不变链（II）和HLA-DM的细胞内蛋白调节的。Ii稳定新合成的MHC Ii，并促进MHC Ii从内质网到核内体的运动，在核内体Ii被分解，在肽结合区留下一个称为CLIP的小片段。HLA-DM的主要功能是从核内体中去除CLIP以交换抗原肽，并编辑II类肽库。我们之前的数据显示，HLA-DQ2与两种不同的CLIP肽相关：传统的CLIP1和不寻常的CLIP2。在目的1中，我将确定这种不寻常的关联是在内质网全长Ii内还是在Ii蛋白水解后的核内体中开始的。为了回答这个问题，我将在Ii中引入突变，消除CLIP1或CLIP2或两者与DQ2的结合。在内质网中与DQ2相关的主要肽种将通过脉冲追踪免疫沉淀和MALDI-TOF/TOF ms鉴定。结果将告诉我们这两种记录是否用于内质网中的Ii结合。如果不是，则意味着CLIP2寄存器是在Ii的内体加工之后产生的，并且可能是通过将凹槽中的CLIP滑动到CLIP2寄存器中而产生的。我们之前的研究也表明，与HLA-DR3和HLA-DQ1相比，HLA-DQ2与HLA-DM的相互作用较弱。为了进一步表征DQ2-DM相互作用，我将在Aim 2中利用突变分析阐明DQ2抵抗DM效应的结构基础。根据以往关于DM与其他MHC II类等位基因相互作用的研究，将在DQ2中引入14种不同的突变，这些突变有望改善DM-DQ2的相互作用。DQ2 WT和突变体在DM+和DM-淋巴细胞中均稳定表达。此外，可溶性DQ2 WT或突变体将在昆虫细胞中表达和纯化。突变对DQ2功能的影响将通过测量1)CLIP的表面表达来研究；2) DQ2中CLIP的释放效率；3) DQ2的肽交换率；4)麦胶蛋白肽与DQ2的结合亲和力；5) dm介导的DQ2构象变化；6) DM与可溶性DQ2之间的结合亲和力。如果上述突变均不能改善DQ2-DM相互作用，则采用DQ2的随机诱变来鉴定增加DQ2对DM功能敏感性的突变。DQ2突变的鉴定将最终用于确定DM-DQ2相互作用在转基因小鼠T细胞选择中的作用。

DESCRIPTION (provided by applicant): It is generally accepted that strong association exists between particular MHC class II molecules and autoimmune diseases, but the mechanisms underlying this association are unknown. The genetic link between the HLA-DQ2 allele and celiac disease and type 1 insulin-dependent diabetes has been appreciated long time ago, the explanation for this association, however, remains unknown. MHC II molecules are cell surface proteins that are able to present antigenic peptides to CD4+ T cells. The loading of peptides into MHC II is regulated by two intracellular proteins called invariant chain (Ii) and HLA-DM. Ii stabilizes newly synthesized MHC II and facilitates the movement of MHC II from the ER to endosomes, where Ii is broken down, leaving a small fragment called CLIP in the peptide-binding region. The major function of HLA-DM is to remove CLIP exchanging for an antigenic peptide from endosomes and edit peptide repertoire presented by class II. Our previous data show that HLA-DQ2 is associated with two different CLIP peptides: the traditional CLIP1 and the unusual CLIP2. In aim 1, I will determine whether this unusual association is initiated within the full-length Ii in ER or in endosomes after some proteolysis of Ii. To answer this question, I will introduce mutations into Ii that eliminate CLIP1 or CLIP2 or both binding to DQ2. The predominant peptide species associated with DQ2 in ER will then be identified by both pulse chase immunoprecipitation and MALDI-TOF/TOF MS. The results will tell us whether both registers are used for Ii binding in the ER. If not, the implication will be that the CLIP2 register is generated after endosomal processing of Ii and likely by sliding of CLIP in the groove to the CLIP2 register. Our previous studies also indicate the interaction between HLA-DQ2 and HLA-DM is weaker compared to HLA-DR3 and HLA-DQ1. To further characterize DQ2-DM interaction, I will elucidate the structural basis of the DQ2 resistance to DM effect using mutational analysis in Aim 2. Fourteen different mutations will be introduced into DQ2 based on previous studies of DM interaction with other MHC class II alleles, which are expected to improve DM-DQ2 interaction. Both DQ2 WT and mutants will be stably expressed in both DM+ and DM- lymphocytes. Additionally, soluble DQ2 WT or mutants will be expressed in and purified from insect cells. The effects of mutations on DQ2 function will be investigated by measuring 1) surface expression of CLIP; 2) efficiency of CLIP release from DQ2; 3) peptide exchange rate of DQ2; 4) binding affinity of gliadin peptides to DQ2; 5) DM-meditated conformational change in DQ2; 6) binding affinity between DM and soluble DQ2. If none of the above mutations will improve DQ2-DM interaction, random mutagenesis of DQ2 will be employed to identify mutations increasing DQ2 sensitivity to DM function. The identification of mutations in DQ2 will be ultimately used to determine the role of DM-DQ2 interaction in T cell selection in transgenic mice. PUBLIC HEALTH RELEVANCE: Genetic and epidemiological data have revealed that the strong genetic link between HLA-DQ2 and celiac disease and type 1 insulin-dependent diabetes, but the mechanisms underlying this association are unknown. I propose to elucidate the mechanisms that HLA-DQ2 predisposes to autoimmune diseases by exploring the defective interaction between DQ2 and DM (and DQ2's association with unusual invariant chain peptides), both of which play central role in regulating antigen presentation by MHC class II. Our results will provide important mechanistic insights into the unique features of DQ2 interaction with the antigen presentation machinery, which may ultimately lead to improved understanding of the association of DQ2 with autoimmunity and may suggest new therapeutic approaches for DQ2-associated autoimmune disorders.