Characterization of HLA-DQ2 and HLA-DM interaction

描述(由申请人提供)：人们普遍认为特定的MHC II类分子与自身免疫性疾病之间存在强烈的关联，但这种关联背后的机制尚不清楚。人类白细胞抗原-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早就被认识到了，然而，这种联系的解释仍然不清楚。MHC II分子是一种细胞表面蛋白，能够将抗原肽呈递给CD4+T细胞。多肽进入MHC II的过程受两种细胞内蛋白的调节，分别称为不变链(II)和人类白细胞抗原-DM。II稳定新合成的MHC II，并促进MHC II从内质网移动到内小体，在内体II被分解，在多肽结合区留下一个称为CLIP的小片段。人类白细胞抗原DQ2的主要功能是去除内噬菌体中抗原肽的片段交换，并编辑由第II类提出的多肽库。我们先前的数据表明，人类白细胞抗原DQ2与两种不同的片段多肽有关：传统的CLIP1和不寻常的CLIP2。在目标1中，我将确定这种不寻常的联系是在内质网的全长II内启动的，还是在II蛋白分解后的内体中启动的。为了回答这个问题，我将在II中引入突变，消除CLIP1和/或CLIP2与DQ2的结合。然后，通过脉冲追逐免疫沉淀和MALDI-TOF/TOF MS鉴定内质网中与DQ2相关的主要多肽物种。结果将告诉我们，这两个寄存器是否都用于内质网中的II结合。如果不是，则意味着CLIP2寄存器是在对II进行内体处理之后产生的，并且很可能是通过将槽中的片段滑动到CLIP2寄存器而产生的。我们以前的研究也表明，与人类白细胞抗原DR3和人类白细胞抗原DQ1相比，人类白细胞抗原-DQ2和人类白细胞抗原-糖尿病之间的相互作用较弱。为了进一步研究DQ2与DM的相互作用，我将在AIM 2中使用突变分析来阐明DQ2对DM效应的抗性的结构基础。在先前DM与其他MHC II类等位基因相互作用的研究的基础上，将14种不同的突变引入DQ2，以期改善DM与DQ2的相互作用。DQ2WT和突变体将在DM+和DM-淋巴细胞中稳定表达。此外，可溶的DQ2WT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；2)CLIP从DQ2释放的效率；3)DQ2的肽交换率；4)醇溶蛋白多肽与DQ2的结合亲和力；5)DM对DQ2的构象变化；6)DM与可溶性DQ2之间的结合亲和力来研究。如果上述突变都不能改善DQ2-DM的相互作用，则将采用DQ2的随机突变来鉴定增加DQ2对DM功能的敏感性的突变。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.