Characterization of HLA-DQ2 and HLA-DM interaction

HLA-DQ2 和 HLA-DM 相互作用的表征

• 批准号: 8082668
• 负责人: Tieying Hou
• 金额: $ 5.13万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8082668
• 关键词: Affinity; Alleles; Anabolism; Antigen Presentation; Aspartic Endopeptidases; Autoimmune Diseases; Autoimmunity; B-Lymphocytes; Binding; CD4 Positive T Lymphocytes; CLIP peptide; Celiac Disease; Cell Surface Proteins; Cells; Complex; Cysteine Protease; Data; Endoplasmic Reticulum; Endosomes; Epidemiologic Studies; Epidemiology; Future; Genetic; Gliadin; HLA-DR3 Antigen; Histocompatibility Antigens Class II; Immune; Immunoprecipitation; Insecta; Insulin-Dependent Diabetes Mellitus; Lead; Left; Length; Link; Lymphocyte; MHC Class II Genes; Measures; Mediating; Movement; Mutagenesis; Mutation; Peptides; Physiologic pulse; Play; Process; Proteins; Proteolysis; Resistance; Role; Shapes; Slide; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Surface; T-Lymphocyte; Time; Transgenic Mice; base; improved; insight; invariant chain; mutant; novel therapeutic intervention; premature; prevent; research study

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.

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关联，但这种关联的机制尚不清楚。HLA-DQ 2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被认识到，然而，这种关联的解释仍然未知。MHC II分子是能够将抗原肽呈递给CD 4 + T细胞的细胞表面蛋白。肽装载到MHC II中受两种称为不变链（Ii）和HLA-DM的细胞内蛋白质调节。Ii稳定新合成的MHC II，并促进MHC II从ER移动到内体，在那里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去除CLIP交换抗原肽并编辑由II类呈递的肽库。我们以前的数据表明，HLA-DQ 2与两种不同的CLIP肽相关：传统的CLIP 1和不寻常的CLIP 2。在目标1中，我将确定这种不寻常的关联是否在ER中的全长Ii或Ii的一些蛋白水解后的内涵体中启动。为了回答这个问题，我将在Ii中引入突变，消除CLIP 1或CLIP 2或两者与DQ 2的结合。然后，通过脉冲追踪免疫沉淀和MALDI-TOF/TOF MS鉴定ER中与DQ 2相关的主要肽种类。结果将告诉我们两个寄存器是否用于ER中的Ii结合。如果不是，则意味着CLIP 2寄存器是在Ii的内体加工之后产生的，并且可能是通过CLIP在凹槽中滑动到CLIP 2寄存器而产生的。我们以前的研究也表明HLA-DQ 2和HLA-DM之间的相互作用比HLA-DR 3和HLA-DQ 1之间的相互作用弱。为了进一步表征DQ 2-DM相互作用，我将使用目的2中的突变分析阐明DQ 2对DM效应的抗性的结构基础。基于先前对DM与其他MHC II类等位基因相互作用的研究，将向DQ 2中引入14种不同的突变，这些突变有望改善DM-DQ 2相互作用。DQ 2 WT和突变体将在DM+和DM-淋巴细胞中稳定表达。此外，可溶性DQ 2 WT或突变体将在昆虫细胞中表达并从昆虫细胞中纯化。将通过测量1）CLIP的表面表达; 2）CLIP从DQ 2释放的效率; 3）DQ 2的肽交换速率; 4）麦胶蛋白肽与DQ 2的结合亲和力; 5）DM介导的DQ 2的构象变化; 6）DM与可溶性DQ 2之间的结合亲和力来研究突变对DQ 2功能的影响。如果上述突变均不会改善DQ 2-DM相互作用，则采用DQ 2的随机诱变来鉴定增加DQ 2对DM功能的敏感性的突变。DQ 2中突变的鉴定将最终用于确定DM-DQ 2相互作用在转基因小鼠中T细胞选择中的作用。