Structure-Based Design of HLA-DQ2 Ligands

描述(由申请人提供)：乳糜泻是一种遗传性小肠自身免疫性疾病，患者从普通食物来源如小麦、黑麦和大麦中摄取谷蛋白后会触发这种疾病。尽管对这种疾病进行了50年的积极研究，但除了终身严格的无麸质饮食外，没有其他治疗方法可供选择。该项目的长期目标是开发抑制II型MHC介导的对乳糜泻患者面筋蛋白的免疫致病反应。一些研究已经暗示了人类白细胞抗原-DQ2的致病作用，这是存在于90%的腹腔癌患者中的第二类MHC等位基因。最近，面筋蛋白中的表位已经被确定，它以DQ2限制性的方式诱导CD4+炎性T辅助细胞的增殖。因此，这一探索性项目的具体目标有两个： (I)解决与高亲和力面筋表位结合的人类白细胞抗原DQ2的X射线晶体结构；以及 (Ii)鉴定与人类白细胞抗原-DQ2紧密结合但抑制人类白细胞抗原-DQ2介导的腹腔炎患者疾病特异性T细胞增殖的合成肽。 为了支持这些目标，我们已经(I)鉴定了一种含有高亲和力的HLA-DQ2结合序列的蛋白水解性谷蛋白多肽；(Ii)基于密切相关的HLA-DQ8异源二聚体的最新晶体结构，开发了该表位与HLA-DQ2活性部位结合的同源模型；(Iii)使用该模型预测了表位上的哪些残基可能影响DQ2结合和T细胞受体结合；(Iv)建立了可溶性HLADQ2的表达系统和纯化程序；以及(V)确定了可重复生长的可溶双折射晶体生长的条件。 如果成功，该项目将为最终确定适用于Celiac Sprue疗法的口服预防药物提供一个强有力的起点。更广泛地说，它提供了一个独特的机会来验证II类MHC蛋白作为有吸引力的自身免疫性疾病的治疗靶点。

DESCRIPTION (provided by applicant): Celiac Sprue is an inheritable autoimmune disorder of the small intestine that is triggered in patients upon exposure to dietary gluten from common food sources such as wheat, rye and barley. Notwithstanding 50 years of active research into this disease, there is no therapeutic alternative to a strict lifelong gluten-free diet. The long-term goal of this project is the development of inhibitors for the Class II MHC-mediated immunopathogenic response to gluten in Celiac Sprue patients. Several studies have implicated a causative role for HLA-DQ2, the class II MHC allele present in >90% of Celiac Sprue patients. More recently, epitopes have been identified in gluten that induces proliferation of CD4+ inflammatory T-helper cells in a DQ2 restricted manner. Therefore, the specific aims of this exploratory project are two-fold: (i) To solve the X-ray crystal structure of HLA-DQ2 bound to a high-affinity gluten epitope; and (ii) To identify synthetic peptides that bind tightly to HLA-DQ2, but inhibit HLA-DQ2 mediated proliferation of disease-specific T cells derived from Celiac Sprue patients. In support of these aims, we have (i) identified a proteolytically resistant gluten peptide that contains a high affinity HLA-DQ2 binding sequence; (ii) developed a homology model for the binding of this epitope to the HLA-DQ2 active site based on the recent crystal structure of the closely related HLA-DQ8 heterodimer; (iii) used this model to predict which residues in the epitope are likely to influence DQ2 binding and T cell receptor binding; (iv) developed an expression system and purification procedure for a soluble form of HLADQ2; and (v) identified conditions for reproducible growth of birefringent crystals of soluble HLA-DQ2. If successful, the project will provide a strong starting point for the eventual identification of an oral prophylactic drug candidate for Celiac Sprue therapy. More generally, it provides a unique opportunity to validate class II MHC proteins as attractive therapeutic targets for autoimmune disorders.