Data from: Enhanced B-cell receptor recognition of the autoantigen transglutaminase 2 by efficient catalytic self-multimerization

A hallmark of the gluten-driven enteropathy celiac disease is autoantibody production towards the enzyme transglutaminase 2 (TG2) that catalyzes the formation of covalent protein-protein cross-links. Activation of TG2-specific B cells likely involves gluten-specific CD4 T cells as production of the antibodies is dependent on disease-associated HLA-DQ allotypes and dietary intake of gluten. IgA plasma cells producing TG2 antibodies with few mutations are abundant in the celiac gut lesion. These plasma cells and serum antibodies to TG2 drop rapidly after initiation of a gluten-free diet, suggestive of extrafollicular responses or germinal center reactions of short duration. High antigen avidity is known to promote such responses, and is also important for breakage of self-tolerance. We here inquired whether TG2 avidity could be a feature relevant to celiac disease. Using recombinant enzyme we show by dynamic light scattering and gel electrophoresis that TG2 efficiently utilizes itself as a substrate due to conformation-dependent homotypic association, which involves the C-terminal domains of the enzyme. This leads to the formation of covalently linked TG2 multimers. The presence of exogenous substrate such as gluten peptide does not inhibit TG2 self-cross-linking, but rather results in formation of TG2-TG2-gluten complexes. The celiac disease autoantibody epitopes, clustered in the N-terminal part of TG2, are conserved in the TG2-multimers as determined by mass spectrometry and immunoprecipitation analysis. TG2 multimers are superior to TG2 monomer in activating A20 B cells transduced with TG2-specific B-cell receptor, and uptake of TG2-TG2-gluten multimers leads to efficient activation of gluten-specific T cells. Efficient catalytic self-multimerization of TG2 and generation of multivalent TG2 antigen decorated with gluten peptides suggest a mechanism by which self-reactive B cells are activated to give abundant numbers of plasma cells in celiac disease. Importantly, high avidity of the antigen could explain why TG2-specific plasma cells show signs of an extrafollicular generation pathway.乳糜泻（celiac disease）作为一种麸质介导的肠病，其核心特征是针对谷氨酰胺转移酶2（transglutaminase 2, TG2）的自身抗体产生——该酶可催化蛋白质间共价交联的形成。TG2特异性B细胞的活化可能依赖于麸质特异性CD4⁺ T细胞，因为此类自身抗体的产生与疾病相关的HLA-DQ等位基因分型及膳食麸质摄入息息相关。在乳糜泻患者的肠道病变部位，存在大量产生低突变率TG2抗体的IgA浆细胞。在启动无麸质饮食后，此类浆细胞及血清中的抗TG2抗体水平会迅速下降，提示该免疫应答属于短期的滤泡外反应或生发中心反应。已知高抗原亲合力可促进此类免疫应答，同时对打破自身免疫耐受也具有关键意义。本研究旨在探究TG2亲合力是否可作为与乳糜泻相关的免疫特征。本研究利用重组酶，通过动态光散射（dynamic light scattering）与凝胶电泳（gel electrophoresis）实验证实：因依赖构象的同型聚集作用（该过程涉及该酶的C端结构域），TG2可高效以自身作为底物，进而形成共价连接的TG2多聚体。外源性底物（如麸质肽）的存在并不会抑制TG2的自身交联，反而会促进TG2-TG2-麸质复合物的形成。通过质谱（mass spectrometry）与免疫沉淀（immunoprecipitation）分析证实：聚集于TG2 N端区域的乳糜泻自身抗体表位在TG2多聚体中保持保守。相较于TG2单体，TG2多聚体可更高效地激活转染了TG2特异性B细胞受体（B-cell receptor）的A20 B细胞；而TG2-TG2-麸质多聚体的摄取则可有效活化麸质特异性T细胞。TG2高效的催化性自身多聚化，以及被麸质肽修饰的多价TG2抗原的生成，揭示了乳糜泻中自身反应性B细胞被活化并产生大量浆细胞的潜在机制。尤为重要的是，抗原的高亲合力可解释为何TG2特异性浆细胞呈现出滤泡外生成通路的特征。