Age-related structural alterations in loss of self-tolerance to a3(IV) collagen

描述(申请人提供)：抗肾小球基底膜抗体(抗-GBM)肾炎是一种自身免疫性疾病，由自身抗体与肾小球基底膜(GBM)中A3(IV)胶原蛋白的NC1域结合。在自然条件下，AutoAbs与成人肾脏的GBM结合，但不与3-5岁儿童肾脏的GBM结合，尽管表达A3(IV)胶原。本研究的目的是探讨A3(IV)胶原结构和免疫反应性的增龄性改变以及对A3(IV)胶原的耐受性丧失在抗GBM病中的作用。我们证明了表位的密码性取决于a3Nc1结构域的四级组织，这些结构域以3a4a5NC1六聚体复合体的形式存在于组织中。我们发现成体GBM含有两种不同的分子形式的a3NC1区：交联型D-六角体惰性抗a3NC1AutoAbs，非交联型M-六角体可被AutoAbs解离。初步研究表明，用自体天然a3NC1六聚体免疫的小鼠产生有限的免疫反应，没有肾脏疾病，但抗原的结构变化引发了强大的自身抗体反应和严重的自身免疫性肾炎。具体目的1是确定A3(IV)胶原结构和免疫反应性随年龄变化的分子基础。我们假设：a)年轻的GBM中所有的A3(IV)胶原蛋白都是由于其四级结构的分子强化而获得免疫特权的，b)随着年龄的增长，GBM免疫反应性的增加是由于A3(LV)胶原蛋白翻译后的变化积累。具体目的2是确定与年龄相关的A3(IV)胶原结构变化在对这种抗原的耐受性丧失中的作用。我们假设，A3(IV)胶原缺乏稳定的交联链或翻译后修饰对于自身免疫病理是必要的，因为它绕过了自身耐受。这将通过体外研究，比较年轻和成熟肾脏的A3(IV)胶原的结构和免疫反应性，以及在小鼠模型中，比较年轻和成熟的GBM自身抗原的免疫原性和致病性来实现。这些发现将增加我们对长寿命细胞外基质抗原免疫耐受建立和丧失所涉及的分子机制，以及翻译后修饰和自身抗原老化在自身免疫病因中的作用。因此，可能会确定预防或治疗干预的新目标。

DESCRIPTION (provided by applicant): Anti-GBM antibody (Ab) nephritis is an autoimmune disease mediated by autoAbs that bind to the NC1 domain of a3(IV) collagen in the glomerular basement membrane (GBM). Under native conditions, autoAbs bind to the GBM of adult kidneys, but not to the GBM of kidneys from children younger than 3-5 years, despite expression of a3(IV) collagen. The objective of this proposal is to investigate the role of age-related changes in the structure and immunoreactivity of a3(IV) collagen and loss of tolerance to a3(IV) collagen in anti-GBM disease. We showed that epitope crypticity depends on the quaternary organization of the a3NC1 domains, which exist in tissues as a3a4a5NC1 hexamer complexes. We found that the adult GBM contains a3NC1 domains in two distinct molecular forms: cross-linked D-hexamers inert toward anti-a3NC1 autoAbs, and non-crosslinked M-hexamers dissociable by autoAbs. Preliminary studies showed that mice immunized with autologous native a3NC1 hexamers develop a limited immune response and no renal disease, but structural alterations of the antigen elicited a robust autoAb response and severe autoimmune nephritis. Specific Aim 1 is to determine the molecular basis of the age-dependent changes in the structure and immunoreactivity of a3(IV) collagen. We hypothesize that: a) all a3(IV) collagen in the young GBM is immunologically privileged by molecular reinforcement of its quaternary structure, b) the increase in GBM immunoreactivity with age is due to an accumulation of post-translational changes in a3(lV) collagen. Specific Aim 2 is to establish the role of age-related changes of a3(IV) collagen structure in the loss of tolerance to this antigen. We hypothesize that the absence of stabilizing cross-links or post-translational modifications of a3(IV) collagen are necessary for autoimmune pathology by circumventing self-tolerance. This will be achieved by in vitro studies, comparing the structure and immunoreactivity of a3(IV) collagen from young versus mature kidneys, and in vivo studies in mice models, comparing the immunogenicity and pathogenicity of self-antigen from young versus mature GBM. The findings will increase our knowledge of the molecular mechanisms implicated in the establishment and loss of immune tolerance to long-lived extracellular matrix antigens, and the role of post-translational modifications and autoantigen "aging" in the autoimmune etiology. New targets for prevention or therapeutic intervention may be identified as a result.