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

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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.