Presentation of a protein antigen to T cells generally requires that the antigen be enzymatically processed into an immunogenic peptide(s). The identification of a protease(s) and its mechanism of action in the proteolysis of such an antigen is therefore a primary goal in the study of antigen processing. We show here that insulin degrading enzyme (IDE), a neutral thiol metalloendoproteinase that is structurally non-homologous to the classical metallo, thiol, acid, or serine proteinases, is relatively specific in its proteolytic activity for insulin and digests human insulin (H(I)) into peptides that are presented by murine TA3 B cell antigen presenting cells (APCs) to HI/I-Ad-reactive T cells. These peptides are, however, not presented by fixed TA3 APCs. Anti-IDE mAbs, after their internalization by TA3 cells, significantly inhibit the presentation of H(I) by these APCs. Immunoblotting experiments demonstrate that this inhibition is mediated by the reactivity of these mAbs with a 110 kDa protein, the known M(r) of IDE. These data show that IDE is an endoproteinase that is involved in the processing of insulin and that this IDE-mediated proteolysis is necessary but not sufficient for the recognition of insulin by T cells. Furthermore, we demonstrate that reduction of the disulfide bonds of a pre-processed A-loop containing heterodimeric insulin peptide is required to further process insulin into a T cell epitope.
Our understanding of how an autoantigen is processed and presented during the development of a major histocompatibility complex (MHC) class II-dependent and T-cell-mediated autoimmune disease, such as IDDM, is incompletely understood. We have used insulin as a model autoantigen in IDDM to address the question of whether MHC class II molecules play a role in the generation and/or preservation of an autoantigen peptide that stimulates T-cell activation. Analyses of the requirement of I-Ad class II molecules in the processing of the partially processed porcine insulin peptide A1-A14/B1-B16 demonstrate that the binding of this peptide to I-Ad is essential for it to be further processed and tailored into a T-cell epitope. Based on our observations, we propose a two-step model for insulin processing in which insulin is first processed by an enzyme(s) into an intermediate peptide that binds to class II and then class II functions as a template to guide the processing of this partially processed peptide by cathepsin D into a T-cell epitope. Our data further underscore the important realization that MHC class II-directed processing of an autoantigen (e.g., insulin) may regulate 1) the relative immunodominance of T-cell determinants in an autoantigen, 2) the self-reactivity to cryptic T-cell epitopes in autoantigens, and 3) the susceptibility to autoimmune disease.
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