Processing and presentation of insulin. III. Insulin degrading enzyme: a neutral metalloendoproteinase that is non-homologous to classical endoproteinases mediates the processing of insulin epitopes for helper T cells

Abstract

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.

Thymic expression of insulin-related genes in an animal model of autoimmune type 1 diabetes

“…Among a number of identified islet b-cell autoantigens, insulin is considered to be a critical target and the most specific autoantigen of b-cells [2][3][4]. Insulin as an immune target has been associated with the development of autoimmune diabetes in humans and NOD mice [1][2][3][4], though its role as an autoantigen in the BB rat remains to be clarified. Based on the data implicating autoreactivity to insulin in the pathogenesis of type 1 diabetes, both ourselves and others have suggested that intrathymic expression of INS and IGF could induce the central T-cell tolerance of the insulin family [11,[16][17][18]20].…”

“…In this cohort of autoantigens, insulin is the most specific autoantigen of the islet b-cell. Insulin has been shown to play an important role in the development of type 1 diabetes both in humans and in animal models of type 1 diabetes [1][2][3][4]. Though the autoimmune nature of type 1 diabetes is well established, animal experiments and human clinical studies have failed to determine the specific origin of the immunological self-reactivity against insulin-secreting islet b-cells.…”

Insulin-degrading Enzyme Regulates Extracellular Levels of Amyloid β-Protein by Degradation

“…Cerebrospinal Fluid-Although there have been reports of the release of IDE from cells (14,15) and the presence of intact, 110-kDa IDE at the cell surface (16), it has generally been assumed that IDE is restricted to the cytosol and peroxisomes and is not normally released from cells (7,17). However, our results in the previous section show that readily detectable amounts of enzymatically active IDE are found in the medium of intact BV-2 cells under normal culture conditions, including in serum-containing cultures.…”

“…Previous studies of CHO cells have reported that IDE is localized to the cytosol and peroxisomes (17), loci in which A␤ has not been found, raising the question of whether and how the protease could degrade endogenous A␤. Although some studies have detected insulin-degrading activ- ity in the conditioned media of cultured cells (14,15) and more recently on the cell surface (16), the extent of cell permeability and thus possible release of IDE from leaky cells, especially when serum is absent from the culture medium, was not specifically assessed in these reports. In our study, extracellular A␤ degradation mediated by IDE was observed both in whole BV-2 cultures and in their CM alone (Fig.…”

Naturally processed heterodimeric disulfide-linked insulin peptides bind to major histocompatibility class II molecules on thymic epithelial cells.

“…We previously demonstrated that heterodimeric disulfidelinked insulin peptides are presented by B cells to CD4+ T cells in vitro (4)(5)(6). Since this suggested that a disulfide-linked peptide binds to MHC class II, we analyzed whether human insulin (HI) is processed into a disulfide-linked peptide that binds to MHC class II in vivo.…”