Autoimmune diabetes in both humans and nonobese diabetic (NOD) mice results from T cell-mediated autoimmune destruction of insulin-producing pancreatic beta cells. Both class I major histocompatibility complex (MHC)-restricted and class II MHC-restricted T cells are involved. However, T cell receptors (TCRs) restricted to recognition of autoantigenic peptides presented by the trimeric class I molecule, consisting of MHC heavy chain, beta2-microglobulin (beta2m), and peptide, are absolutely required for the initiation of disease in the NOD mouse. Autoimmune diabetes is a polygenic disease, with particular MHC haplotypes providing the primary genetic component of susceptibility in both humans and NOD mice. Recently, (beta2m) was identified as a diabetes susceptibility gene in NOD mice, the first such gene to be identified that maps outside of the MHC region, lying within the Idd13 locus on Chromosome 2. Two beta2m alleles are widespread throughout the common laboratory mouse strains. When the a2ma allele found in NOD mice is replaced by the a2mb allele, development of diabetes is prevented. The amino acid sequence difference between these two allelic proteins resides in a single exchange of Asp (""""""""a"""""""" isoform) for Ala (""""""""b"""""""" isoform) at position 85. A number of previous serological and T cell recognition studies, using a variety of MHC allelic products and a2m proteins from different species, have suggested significant conformational flexibility of the class I molecule dependent on the particular beta2m present in the complex. Based on these findings, it can be hypothesized that class I MHC molecules containing beta2ma might exhibit an altered conformation as compared to those containing beta2mb. Such changes could exert effects on both selection of autoreactive T cells and presentation of autoantigenic peptides. The overall goal of this proposal is to test this hypothesis by systematically examining the structural, biochemical, and biological properties of disease-relevant MHC-peptide complexes containing the two beta2m isoforms.
Four Specific Aims are proposed: (1) Structural, thermodynamic, and dynamic characterization of the isolated allelic beta2m proteins, using X-ray diffraction analysis, chemical and thermal denaturation, and amide proton exchange; (2) Similar characterization of the diabetes-related MHC/peptide complexes, including measurements of beta2m and peptide exchange rates; (3) Structural, biochemical, and dynamic characterization of the diabetes-related TCR/MHC-peptide complexes containing the two isoforms Of beta2m, using X ray diffraction analysis, amide proton exchange, and surface plasmon resonance; and (4) Cellular analysis of T Cell recognition and TCR dwell time using MHC-peptide complexes containing the two different beta2m isoforms. Completion of the proposed Aims should allow identification of the molecular and atomic determinants that result in beta2m-dependent susceptibility or protection against disease.
