Peptide analysis, stability studies, and structural modeling explain contradictory peptide motifs and unique properties of the NOD mouse MHC class II molecule H2-A^g7

The MHC class II molecule H2-A^g7 is the chief genetic determinant in insulin-dependent diabetes mellitus of the non-obese diabetic (NOD) mice. Poor peptide binding ability, as well as presentation of a unique subset of peptides by this molecule was suggested to promote autoimmunity in this strain. However, several laboratories have presented results in favor of an H2-A^g7 molecule that can avidly bind many different peptides. The crystal structures of H2-A^g7 in complex with two different peptides did not completely resolve this issue. To analyze the peptide binding capacity and the motif requirements of H2-A^g7, we eluted natural ligands from purified H2-A^g7 molecules isolated from the H2-A^g7 -transfected M12-C3 cells. A low peptide yield dominated by a few peptide ligands was found. Pool sequencing and alignment of individual ligands on the basis of molecular modeling revealed a peptide-binding motif with basic/aliphatic/small hydrophilic amino acids at relative position 1 (p1), aliphatic amino acids at p4, Ala at p6, and acidic amino acids and Ser/Gly at p9, as well as acidic residues at p10/11. Though weak, the binding of individual ligands, as well as the importance of an acidic C-terminal residue was confirmed by peptide binding studies to isolated H2-A^g7 molecules. Furthermore, the H2-A^g7 molecule incompletely dissociated into its constituent chains in SDS-electrophoresis under nonreducing conditions. This provides additional evidence of its weak affinity for peptides, which probably arises from the combination of β56His/β57Ser/β78Ala and other unique H2-A^g7 residues in contact with the antigenic peptide. These results allow a better understanding of the role of this molecule in the development of autoimmunity and the identification of epitopes relevant to diabetes.