Peptide analysis, stability studies, and structural modeling explain contradictory peptide motifs and unique properties of the NOD mouse MHC class II molecule H2-Ag7

Abstract

The MHC class II molecule H2‐Ag7 is the chief genetic determinant in insulin‐dependent diabetes mellitus of the non‐obese diabetic (NOD) mice. Poor peptide binding ability, as well aspresentation 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‐Ag7 molecule that can avidly bind many different peptides. The crystal structures of H2‐Ag7 in complex with two different peptides did not completely resolve this issue. To analyze the peptide binding capacity and the motif requirements of H2‐Ag7, we eluted natural ligands from purified H2‐Ag7 molecules isolated from the H2‐Ag7‐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‐Ag7 molecules. Furthermore, the H2‐Ag7 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‐Ag7 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.

Do the peptide-binding properties of diabetogenic class II molecules explain autoreactivity?

“…These conclusions were further bolstered by previous work from different laboratories, demonstrating that the diabetogenic class II MHC molecules, were unstable in SDS, i.e. they dissociated into the α-and β-subunits under non-denaturing conditions [13,[16][17][18][19][20][21]. Prior work with other alleles, such as I-A k or I-A b , had established a clear correlation between SDS stability and peptide quality: high affinity peptides formed SDSstable heterodimers, while low affinity peptides did not [16,22,23].…”

“…We and others have noted that I-A g7 -bound peptides contain multiple C-termini acidic residues that contribute towards binding affinity [17,24]. More importantly, >80-90% of peptides isolated from I-A g7 contain C-terminal stretches of two or three acidic residues [24,25] (Figure 1).…”

“…Since I-A g7 and DQ8 were noticeably SDS-unstable it was proposed that they must bind promiscuously and to low affinity peptides [16]. However, mass-spectrometry based identification of naturally processed peptides selected by I-A g7 and DQ8 generated very different results: both the murine and human alleles selected for similar peptides with a very well defined binding motif characterized by multiple C-termini acidic amino acids [17,[24][25][26].…”

Functional consequences of HLA-DQ8 homozygosity versus heterozygosity for islet autoimmunity in type 1 diabetes

“…Although in appearance IA g7 and HLA-DQ8 appear similar, yet there are subtle differences that may affect peptide binding and T-cell recognition. 42 IA g7 has a pH-dependent peptidebinding motif concerning pockets 6 and 9 arising largely from the presence of three histidines in proximity of these pockets (a68, b9 and b56), whereas DQ8 shows a higher binding affinity for peptides at endosomal pH, but no change in motif.…”

The Insulin-Specific T Cells of Nonobese Diabetic Mice Recognize a Weak MHC-Binding Segment in More Than One Form

“…DQ8 in comparison to I-A g7 has some distinct features, particularly at P4 in which large residues are favored (26). Examination of naturally processed peptides bound to I-A g7 of APCs showed that ϳ80 -90% contained from one to three acidic residues at the carboxyl end (23,24,27,28). As predicted from the structural analyses (19,25), there was binding cooperativity among acidic residues at P9, P10, or P11 (23).…”