Natural peptides selected by diabetogenic DQ8 and murine I-A(g7) molecules show common sequence specificity

Abstract

In this study, a large number of naturally processed peptides was isolated and identified from the human diabetes-susceptible class II MHC molecules HLA-DQ8 (DQA1*0301,DQB1*0302) and from murine I-A species, both of which are expressed in genetically identical APC lines. The peptides presented during the processing of autologous proteins were highly selective in showing sequence specificity, mainly consisting of 1 or more acidic residues at their C terminus. Testing for binding to the MHC molecules revealed that the position 9 (P9) acidic residues of the peptides contributed decisively to binding. For HLA-DQ8, the P1 residue, which was also an acidic amino acid, influenced binding positively. Both HLA-DQ8 and I-A(g7) selected for common peptides that bound in the same register. There was no evidence for selection of peptides having nonspecific or promiscuous binding. Thus, diabetogenic class II MHC molecules are highly selective in terms of the peptides presented by their APCs, and this is governed by the features of their P9 anchor pocket. These results are in striking contrast to those from studies examining synthetic peptide or phage display libraries, in which many peptides were shown to bind.

Figure 1

Class II MHC expression among APC lines. NOD.C3 and NOD.DQ8 cells were stained with anti–I-A^g7 or anti-DQ8 mAb (gray lines), respectively, and then analyzed by FACS. Black lines indicate the isotype control for each cell line.

Figure 2

Acidic amino acid distribution among naturally processed peptides selected by I-A^g7 and DQ8. Each peptide was scored for the presence of either an aspartic acid or glutamic acid residue starting at the most C-terminal position, which was assigned position 1 in the figure. Thus, by this scheme, the second most C-terminal residue would be position 2, the third would be position 3, and so on. Note that peptides selected by both I-A^g7 and DQ8 contained acidic amino acids toward their C termini. In addition, peptides selected by DQ8 also exhibited the biased presence of acidic amino acid toward their N terminus. The dotted line represents the background frequency of the presence of acidic amino acids.

Figure 3

Distribution of amino acids at the P1, P4, P6, and P9 positions. (A) Peptide binding motif for DQ8. The P1 through P9 amino acids of the naturally processed peptides listed in Supplemental Table 1 were aligned to identify the preferred residues at P1, P4, P6, and P9. (B) Peptide binding motif for I-A^g7. The P1 through P9 amino acids of the naturally processed peptides listed in Supplemental Table 2 were aligned to identify the preferred residues at P1, P4, P6, and P9.