A Molecular Analysis of the Shared Epitope Hypothesis Binding of Arthritogenic Peptides to DRB1*04 Alleles

Objective. The shared epitope hypothesis posits that amino acids QR/KRAA in positions 70-74 of the DRB1 chain are responsible for rheumatoid arthritis susceptibility. However, even DRB1*04 alleles containing the shared epitope vary greatly with respect to degrees of susceptibility. This study was undertaken to conduct a molecular examination of the shared epitope hypothesis by measuring binding of arthritogenic peptides to susceptibility and resistance alleles. Methods. We measured binding of native and citrullinated forms of vimentin(66-78) and alpha-enolase(11-25) and noncitrullinated type II collagen(258-272) to 88 class II alleles on Luminex beads (which includes alleles of many varying degrees of susceptibility and resistance). We expressed DRB1*04: 01, *04: 02, and *08: 01 in T2 cells and mutated DRB1*04: 01 at positions 67, 70, 71, 74, and 86 to corresponding residues in DRB1*04: 02, *04: 03, *04: 04, *04: 05, and *08:01. Finally, we measured responses of 4 DRB1*04: 01 restricted collagen(258-272) T cell hybridomas against wild-type DRB1*04: 01, *04: 02, and all mutated alleles. Results. The most susceptible allele, DRB1*04: 01, preferentially bound citrullinated vimentin(66-78) and citrullinated a-enolase(11-25) over the native forms. DRB1*04: 02 exhibited no preference for citrullinated peptides, and *08: 01 preferred native peptides. Similarly, DRB1*04: 01 bound collagen(258-272), but *04: 02 and *08: 01 did not. Mutating DRB1*04: 01 at positions 70, 71, 74, and 86 to the corresponding residues in DRB1*04: 02 or *08: 01 dramatically reduced the specificity for citrullinated peptides and collagen(258-272) binding. Conclusion. These observations demonstrate that while amino acids at positions 70, 71, and 74 within the shared epitope in DRB1 mediate binding and T cell responses of arthritogenic peptides, position 86 outside the shared epitope also plays a critical role.