The critical amino acids of a nephritogenic epitope on human Goodpasture autoantigen for binding to HLA-DRB1*1501.

BACKGROUND

Anti-GBM disease is caused by autoimmunity to Goodpasture antigen on α3(IV)NC1 and had strong associations with HLA-DRB11501. Previous studies identified α3 (P14: TDIPPCPHGWISLWKGFSFIMF) as a T cell epitope. The present study was aimed to investigate the binding capacity of P14 to HLA-DRB11501 and the critical amino acids for this binding.

METHODS

A line of EBV-transformed human B cells homozygous for HLA-DRB11501 was used to detect the binding capacity of peptides to HLA-DRB11501 using flow cytometry analysis. P14 was sequentially truncated into 8 peptides with 15 amino acids to identify the core binding motif. A set of alanine substituted peptides of P14-2 was then synthesized to identify its critical residues for binding to HLA-DRB1*1501. The structure of HLA-DR2b-Peptide-TCR complex was constructed by modeling to analyze the interaction of each amino acids of P14-2 with the HLA-DR2b molecule.

RESULTS

P14 could bind to HLA-DRB11501 expressed on B cell surface. The N-terminus of P14 was the core binding motif and the truncated peptide P14-2 (DIPPCPHGWISLWKG) had the strongest binding capacity. After sequential amino acid substitution, we found the binding capacity of P14-2 was completely lost by the substitution of cysteine (C) and significantly decreased by the substitution of tryptophan (W) , lysine (K) , or glycine (G) , but still at a high level. The modeling showed that (C) had a strong interaction with pocket 4 on the β chain of DR2b. Thus, C, W , K, and G were defined as the critical amino acid residues for the binding capacity of P14 to HLA-DRB11501.

CONCLUSION

We identified α3 (DIPPCPHGWISLWKG) as the core binding motif of P14 to HLA-DRB1*1501 molecule. And the critical amino acid residues for this binding were further defined as C, W , K , and G .