Peptide phage display and epitope mapping of a fibril-related conformational epitope recognized by the 11-IF4 monoclonal antibody

Amyloid fibrils and partially unfolded intermediates may be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we previously had reported that the mAb 11-1F4 reacted specifically with light chain (LC) fibrils, irrespective of kappa or lambda, isotype, but notably did not with native molecules (1). To elucidate the molecular basis of this specificity, we have characterized 11-1F4 binding to LC peptides of its immunogen, a kappa 4 Bence Jones Protein, Len, and to 12-mer phage displayed peptides. We demonstrated that the antibody recognizes a conformational determinant on misfolded Len that is within the first (N-terminal) 15 amino acids of framework 1. Furthermore, a sequence alignment of 11-1F4-binding phage peptides suggests that the epitope also relies on LC sequence complementarily. Interestingly, the phage peptides contain an invariant proline that corresponds to Pro8 in light variable domain (V-L) of Len (LCs also contain a conserved proline at positions 7 and/or 8), and a high propensity for the preceding residue to be aromatic. The presence of an aromatic residue adjacent to proline in the phage peptides strongly suggests that the antibody's non-native binding site is anchored by a cis-proline beta-turn since the cis-conformation is stabilized by ring stacking interactions involving aromatic and pyrrole ring systems.