MIMICKING OF DISCONTINUOUS EPITOPES BY PHAGE-DISPLAYED PEPTIDES .1. EPITOPE MAPPING OF HUMAN H-FERRITIN USING A PHAGE LIBRARY OF CONSTRAINED PEPTIDES

We have constructed a random nonapeptide library in the N-terminal reg ion of the major coat protein VIII of bacteriophage f1, with two cyste ines flanking the insert, and preliminary data suggest that many of th e clones display at least some of their peptides in cyclized form. Thi s library was used to select oligopeptides binding to the monoclonal a ntibody (mAb) H107, recognising the assembled native conformation of r ecombinant human H-subunit ferritin (H Fer), whose three-dimensional s tructure is known. Comparison of the selected oligopeptides with one a nother allowed us to derive two consensus sequences characterized by c onserved amino acid (aa) residues. Analysis of the distribution of the aa side chains exposed on the surface of H Fer reveals that most of t he aa defining both consensus sequences are present either at the end of the big loop or at the end of the A helix. These two regions of the H Fer, though separated in the linear sequence, are very close in the folded molecule. Interestingly, each consensus sequence derived from the selected phage-displayed peptides is characterized by aa present b oth at the end of the big loop and at the end of the A helix. These tw o H Fer regions are good candidates for mimicry by the selected peptid es and therefore for constituting part of the H107 epitope. To provide support to this hypothesis, we constructed several H Fer mutants carr ying point mutations in different positions of these two regions. The point mutations did not affect either the assembly of the Fer mutants or their reactivity with a different mAb, but greatly reduced or aboli shed the binding to the H107 mAb. These results indicate that both dis continuous regions of the epitope are necessary for binding.