Mimicking of discontinuous epitopes by phage-displayed peptides, I. Epitope mapping of human H ferritin using a phage library of constrained peptides.
We have constructed a random nonapeptide library in the N-terminal region of the major coat protein VIII of bacteriophage f1, with two cysteines flanking the insert, and preliminary data suggest that many of the clones display at least some of their peptides in cyclized form. This library was used to select oligopeptides binding to the monoclonal antibody (mAb) H107, recognising the assembled native conformation of recombinant human H-subunit ferritin (H Fer), whose three-dimensional structure is known. Comparison of the selected oligopeptides with one another allowed us to derive two consensus sequences characterized by conserved amino acid (aa) residues. Analysis of the distribution of the aa side chains exposed on the surface of H Fer reveals that most of the 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 both at the end of the big loop and at the end of the A helix. These two H Fer regions are good candidates for mimicry by the selected peptides and therefore for constituting part of the H107 epitope. To provide support to this hypothesis, we constructed several H Fer mutants carrying point mutations in different positions of these two regions.(ABSTRACT TRUNCATED AT 250 WORDS)
Luzzago
A
A
Istituto di Ricerche di Biologia Molecolare P. Angeletti, Pomezia, Rome, Italy.
Felici
F
F
Tramontano
A
A
Pessi
A
A
Cortese
R
R
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics; D003090:Coliphages / Q000235:genetics; D000939:Epitopes / Q000032:analysis*; D005293:Ferritins / Q000235:genetics / Q000276:immunology*; D015723:Gene Library; D006801:Humans; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides / Q000276:immunology; D017433:Protein Structure, Secondary; D014170:Transformation, Genetic
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D005293:Ferritins