Mimotopes of the nicotinic receptor binding site selected by a combinatorial peptide library.
Peptide libraries allow selecting new molecules, defined as mimotopes, which are able to mimic the structural and functional features of a native protein. This technology can be applied for the development of new reagents, which can interfere with the action of specific ligands on their target receptors. In the present study we used a combinatorial library approach to produce synthetic peptides mimicking the snake neurotoxin binding site of nicotinic receptors. On the basis of amino acid sequence comparison of different alpha-bungarotoxin binding receptors, we designed a 14 amino acid combinatorial synthetic peptide library with five invariant, four partially variant, and five totally variant positions. Peptides were synthesized using SPOT synthesis on cellulose membranes, and binding sequences were selected using biotinylated alpha-bungarotoxin. Each variant position was systematically identified, and all possible combinations of the best reacting amino acids in each variant position were tested. The best reactive sequences were identified, produced in soluble form, and tested in BIACORE to compare their kinetic constants. We identified several different peptides that can inhibit the binding of alpha-bungarotoxin to both muscle and neuronal nicotinic receptors. Peptide mimotopes have a toxin-binding affinity that is considerably higher than peptides reproducing native receptor sequences.
Bracci
L
L
Department of Molecular Biology, Division of Biological Chemistry, University of Siena, Via Fiorentina 1, 53100 Siena, Italy. braccil@unisi.it
Lozzi
L
L
Lelli
B
B
Pini
A
A
Neri
P
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001667:Binding, Competitive; D015374:Biosensing Techniques; D002038:Bungarotoxins / Q000138:chemical synthesis / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051379:Mice; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D051381:Rats; D011978:Receptors, Nicotinic / Q000378:metabolism*; D012995:Solubility; D014407:Tumor Cells, Cultured
D002038:Bungarotoxins; D010446:Peptide Fragments; D019151:Peptide Library; D011978:Receptors, Nicotinic