Mimicking the nicotinic receptor binding site by a single chain Fv selected by competitive panning from a synthetic phage library.
We have developed a novel competitive method to select from a phage display library a single chain Fv which is able to mimic the alpha-bungarotoxin binding site of the muscle nicotinic receptor. The single chain Fv was selected from a large synthetic library using alpha-bungarotoxin-coated magnetic beads. Toxin-bound phages were then eluted by competition with affinity purified nicotinic receptor. Recognition of the toxin by the anti-alpha-bungarotoxin single chain Fv was very similar to that of the receptor, such as indicated by the epitope mapping of alpha-bungarotoxin through overlapping synthetic peptides. Moreover, several positively charged residues located in the toxin second loop and in the C-terminal region were found to be critical, to a similar extent, for toxin recognition by the single chain Fv and the receptor. However, although the anti-alpha-bungarotoxin single chain Fv seems to mimic the toxin binding site of the nicotinic receptor, it does not bind other nicotinic agonists or antagonists. Our results suggest that competitive selection of anti-ligand antibody phages can allow the production of receptor-mimicking molecules directly and exclusively targeted at one specific ligand. Since physiologically and pharmacologically different ligands can produce opposite effects on receptor functions, such selective ligand decoys can have important therapeutic applications.
Bracci
L
L
Dipartimento di Biologia Molecolare, Sez. Chimica Biologica, Università di Siena, Via Fiorentina 1, 53100 Siena, Italy. braccil@unisi.it
Pini
A
A
Lozzi
L
L
Lelli
B
B
Battestin
P
P
Spreafico
A
A
Bernini
A
A
Niccolai
N
N
Neri
P
P
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000276:immunology; D000939:Epitopes; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008722:Methods; D019151:Peptide Library; D011978:Receptors, Nicotinic / Q000378:metabolism*; D014101:Torpedo
D002038:Bungarotoxins; D000939:Epitopes; D007128:Immunoglobulin Fragments; D008024:Ligands; D019151:Peptide Library; D011978:Receptors, Nicotinic; C055966:immunoglobulin Fv