IDENTIFICATION OF EPITOPES WITHIN A HIGHLY IMMUNOGENIC REGION OF ACETYLCHOLINE-RECEPTOR BY A PHAGE EPITOPE LIBRARY

We have employed a hexapeptide phage-epitope library to identify epito pes for a mAb (mAb 5.14), which is directed to a determinant within a highly immunogenic, cytoplasmic region of the alpha-subunit of acetylc holine receptor (AChR). We have selected two different peptide-present ing phages (SWDDIR-phage and LWILTR-phage) which interact specifically with mAb 5.14. This interaction is specifically inhibited by AChR and by synthetic peptides corresponding to the hexapeptides presented by the selected phages. Although mAb 5.14 binds to AChR in its native as well as its denatured form, the selected hexapeptides do not exist as such in the AChR molecule. However, three amino acid sequence homologi es with these hexapeptides were shown to be present in the cytoplasmic region of Torpedo AChR. By extending the selected hexapeptides, at on e or both ends, with amino acid residues flanking the hexapeptides in the phage, we obtained mimotopes with an up to two order of magnitude higher affinity to the Ab. These extended peptides were able to effici ently block the binding of mAb 5.14 to both peptide-presenting phages, and to AChR.