EPITOPE MAPPING OF FORM-SPECIFIC AND NONSPECIFIC ANTIBODIES TO ACETYLCHOLINESTERASE

We have mapped the epitopes to which two monoclonal antibodies against acetylcholinesterase (AChE) from Torpedo caifornica are directed. One antibody, 2C9, has equivalent affinity for both the 5.6S (amphiphilic ) and 11S (hydrophilic) enzyme forms; the other, 4E7, recognizes only the amphiphilic form and has been shown previously to require an N-lin ked oligosaccharide residue on the protein. Isolation of cyanogen brom ide peptides from the amphiphilic form and assay by a competition ELIS A for 2C9 and by a direct binding ELISA for 4E7 identified the same pe ptide, residues 44-82, as containing epitopes against both antibodies. The epitope for 4E7 includes the oligosaccharide conjugated to Asp59, an N-linked glycosylation site not present in mouse AChE. A 20-amino- acid synthetic peptide, RFRRPEPKKPWSGVWNASTY, representing residues 44 -63, was synthesized and found to inhibit completely 2C9 binding to 5. 6S enzyme at molar concentrations comparable to those of the cyanogen bromide peptide. It was unreactive with 4E7. Fractionation of the synt hetic peptide further localized the 2C9 epitope. Peptides RFRRPEPKKPW and KPWSGVWNASTY both reacted but less so than the entire synthetic pe ptide at equivalent molar concentrations, whereas the peptide RPEPKKPW SGVWNASTY was as effective as the larger synthetic peptide. The crysta l structure of AChE shows the peptide to be on the surface of the mole cule as part of a convex hairpin loop starting before the first alpha- helix.