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 californica 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-linked oligosaccharide residue on the protein. Isolation of cyanogen bromide peptides from the amphiphilic form and assay by a competition ELISA for 2C9 and by a direct binding ELISA for 4E7 identified the same peptide, 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 synthetic peptide further localized the 2C9 epitope. Peptides RFRRPEPKKPW and KPWSGVWNASTY both reacted but less so than the entire synthetic peptide at equivalent molar concentrations, whereas the peptide RPEPKKPWSGVWNASTY was as effective as the larger synthetic peptide. The crystal structure of AChE shows the peptide to be on the surface of the molecule as part of a convex hairpin loop starting before the first alpha-helix.
Wasserman
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L
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Doctor
B P
BP
Gentry
M K
MK
Taylor
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P
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003196:Computer Graphics; D003488:Cyanogen Bromide; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D014101:Torpedo
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D010455:Peptides; D000110:Acetylcholinesterase; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D003488:Cyanogen Bromide