The intact human acetylcholinesterase C-terminal oligomerization domain is alpha-helical in situ and in isolation, but a shorter fragment forms beta-sheet-rich amyloid fibrils and protofibrillar oligomers.
A 14-residue fragment of the C-terminal oligomerization domain, or T-peptide, of human acetylcholinesterase (AChE) shares sequence homology with the amyloid-beta peptide implicated in Alzheimer's disease and can spontaneously self-assemble into classical amyloid fibrils under physiological conditions [Greenfield, S. A., and Vaux, D. J. (2002) Neuroscience 113, 485-492; Cottingham, M. G., Hollinshead, M. S., and Vaux, D. J. (2002) Biochemistry 41, 13539-13547]. Here we demonstrate that the conformation of this AChE(586-599) peptide, both before and after fibril formation, is different from that of a longer peptide, T(40), corresponding to the entire 40-amino acid T-peptide (residues 575-614 of AChE). This peptide is prone to homomeric hydrophobic interactions, consistent with its role in AChE subunit assembly, and possesses an alpha-helical structure which protects against the development of the beta-sheet-rich amyloidogenic conformation favored by the shorter constituent AChE(586-599) fragment. Using a conformation-sensitive monoclonal antibody raised against the alpha-helical T(40) peptide, we demonstrate that the conformation of the T-peptide domain within intact AChE is antigenically indistinguishable from that of the synthetic T(40) peptide. A second monoclonal antibody raised against the fibrillogenic AChE(586-599) fragment recognizes not only beta-sheet amyloid aggregates but also SDS-resistant protofibrillar oligomers. A single-antibody sandwich ELISA confirms that such oligomers exist at micromolar peptide concentrations, well below that required for formation of classical amyloid fibrils. Epitope mapping with this monoclonal antibody identifies a region near the N-terminus of the peptide that remains accessible in oligomer and fibril alike, suggesting a model for the arrangement of subunits within AChE(586-599) protofibrils and fibrils.
Cottingham
Matthew G
MG
Sir William Dunn School of Pathology, University of Oxford, UK.
Voskuil
Jan L A
JL
Vaux
David J T
DJ
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002478:Cells, Cultured; D002942:Circular Dichroism; D018604:Epitope Mapping / Q000379:methods; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D014162:Transfection
D000682:Amyloid; D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000110:Acetylcholinesterase