The Intact Human Acetylcholinesterase C-Terminal Oligomerization Domain Is -Helical in Situ and in Isolation, but a Shorter F

详细信息    查看全文

• 作者：Matthew G. Cottingham ; Jan L. A. Voskuil ; and David J. T. Vaux
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：September 16, 2003
• 年：2003
• 卷：42
• 期：36
• 页码：10863 - 10873
• 全文大小：194K
• 年卷期：v.42,no.36(September 16, 2003)
• ISSN：1520-4995

文摘

A 14-residue fragment of the C-terminal oligomerization domain, or T-peptide, of humanacetylcholinesterase (AChE) shares sequence homology with the amyloid- peptide implicated inAlzheimer's disease and can spontaneously self-assemble into classical amyloid fibrils under physiologicalconditions [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 thatthe conformation of this AChE_586-599 peptide, both before and after fibril formation, is different from thatof a longer peptide, T₄₀, 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 subunitassembly, and possesses an -helical structure which protects against the development of the -sheet-richamyloidogenic conformation favored by the shorter constituent AChE_586-599 fragment. Using a conformation-sensitive monoclonal antibody raised against the -helical T₄₀ peptide, we demonstrate that theconformation of the T-peptide domain within intact AChE is antigenically indistinguishable from that ofthe synthetic T₄₀ peptide. A second monoclonal antibody raised against the fibrillogenic AChE_586-599fragment recognizes not only -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 monoclonalantibody identifies a region near the N-terminus of the peptide that remains accessible in oligomer andfibril alike, suggesting a model for the arrangement of subunits within AChE_586-599 protofibrils and fibrils.