title: Identification and structural characterisation of carboxy-terminal polypeptides and antibody epitopes of Alzheimer's amyloid precursor protein using high-resolution mass spectrometry.
abstract: Alzheimer's disease (AD) is the most common cause for human age-related dementia, characterised by formation of diffuse plaques in brain that are directly involved in AD pathogenesis. The major component of AD plaques is beta-amyloid, a 40 to 42 amino acid polypeptide derived from the amyloid precursor protein (APP) by proteolytic degradation involving the specific proteases, beta-and gamma-secretase acting at the N- and C- terminal cleavage site, respectively. In this study we have prepared polypeptides comprising the carboxy-terminal and transmembrane sequences of APP, by bacterial expression and chemical synthesis, as substrates for studying the C-terminal processing of APP and its interaction with the gamma-secretase complex. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used as a major tool for structure analysis. Immunisation of transgenic mouse models of AD with Abeta42 has been recently shown to be effective to inhibit and disaggregate Abeta-fibrils, and to reduce AD-related neuropathology and memory impairments. However, the mechanism underlying these therapeutic effects has been as yet unclear. Using proteolytic epitope excision from immune complexes in combination with FT-ICR-MS, we identified the epitope recognised by the therapeutically active antibody as the N-terminal Abeta(4-10) sequence; this soluble, nontoxic epitope opens new lead structures for AD vaccine development. A monoclonal antibody (Jonas; JmAb) directed against the cytosolic APP domain was used in studies of APP biochemistry and metabolism. Here we report the identification of the epitope recognised by the JmAb, using the combination of epitope excision and peptide mapping by FT-ICR-MS. The epitope was determined to be located at the C-terminal APP(740-747) sequence; it was confirmed by ELISA binding assays and authentic synthetic peptides and will be an efficient tool in the development of new specific vaccines. These results demonstrate high-resolution FT-ICR-MS as a powerful method for characterising biochemical pathways and molecular recognition structures of APP.
authors.affiliation: Department of Chemistry, Analytical Chemistry, University of Konstanz, 78457 Konstanz, Germany. xiaodan.tian@uni-konstanz.de
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Tian
authors.forename: Roxana
authors.initials: R
authors.lastname: Cecal
authors.forename: JoAnne
authors.initials: J
authors.lastname: McLaurin
authors.forename: Marilena
authors.initials: M
authors.lastname: Manea
authors.forename: Raluca
authors.initials: R
authors.lastname: Stefanescu
authors.forename: Sandra
authors.initials: S
authors.lastname: Grau
authors.forename: Mona
authors.initials: M
authors.lastname: Harnasch
authors.forename: Sarah
authors.initials: S
authors.lastname: Amir
authors.forename: Michael
authors.initials: M
authors.lastname: Ehrmann
authors.forename: Peter
authors.initials: P
authors.lastname: St George-Hyslop
authors.forename: Markus
authors.initials: M
authors.lastname: Kohlmann
authors.forename: Michael
authors.initials: M
authors.lastname: Przybylski
mesh_terms: D000544:Alzheimer Disease / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000818:Animals; D017357:Cyclotrons; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D005583:Fourier Analysis; D006801:Humans; D007477:Ions; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D016564:Amyloid beta-Protein Precursor; D000939:Epitopes; D007477:Ions; D011994:Recombinant Proteins