Identification and structural characterisation of carboxy-terminal polypeptides and antibody epitopes of Alzheimer's amyloid precursor protein using high-resolution mass spectrometry
Alzheimer's disease (AD) is the most common cause for human age-related dementia, characterised by formation of diffuse plaques in the brain that are directly involved in AD pathogenesis. The major component of AD plaques is β-amyloid, a 40 to 42 amino acid polypeptide derived from the amyloid precursor protein (APP) by proteolytic degradation involving the specific proteases, β-and γ-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 γ-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 Aβ42 has been recently shown to be effective to inhibit and disaggregate Aβ-fibrils, and to reduce AD-related neuropathology and memory impairments. However, the mechanism underlying these therapeutic effects as yet has been 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 Aβ(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.
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