Molecular characterization of the -amyloid(4-10) epitope of plaque specific A antibodies by affinity-mass spectrometry using alanine site mutation

Alzheimer disease is a neurodegenerative disease affecting an increasing number of patients worldwide. Current therapeutic strategies are directed to molecules capable to block the aggregation of the -amyloid(1-42) (A) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. A-specific antibodies have been recently developed as powerful antiaggregation tools. The identification and functional characterization of the epitope structures of A antibodies contributes to the elucidation of their mechanism of action in the human organism. In previous studies, the A(4-10) peptide has been identified as an epitope for the polyclonal anti-A(1-42) antibody that has been shown capable to reduce amyloid deposition in a transgenic Alzheimer disease mouse model. To determine the functional significance of the amino acid residues involved in binding to the antibody, we report here the effects of alanine single-site mutations within the A-epitope sequence on the antigen-antibody interaction. Specific identification of the essential affinity preserving mutant peptides was obtained by exposing a Sepharose-immobilized antibody column to an equimolar mixture of mutant peptides, followed by analysis of bound peptides using high-resolution MALDI-Fourier transform-Ion Cyclotron Resonance mass spectrometry. For the polyclonal antibody, affinity was preserved in the H6A, D7A, S8A, and G9A mutants but was lost in the F4, R5, and Y10 mutants, indicating these residues as essential amino acids for binding. Enzyme-linked immunosorbent assays confirmed the binding differences of the mutant peptides to the polyclonal antibody. In contrast, the mass spectrometric analysis of the mutant A(4-10) peptides upon affinity binding to a monoclonal anti-A(1-17) antibody showed complete loss of binding by Ala-site mutation of any residue of the A(4-10) epitope. Surface plasmon resonance affinity determination of wild-type A(1-17) to the monoclonal A antibody provided a binding constant K-D in the low nanomolar range. These results provide valuable information in the elucidation of the binding mechanism and the development of A-specific antibodies with improved therapeutic efficacy.