Epitope structure and binding affinity of single chain llama anti-ß-amyloid antibodies revealed by proteolytic excision affinity-mass spectrometry

Abstract

ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer’s Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aß epitope recognized by the variable domain of single chain llama anti-Aß-antibodies, termed Aß-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aß-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin,GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization – mass spectrometric analysis of the affinity – elution fraction provided the epitope, Aß(17–28), in the mid- to carboxy-terminal domain of Aß, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aß(17–28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aß(1–40) or Aß-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aß-nanobodies and Aß(1–40) and the Aß(17–28) epitope provided KD values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aß-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors.