Recent advances in immunology and molecular biology have lead to the development of therapeutic vaccines which are of potential use in chronic diseases such as cancer, cardiovascular disorders and neurodegenerative diseases, where efficacies of available therapies are poor. Future advances in vaccine development will rely substantially on a more complete understanding of the structural basis of immune response. Mass spectrometry has emerged as a widespread technique for the study of protein structure, function and interaction with other biomolecules.
Important features of the mass spectrometric protein analysis are the high sensitivity, high mass accuracy, short analysis time and low sample consumption. To obtain information on complex protein mixtures and to dissect the structure of the molecular recognition domains diverse applications have been developed in conjunction with mass spectrometry. These methods include chromatographic and electrophoretic separations, proteolytic assays, differential chemical modification of specific amino acid functions and bioinformatic tools for data analysis.
One of the hallmarks of Alzheimer´s Disease is the accumulation in the human brain of extracellular plaques containing aggregates of the neurotoxic ß-amyloid peptide.
The immunotherapeutical approaches capable of triggering the clearance of amyloid plaques and preventing Aß aggregation have gained increasing interest in recent years. The first two parts of the thesis are focused on the development and application of mass spectrometric and immuno-analytical methods to the identification of epitopes on Aß recognized by anti-Aß antibodies.
The first part of the thesis was focused on the detailed characterization of the ß-amyloid (4-10) FRHDSGY interaction with cognate antibodies. The sequence has been previously identified as a structural epitope for two antibodies, a polyclonal anti-Aß(1-42) and a monoclonal anti-Aß(1-17) antibody. In order to determine the functional significance of these residues to the antibodies, site-directed mutagenesis was performed using synthetic ß-amyloid (4-10) mutants as model substrate peptides.
Selective identification of the affinity preserving mutant peptides was achieved by comparative ELISA binding studies. While the interaction to the polyclonal antibody
was preserved in the D7A, S8A, G8A and Y10A mutants indicating F4, R5 and H6 as essential residues, for the monoclonal antibody all amino acid residues were essential for binding. The binding of Aß(1-16) to mAb- anti(1-17), clone 6E10 and mAb-anti(1-40) clone Bam-10 was studied in the presence of Zn2+ ions. Previous mass spectrometric studies mapped Zn2+ binding sites to His6, His13 and His14.
Aß(1-10) and Aß(1-16) were synthesized by solid phase peptide synthesis according to the Fmoc strategy with a pentaglycine spacer and biotin at the N-terminal end.
Both peptides reacted in a dose-dependent manner with the 6E10 and Bam-10 mAbs.
The presence of Zn2+ triggered a 4- and 10-fold increase in ELISA response of biotin-G5-Aß(1-16) to both mAb 6E10 and Bam-10. By contrast, the presence of Zn2+ was without effect on the ELISA response of biotin-G5-Aß(1-10). The cations Co2+ and Ni2+ had no effect on biotin-G5-Aß(1-16) recognition. The presence of Cu2+ ions did not influence the recognition of biotin-G5-Aß(1-16) by 6E10 mAb but resulted in a higher ELISA response with Bam-10. These results show a zinc induced conformational effect on the N-terminal region Aß(1-16) of the ß-amyloid peptide which may result in an enhanced accessibility of the F4-Y10 epitope to anti-Aß antibodies.
The second and major part of the thesis was focused on the identification of the epitope recognized by anti-Aß-autoantibodies naturally occurring in human blood.
The antibodies were isolated by affinity chromatography from human immunoglobulin preparations, and serum samples of Alzheimer´s disease patients. An affinity column for antibody isolation was prepared by immobilising Cys-Aß(1-40) on a iodoacetyl-support. For mass spectrometric epitope identification, an affinity column was prepared using the purified antibodies. In epitope excision, selective proteolytic cleavage of the intact Aß affinity- bound to the immobilised antibody was performed using trypsin or endoproteinase V8, followed by MALDI-TOF mass spectrometric analysis of the epitope- and non-epitope fractions, and provided direct information that the epitope is located within the sequence Aß(12-40). A consistent result was obtained by epitope extraction-mass spectrometry. The use of pronase provided the identification of Aß(21-37) as the minimal epitope structure for recognition.
Comparative binding studies of human Aß-antibodies with Aß(1-16), Aß(1-40), Aß(12-40) and Aß(17-28), each synthesized with a pentaglycine spacer and biotin at the N-terminal end, were performed by indirect ELISA. The results showed that
Aß(1-16) and Aß(17-28) do not interact with the antibodies while Aß(12-40) and Aß(1-40) reacted with the autoantibodies in a concentration-dependent manner.
Similar results were obtained by analyzing samples of anti-Aß autoantibodies isolated from AD patients. The separation by 2D gel electrophoresis revealed the polyclonality of the antibodies. MALDI-FT-ICR mass spectrometric analysis of the tryptic mixture resulting from in-gel digestion of the protein spots led to the identification of γ1 and γ2-heavy chains, and κ-light chains.
A further part of the dissertation was focused on the serine protease HtrA1 which has been implicated in amyloid precursor protein processing. Astrocytes produce significant levels of HtrA1 and Aß and application of an HtrA1 inhibitor leads to the accumulation of Aß in cell culture supernatants. ß-secretase cleaves amyloid precursor protein (APP) before Asp672 producing APP(672-770) which is further cleaved by γ-secretase resulting in APP(672-711/713) referred as Aß, and a C-terminal fragment. To provide quality assurance for the investigation of the proteolytic specificity of HtrA1, the primary structure of the recombinant APP(672-770) was characterised by a combination of 1D-gel electrophoresis and mass spectrometry.
Proteolytic digestion by HtrA1 was analysed for APP(672-770) in comparison to APP(672-711), APP(672-713), APP(724-770) and APP(661-687), and digestion products were identified directly by high-resolution MALDI-FT-ICR. Digestion of APP(672-770) was established to occur after residues Val-683, Gln-686, Asn-755, and Asp-672 providing degradation products of approximately equal sequence lengths. A consistent molecular fragmentation pattern for the C-terminal end of C99 was found when using APP(724-770) and Aß as substrates. However, additional cleavage sites were observed at similar sequence lengths ranging between 10-20 residues.
The last part of the thesis was focused on mass spectrometric epitope elucidation of a specific antibody to the H1-carbohydrate recognition domain. ESI-FT-ICR mass spectrum of the intact H1CRD (17 kDa) and LC-MS of antigen tryptic mixtures provided information on the structure of the antigen. The Met-1 residue was found to be missing. A comparison between the MALDI-FT-ICR mass spectra of the tryptic mixture of the antigen in native and reduced/alkylated form led to the identification of 2 disulfide bridges. The antigen was found to bind to the immobilised antibody in
both native and alkylated form. Epitope excision and extraction using trypsin led to the identification of 2 N-terminal epitope peptides which exhibit affinity to the antibody.