Autoimmune-related diseases have become a major medical concern in recent years due to the increasing number individuals affected, poor understanding of disease etiology, and the lack of an efficient therapy. In the last years, evidence suggests the implication of the immune system in the pathogenesis and progression of autoimmune diseases, and new therapeutic approaches targeting the immune system are being developed. Future advances in the design of effective vaccines will substantially depend on a more complete understanding of the structural basis of immune responses. Mass spectrometry has emerged as a high performance tool for protein structure determination and characterization of protein – protein interactions, because of its sensitivity, high mass accuracy, short analysis time and applicability to mixtures. The successful development of analytical strategies of mass spectrometry, such as differential chemical modification of proteins and proteolytic epitope excision, have improved the level of structural information provided by classical techniques by revealing information about molecular recognition structures in antigen-antibody interactions. In particular, tandem mass spectrometry, producing different types of fragment ions by various dissociation techniques, has been developed and applied as a major tool in this dissertation for determination of protein structures and post-translational modifications .
Alzheimer's disease related neurodementia has become a great medical threat among the aging population, with the histopathological hallmark being the deposition of ß-amyloid plaques in brain. Innovative immunotherapeutic approaches, aimed at reducing the plaques and preventing neurotoxic effects and aggregation of Aß, are based on the epitope structures recognized by Aß-specific antibodies. The first two chapters of this thesis are focused on the determination of complete primary structures, and elucidation of the N-glycosylation structures of Aß-specific antibodies. Plaque-specific antibodies produced by active immunization with Aß recognize an epitope located at the Aß N-terminus (4FRHDSGY10) and were shown to reduce the amyloid burden and to improve cognitive functions. Using high performance liquid chromatography – tandem mass spectrometry LC-MSMS, the complete structural characterization of a plaque-specific mouse monoclonal antibody was performed. Using both database search and de novo sequence analysis, near-complete sequence determinations were obtained for both heavy and light chains, and the amino acid sequences of five of the six complementary
determining regions were completely elucidated. Several sequence microheterogeneities were identified in the region spanning the CDR2 of the light chain, which may provide key information for the antigen recognition arising during the B-cell affinity maturation. The major glycan structure attached at the heavy chain constant region was identified to be of complex type with zero to four terminal galactose residues and minimal amounts of N-glycolyl neuraminic acid.
Human physiological Aß-antibodies (“plaque-protective" antibodies) were identified in plasma of healthy individuals and recognize an epitope located in the C-terminal region of Aß (residues (21-37)), and this feature is suggested to prevent accumulation and deposition of Aß. The complete primary structure and glycosylation of a plaque-protective mouse monoclonal antibody, raised against an epitope located at residues (17-24) of Aß, was obtained by LC-MS/MS, in combination with collision induced dissociation and electron-transfer dissociation (CID and ETD) fragmentation. In this study, the structures of all six CDRs, and an unexpected consensus amino acid sequence of N-linked glycosylation were elucidated in the light chain. This glycosylation site was identified to carry partially hybrid glycans having one antenna of high mannose type, and a second one of complex type, terminated with N-acetyl and N-glycolyl neuraminic acid. Furthermore, small amounts of complex type structures were identified in the variable region, containing up to two galactose residues and terminated with N-glycolyl neuraminic acid. Glycosylation of the heavy chain constant region was found less heterogeneous than that of the variable region, consisting of complex type structures with zero to four galactose residues.
Epitope-specific affinity purification of plaque-protective, human Aß-autoantibodies from commercial immunoglobulins was performed using immobilized Cys-Aß(12-40), and the determination of their primary structures indicated the presence of all four IgG subclasses.
Subclass- specific glycosylation analysis, performed at the glycopeptide level, revealed elevated amounts of IgG2/IgG3 and IgG4, respectively. The glycan structure of each subclass indicated comparable amounts of the individual glycoforms in the Aß-autoantibody and pooled immunoglobulins, with a slight tendency towards increased agalactosylation of the autoantibody.
The ß-amyloid peptide is derived by proteolytic processing of the amyloid precursor protein APP, a transmembrane protein of hitherto largely unknown function. Although the
importance of N- and O-glycosylation in the secretory pathway of APP was previously suggested, no sites of glycosylation have been reported. The elucidation of the O-glycosylation structures of APP at three specific sites, Thr291, Thr292 and Thr576, was obtained using LC-MS and a combination of ETD and CID fragmentation. The O-glycosylation sites, Thr291, Thr292 and Thr576, were found to comprise multiple short Core-1 type glycans. The minimal O-glycan structure was N-acetyl galactosamine (GalNAc), while elongated structures were identified to contain the GalNAc – Gal core terminated with sialic acid, attached either in a linear fashion to galactose, or branched with attachment to GalNAc.
In the last chapter of this thesis, the determination of subclass specific glycosylation structures of total plasma IgG from myositis patients was performed as part of a clinical study aimed at delineating genetic and environmental risk factors for pathogenesis of systemic rheumatic disorders. Total plasma IgG from patients, healthy twins/siblings and unrelated age matched controls was isolated using protein G affinity chromatography.
Analysis of glycopeptides from each subclass revealed elevated amounts of core-fucosylated agalaytosyl glycoform (G0F) in patients compared to unrelated controls, and these two groups were found to be statistically different. In contrast, statistical analysis of patients – siblings and siblings – controls groups suggested a random structure distribution of antibody glycosylation. This indicated the existence of a genetic predisposition of healthy siblings towards development of an autoimmune condition, possibly as a result of various environmental exposures.