Integrating glycomics, proteomics and glycoproteomics to understand the structural basis for influenza a virus evolution and glycan mediated immune interactions
MetadataShow full item record
Glycosylation modulates the range and specificity of interactions among glycoproteins and their binding partners. This is important in influenza A virus (IAV) biology because binding of host immune molecules depends on glycosylation of viral surface proteins such as hemagglutinin (HA). Circulating viruses mutate rapidly in response to pressure from the host immune system. As proteins mutate, the virus glycosylation patterns change. The consequence is that viruses evolve to evade host immune responses, which renders vaccines ineffective. Glycan biosynthesis is a non-template driven process, governed by stoichiometric and steric relationships between the enzymatic machinery for glycosylation and the protein being glycosylated. Consequently, protein glycosylation is heterogeneous, thereby making structural analysis and elucidation of precise biological functions extremely challenging. The lack of structural information has been a limiting factor in understanding the exact mechanisms of glycan-mediated interactions of the IAV with host immune-lectins. Genetic sequencing methods allow prediction of glycosylation sites along the protein backbone but are unable to provide exact phenotypic information regarding site occupancy. Crystallography methods are also unable to determine the glycan structures beyond the core residues due to the flexible nature of carbohydrates. This dissertation centers on the development of chromatography and mass spectrometry methods for characterization of site-specific glycosylation in complex glycoproteins and application of these methods to IAV glycomics and glycoproteomics. We combined the site-specific glycosylation information generated using mass spectrometry with information from biochemical assays and structural modeling studies to identify key glycosylation sites mediating interactions of HA with immune lectin surfactant protein-D (SP-D). We also identified the structural features that control glycan processing at these sites, particularly those involving glycan maturation from high-mannose to complex-type, which, in turn, regulate interactions with SP-D. The work presented in this dissertation contributes significantly to the improvement of analytical and bioinformatics methods in glycan and glycoprotein analysis using mass spectrometry and greatly advances the understanding of the structural features regulating glycan microheterogeneity on HA and its interactions with host immune lectins.