Method Development for O-glycan Analysis and its Application to Parkinson's Disease

Glycosylation is the most common post-translational modification of proteins with glycoproteins performing roles in cell-cell interactions, protein folding and function. By observing glycosylation changes between control and diseased tissues, biomarkers may be identified and regenerative bio-implants created such as glycan-functionalized implantable hydrogels. Two main classes of glycans present on proteins are N-glycans (bound to asparagine residues) and O-glycans (bound to serine and threonine residues). Due to the availability of endoglycosidases such as PNGase F which cleaves common oligomannose, complex and hybrid N-glycans from glycoproteins, there is a strong understanding of N-glycan heterogeneity, however, no single approach exists to release O-glycans with the same level of proficiency due in-part to a lack of common O-glycan core. Enzymatic methods may be used to selectively cleave only the most basic O-glycan disaccharides with no further saccharide modification, however, this method is severely limited. Alternative chemical methods are used (typically under alkaline conditions where O-glycosylation sites are labile) to release the vast array of more complex O-glycan structures. The downside to this is a process termed “peeling” in which the glycan undergoes rapid stepwise monosaccharide degradation from the cleaved end of the structure. The objectives of this Research Masters were (i) to critically evaluate the state-of-the-art of O- glycosylation, addressing strengths and weaknesses of the various forms of release and analysis, (ii) to develop and optimize an O-glycan release protocol by applying a mildly alkaline reaction solution to glycoprotein standards, under microwave radiation, in a process termed microwave-assisted ammonia-based non-reductive ß-elimination to liberate O-glycans from the protein for subsequent fluorescence and mass spectrometric analysis; and (iii) to apply this optimized release and analysis protocol to characterize and statistically evaluate O-glycan changes between healthy and diseased patients with Parkinson’s Disease (PD) and Incidental Lewy Body Disease (ILBD; a progenitor disease or “pre-diagnosis” to PD). The optimization of the O-glycan release protocol was successful in achieving reproducibility of release over 10 replicates of bovine fetuin standard glycoprotein, with peeling reduced to levels typically seen in commercially available released O-glycan standards. This instilled confidence in O-glycan release and analysis of PD, where 77 O-glycans were identified. It was discovered that sialylation upregulation was observed in nigrostriatal regions (striatum and substantia nigra) in PD. Sulfation was also observed to decrease in striatum upon PD pathogenesis, and total glucuronylation and presence of mannose core structures were found to be decreased in ILBD patients. The work set out here identified several suitable biomarkers, not yet described in the literature, for further studies into glycan therapeutics and PD research.