Glycomics analysis of heparan sulfate domain structure.

摘要

Heparan sulfate (HS) is a ubiquitous cell surface molecule that regulates a multitude of biochemical events including cellular proliferation, differentiation, and pathogenesis. HS polysaccharides display an extraordinary degree of structural heterogeneity, a result of the fact that the sulfation, epimerization, and acetylation reactions that modify the chains do not occur at every possible site. The HS population on a given cell surface is thus a mixture of glycoforms. Despite the inherent heterogeneity, there are several conserved elements of HS structure, including domains of high, low, and intermediate sulfation. It has become clear that the protein-binding ability of HS, and consequently its role in cell-signaling events, is dependent on the organization of its structural domains and on their modification patterns. Mass spectrometry (MS) is a powerful tool for the analysis of complex mixtures, including carbohydrates, but the ability to perform HS glycomics by MS has been hampered due to analytical difficulties specific to this glycan class. To address this, a normal phase, chip-based liquid chromatography-MS system was optimized for analysis of HS oligosaccharides, with particular focus placed on the highly sulfated domains known to interact with growth factors. The result was a robust, reproducible method for heparinoid analysis successful for profiling highly sulfated oligosaccharides up to 18-mer in length. The platform was subsequently applied to comparative glycomics of HS structures from different mammalian tissue sources. The results indicate that extended, highly sulfated domains reside at the end of HS chains. In separate experiments, a size exclusion chromatography MS system was utilized to investigate the Sulf family of enzymes, known to edit HS structure by releasing a subset of 6O-sulfates in a domain specific manner. The action of Sulf2 on an array of HS substrates was determined, and the results indicate that these enzymes uniquely modify HS chain termini. The work presented in this thesis contributes significantly to the knowledge of HS domain structure, especially with respect to novel features of HS termini that are likely to influence signaling events dependent on this region of the chain.