Development and application of methods for structural characterization of xyloglucans and other complex glycans
Tuomivaara, Sami Tapio
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Plant cell walls are extraplasmalemmal organelles whose structural complexity and mechanical robustness render them especially challenging for detailed structural and functional scrutiny. Xyloglucans are a major class of complex polysaccharides with structural, metabolic and regulatory functions in the cell walls of land plants. The amounts and fine structures of xyloglucans vary among plant taxa, tissue and cell type, and according to the developmental stage of the cell. The range of xyloglucan functions and the functional significance of their structural diversity are currently not well understood. Technological advances are now making it possible to begin understanding these aspects of xyloglucan biology. Towards this end, I generated and purified twenty-eight xyloglucan oligosaccharides by a combination of enzymatic hydrolyses and preparative scale chromatography, and demonstrated their utility as mass spectrometry and chromatography standards and as probes in epitope characterization of thirty-two xyloglucan-binding monoclonal antibodies. Using these oligosaccharides, I first compared the utility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and high-pH anion exchange chromatography with pulsed amperometric detection as quantitative methods to study xyloglucan oligosaccharide mixtures. Both methods are suitable for routine analyses of xyloglucan oligosaccharide mixtures, but due to the presence of isomeric structures that leads to ambiguity in the interpretation of mass spectrometry data, and co-elution that leads to ambiguity in the interpretation of chromatography data, these methods should be used in conjunction. Immunofluorescence methods on plant tissue sections are currently one of the most powerful approaches to study the abundances, structures and localizations of xyloglucans in the plant cell walls. Despite their popularity, there has been substantial uncertainty in the interpretation of the observed binding patterns since the precise epitopes of these antibodies had not been determined. The availability of this xyloglucan oligosaccharide collection allowed me to establish the minimum epitopes of these antibodies in great chemical detail using enzyme-linked immunosorbent assays. Knowledge of the binding specificities allows structural interpretation of data from immunofluorescent and other monoclonal antibody utilizing experiments. Collectively, these contributions provide fundamental information that will facilitate our understanding of the biological roles of xyloglucans in the plant cell walls.