Isolation and quantification of the cortical granule lectin ligand oligosaccharides and elucidation of their role in the block to polyspermic fertilization in Xenopus laevis

Thesis (M.S., Biological Sciences (Molecular and Cellular Biology)) -- California State University, Sacramento, 2010.The block to polyspermic fertilization in Xenopus laevis is mediated by a calcium-dependent, galactose specific binding reaction between a lectin derived from the cortical granules and its ligand partners located in the immediate surroundings within the egg extracellular matrix. The cortical granule lectin (CGL) ligands have been shown to possess O-linked oligosaccharides as the functional moieties when binding to the CGL. However, it is unknown as to which particular ligand oligosaccharides are the functional moieties in the binding interaction. Elucidation of the functional oligosaccharides will be valuable to our understanding of fertilization and cell-cell binding interactions involving this lectin family of CGL-like proteins.\ud \ud In the current work, an HPLC based method was developed to profile the oligosaccharides found on the ligands and to isolate and quantify them for subsequent functional binding assays. A novel method was developed without derivatization agents or exact standards utilizing the HPLC-CAD (Charged Aerosol Detection) system interfaced with an amino Prevail Carbohydrate ES HPLC column. The hypothesis for this study was that the HPLC-CAD methodology will allow the isolation and quantification of O-linked oligosaccharides released from the CGL ligand which can then be utilized in functional binding assays to assess which oligosaccharides function in the lectin-ligand binding interaction during the X. laevis block to polyspermy.\ud \ud In testing the methodology, it was demonstrated to be sensitive in the picomolar range or mass detection limits 0.3 to 0.9 ng with regard to oligosaccharides separated under optimized conditions. Commercially available standard oligosaccharides were accurately quantified to within less than a 19% average error with excellent reproducibility. The X. laevis CGL ligand oligosaccharides were chemically released and profiled using the HPLC-CAD demonstrating the presence of four predominate oligosaccharide peaks. Oligosaccharides were subsequently separated, isolated and quantified utilizing the developed quantification methodology employing the Prevail Carbohydrate ES HPLC column. \ud \ud A plate binding assay was developed (enzyme-linked lectin assay) to test whether particular compounds could competitively inhibit the binding interaction of purified CGL and ligands thereby implicating a functional role in binding. The data indicated that binding could be inhibited by galactose, fucose and the galactose containing disaccharides lactose and melibiose. In addition, it was found that whole CGL ligand oligosaccharide fractions possessed strong inhibition properties. The neutral CGL ligand oligosaccharide fraction demonstrated strong lectin-ligand inhibition characteristics eliciting a 95% reduction in binding, whereas the acidic CGL ligand fraction elicited only a 35% reduction. However, when the isolated and quantified CGL ligand oligosaccharide fractions were tested at concentrations of 10 and 45 ??M, they were unable to elicit an inhibitory response of the lectin-ligand binding interaction. Based on these observations, the evidence suggests that there are oligosaccharides that do inhibit lectin-ligand binding that are derived from the ligands, but that the particular purified oligosaccharides tested were not effective at inhibiting binding at the concentrations used. Future studies will need to test these compounds at higher concentrations and to test other oligosaccharide fractions to elucidate which oligosaccharides are the functional binding moieties on the ligand.Biological Sciences (Molecular and Cellular Biology