| Glycosaminoglycans (GAGs) are highly complex linear heterogeneous polysaccharides present at the cell-extracellular matrix (ECM) interface where they interact with several of proteins modulating fundamental biological processes such as cell growth, adhesion, and development. Despite the importance of GAGs in many biological processes, the structural determination of GAGs is a challenging task. NMR spectroscopy has been the most reliable and vital tool for the structural characterization of heterogeneous mixtures of GAGs. In the first part of this study, we used the combination of one-dimensional and two-dimensional sophisticated NMR techniques to determine the structure of an unknown GAG that was named as "testosteronan". Although the testosteronan consists of the sugars D-glucuronic acid and N-acetyl- D-glucosamine, it shows unexpected resistance to GAG-degrading enzymes. 2D-NOESY experiments subsequently demonstrated that the polysaccharide backbone was [-4-D-GlcUA-alpha 1,4-D-GlcNAc-alpha1-]n. This is a linkage for a GAG and explains its resistance to GAG-degrading enzymes.;The second and the main portion of this thesis focused on developing an ultrasensitive analytical method for GAGs and N-glycans. GAGs have remarkably high information content; however, unlike nucleic acids and proteins their biosynthesis is a non-template driven process. Also, GAG species are usually found in low abundance in tissue or cell samples and most critically the oligosaccharide motif that is recognized by a protein is often found in a very low abundance. Thus, there is an urgent need for analytical methods capable of detecting very low abundance GAGs extracted from biological samples. A novel glyco-quantitative polymerase chain reaction (Glyco-qPCR) assay platform for acidic carbohydrates is described. Using chondroitin sulfate (CS) and silaylated N-glycans as model carbohydrates, Glyco-qPCR affords ultrasensitive glycan detection. The Glyco-qPCR approach requires glycans that contain a free reducing end and a carboxyl group, allowing the introduction of a double label of biotin, a capture tag and DNA, a detection tag. Using this technique, we were able to detect as little as 1 zeptomole (10-21 mole) pure biotynlated CS disaccharides. Also, preparative capillary electrophoresis was used to separate and collect different type of chondroitin sulfate GAG-derived disaccharides obtained from Chinese hamster ovary (CHO) cells prior to coupling reactions and qPCR detection. By combining CE separation and collection with Glyco-qPCR, we were able to detect amounts of CS disaccharides (CS-0S and CS-4S) isolated from as few as 500 CHO cells. Next we applied this technique for the detection of N-glycans purified from decorin proteoglycan (PG). We compared the detection limit of Western blot analysis and Glyco-qPCR. The results indicated that 1 fg of decorin PG was required for detection. This makes Glyco-qPCR 106-fold more sensitive than Western blot analysis. |
