Complex carbohydrates or glycans are involved in almost every physiological or pathological process. A major obstacle to advances in Glcyoscience is the lack of pure and structurally well-defined carbohydrates and glycoconjugates. These compounds are often found in low concentrations and in microheterogeneous forms greatly complicating their isolation and characterization. In many cases, well-defined oligosaccharides can only be obtained by chemical- or enzymatic approaches. Despite progress, the chemical synthesis of complex oligosaccharides remains very time consuming. The need for more efficient approaches has stimulated the development of chemo-enzymatic methods in which a synthetic oligosaccharide precursor is modified by a range of glycosyltransferases. A serious limitation of such an approach is that it only provides readily access to symmetrically branched oligosaccharides. A novel chemoenzymatic synthetic approach will be developed to produce highly complex symmetrically and asymmetrically branched /V-linked glycans. The approach will exploit a core pentasaccharide that at key branching positions is modified by orthogonal protecting groups that make it possible to selectively attach unique saccharide moieties by chemical glycosylation. The appendages will be selected in such a way that the antenna of the resulting deprotected compounds can be uniquely extended by glycosyltransferases to give large numbers of asymmetrical multi-antennary glycans. Furthermore, enzyme specificities uncovered in project 1 will be exploited for selective modifications to provide oligosaccharides that otherwise cannot be synthesized. The new chemo-enzymatic methodology will be employed to prepare a panel oligosaccharides found on human oocytes that have been implicated in sperm-egg binding. Furthermore probes will be prepared to identify the glycan binding protein of spermazoae that recognizes SLe''. The new methodology will also be employed to synthesize N-glycans found on primary swine respiratory epithelial cells that have been implicated in influenza virus infections. The compounds will be used in binding studies to uncover information about glycan topology and human and swine infectivity. Finally, the synthetic glycans will be employed as standard for glycan identification in complex biological mixtures. Ultimately, these studies may lead to the identification of novel biomarkers for various diseases.
Most cell surface proteins are modified by complex glycans (carbohydrates) giving rise to glycoproteins. These glycoproteins have been implicated as essential mediators of health and disease. The proposed project will develop new methods for the chemo-enzymatic synthesis of well-defined glycans of biological importance for biomedical research.
|Praissman, Jeremy L; Wells, Lance (2014) Mammalian O-mannosylation pathway: glycan structures, enzymes, and protein substrates. Biochemistry 53:3066-78|
|Bullard, Whitney; Lopes da Rosa-Spiegler, Jessica; Liu, Shuo et al. (2014) Identification of the glucosyltransferase that converts hydroxymethyluracil to base J in the trypanosomatid genome. J Biol Chem 289:20273-82|
|Teo, Chin Fen; Wells, Lance (2014) Monitoring protein O-linked ?-N-acetylglucosamine status via metabolic labeling and copper-free click chemistry. Anal Biochem 464:70-2|
|Praissman, Jeremy L; Live, David H; Wang, Shuo et al. (2014) B4GAT1 is the priming enzyme for the LARGE-dependent functional glycosylation of ?-dystroglycan. Elife 3:|
|Prudden, Anthony R; Chinoy, Zoeisha S; Wolfert, Margreet A et al. (2014) A multifunctional anomeric linker for the chemoenzymatic synthesis of complex oligosaccharides. Chem Commun (Camb) 50:7132-5|