Establishing the Molecular Basis of Glycoconjugate Glycosylation

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P R O J E C T I This project is a part of a program project directed at turning a platform for the expression of highly active glycan processing enzymes into new high-specificity tools for the synthesis of complex glycans, the detection of their existence on glycoproteins and glycolipids in cellular environments, and the monitoring of redistribution in the course of disease. This is no small task as there are an estimated 7000 distinct glycans in mammalian systems and ~200 glycosyltransferases (GTs) contributing to their synthesis in humans. We will take an important step toward accomplishing this task by building a molecular level understanding ofthe origin of GT specificity. The initial focus is a subset of GTs important in modifying the termini ofthe glycans on glycoproteins and glycolipids, members ofthe sialyltransferase and fucosyltransferase families, and building a protocol for the study of other families. Pursuit of three general aims will contribute to this understanding. 1) Substrate specificity of GTs will be characterized using glycan arrays to identify sugar residues and linkages where primary recognition occurs. Novel enzyme based technology will be developed to allow sensitive detection of recognized glycans and the results will be fed to internal and external collaborators to allow development of new synthetic procedures and tagging strategies that will further expand arrays. 2) Atomic level structures of GTs and their bound sugar donors and acceptors will be produced. A combination of X-ray crystallography and NMR methodology will be applied, taking particular advantage ofthe expression platform's ability to produce homogeneously glycosylated GTs for crystallography and sparse isotope labeled GTs for NMR investigation. The resulting structures will provide information needed forthe rational design of functionalized acceptor and donor analogs, as well as future GT inhibitors and genetically modified enzymes. 3) The efficiency and specificity of glycosyltransferase reactions will be examined in cellular environments using a glycoprotein substrate in order to assess the influence of a protein context and cellular location on GT specificity. The information will be essential in extrapolating molecular level specificities to action of enzyme-based reagents in vivo.