CORE A This project is a Resource Core directed at the expression and purification of soluble catalytic domains for numerous glycan biosynthetic enzymes in mammalian suspension culture cells as reagents for the three Projects in this application. This Core activity extends from prior efforts to develop a """"""""Repository of Glyco- enzyme Expression Constructs"""""""" for production of all human glycosyltransferases (GTs) and other glycosylation enzymes. These coding regions were captured and transferred into custom expression vectors for production in mammalian cells and other recombinant hosts. The successful multi-milligram expression of the GTs in mammalian cells is the foundation for Aim 1, where large-scale enzyme expression and purification of the GTs is described for studies on the enzymology and structural biology of the enzymes (Project 1), use ofthe enzymes to aid in synthesis of larger symmetric and assymetric glycan structures (Project 2), and application ofthe well-characterized enzymes toward selective cell surface tagging techniques that will allow glycan and glycoprotein acceptor identification, as well as monitoring traffic and recycling ofthe glycosylated molecules (Project 3). Continued development of novel methods for fusion tag and glycan removal is proposed in Aim 2 for structural studies in Project 1 based on preliminary data with a small number of model enzymes. Strategies for 13C and 15N- amino acid incorporation as well as seleno- Met labeling are also being developed based on initial success in production of labeled recombinant enzymes for NMR and X-ray crystallography studies.
Aim 3 focuses on specificity studies on larger glycoprotein substrates both in vitro and in cultured cells to examine enzyme competition and other factors in the secretory pathway. The initial focus is on a subset of GTs important in modifying the termini of the glycans on glycoproteins and glycolipids, the sialyltransferases and fucosyltransferases, with the development of protocols that can be applied more widely to other enzyme families. These enzymes will provide reagents and greater insights that will enable the more effective use of these enzymatic catalysts in glycan synthesis and as tools for biological applications and studies on disease models.
Glycosylation enzymes are the biosynthetic machinery that generate protein- and lipid-bound glycan structures that influence biological processes from cell development to cell-cell interactions and the life time of signaling molecules in serum. Production of the enzymes will foster their use in enzymatic synthesis, diagnosis, and understanding disease.
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