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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM107012-02
Application #
8740510
Study Section
Special Emphasis Panel (ZRG1)
Project Start
Project End
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Georgia
Department
Type
DUNS #
City
Athens
State
GA
Country
United States
Zip Code
30602
Urbanowicz, Breeanna R; Bharadwaj, Vivek S; Alahuhta, Markus et al. (2017) Structural, mutagenic and in silico studies of xyloglucan fucosylation in Arabidopsis thaliana suggest a water-mediated mechanism. Plant J 91:931-949
Aarnio-Peterson, Megan; Zhao, Peng; Yu, Seok-Ho et al. (2017) Altered Met receptor phosphorylation and LRP1-mediated uptake in cells lacking carbohydrate-dependent lysosomal targeting. J Biol Chem 292:15094-15104
Capicciotti, Chantelle J; Zong, Chengli; Sheikh, M Osman et al. (2017) Cell-Surface Glyco-Engineering by Exogenous Enzymatic Transfer Using a Bifunctional CMP-Neu5Ac Derivative. J Am Chem Soc 139:13342-13348
Hanes, Melinda S; Moremen, Kelley W; Cummings, Richard D (2017) Biochemical characterization of functional domains of the chaperone Cosmc. PLoS One 12:e0180242
Benedetti, Elisa; Pu?i?-Bakovi?, Maja; Keser, Toma et al. (2017) Network inference from glycoproteomics data reveals new reactions in the IgG glycosylation pathway. Nat Commun 8:1483
Sheikh, M Osman; Halmo, Stephanie M; Patel, Sneha et al. (2017) Rapid screening of sugar-nucleotide donor specificities of putative glycosyltransferases. Glycobiology 27:206-212
Liu, Lin; Prudden, Anthony R; Bosman, Gerlof P et al. (2017) Improved isolation and characterization procedure of sialylglycopeptide from egg yolk powder. Carbohydr Res 452:122-128
Halmo, Stephanie M; Singh, Danish; Patel, Sneha et al. (2017) Protein O-Linked Mannose ?-1,4-N-Acetylglucosaminyl-transferase 2 (POMGNT2) Is a Gatekeeper Enzyme for Functional Glycosylation of ?-Dystroglycan. J Biol Chem 292:2101-2109
Ding, Ning; Li, Xiuru; Chinoy, Zoeisha S et al. (2017) Synthesis of a Glycosylphosphatidylinositol Anchor Derived from Leishmania donovani That Can Be Functionalized by Cu-Catalyzed Azide-Alkyne Cycloadditions. Org Lett 19:3827-3830
Prudden, Anthony R; Liu, Lin; Capicciotti, Chantelle J et al. (2017) Synthesis of asymmetrical multiantennary human milk oligosaccharides. Proc Natl Acad Sci U S A 114:6954-6959

Showing the most recent 10 out of 29 publications