Resource for Integrated Glycotechnology

Resource for Integrated Glycotechnology

Research Emphasis

The central goal of the Resource for Integrated Glycotechnology is to increase understanding of the molecular basis of the involvement of carbohydrates in protein-carbohydrate interactions and to develop more powerful technologies necessary to achieve this goal. Complex carbohydrates play an important role in many biomedical processes, including inflammatory response, hormone action, malignancy, viral and bacterial infections, and cell differentiation. As new technologies are developed, application of these processes is pursued through collaborative and service projects.

Current Research

Characterization of protein-carbohydrate interactions is a major challenge that can only be met through integration and application of diverse technologic methods. At this resource, technologies include those for the production of characterizable amounts of the proteins responsible for the synthesis and recognition of cellular carbohydrates, synthesis of carbohydrates with sufficient diversity and purity to probe the molecular basis of affinity, structural characterization of the interactions, and integration of data under a unifying computational platform. In terms of protein production, emphasis is currently placed on production of the glycosyltransferases required for eukaryotic oligosaccharide synthesis. Strategies for ¹⁵N, ¹³C, and SeMet labeling needed in nuclear magnetic resonance (NMR) and X-ray-based structural methodology are developed. In terms of synthetic methodology, emphasis is currently placed on the systematic production of carbohydrates and the development of micro-array technology for ligand identification. Initial targets include both ligands for glycosyltransferases and glycosaminoglycans of the heparan sulfate family. In terms of structural methodology, both mass spectrometry (MS) and NMR methods are developed. MS methodology focuses on the use of deuterium exchange MS to identify the regions on proteins that are protected by the binding of the carbohydrate ligands. NMR methodology focuses on the determination of bound ligand geometry and new methods to structurally characterize sparsely labeled proteins from eukaryotic expression systems. In terms of computational methodology, emphasis is on development of a framework for integrating structural and functional information. It includes continued refinement and expansion of the GLYCAM force field parameters for oligosaccharides and glycoproteins. The effect of technology development projects is enhanced through effective collaboration, service, training, and dissemination activities.

Resource Capabilities

Methods

The following methods are integrated in pursuit of the functional and structural characterization of carbohydrate-protein interactions: protein expression in eukaryotic hosts, synthesis of carbohydrate ligands, MS of proteins and carbohydrates, NMR of proteins and carbohydrates, and molecular dynamics simulations of protein-carbohydrate interactions.

Instruments

Fourier transform and Quadrupole time-of-flight mass spectrometers, high-field NMR (800 and 900 MHz), linux cluster computational servers.

Software

GLYCAM software for carbohydrate simulations. REDCAT software for NMR-residual dipolar cooling analysis.

Special Features

Tools for the preparation of input files for carbohydrate and glycoprotein computer simulations are Web accessible.

Available Resources

Routine analytic service for the characterization of carbohydrates and glycoproteins is available by contacting the Resource Technical Director. Additional NMR, MS, and custom synthesis resources are available through both service and collaborative channels.

Training Opportunities and Workshops

The resource offers training courses for scientists who wish to learn analytical methods in complex carbohydrate science. Two one-week laboratory courses, "Analytical Techniques for Carbohydrates Structure Determination and Separation" and "Characterization of Glycoconjugate Oligosaccharides," are offered annually. These are often supplemented with shorter workshops specializing in NMR and MS of carbohydrates.

Publications

1. Gonzalez-Outeirino, J., Kirschner, K. N., Thobhani, S., and Woods, R.W., Reconciling solvent effects on rotamer populations in carbohydrates: A joint MD and NMR analysis. Canadian Journal of Chemistry (in press).

2. Karaveg, K., Siriwardena, A., Tempel, W., et al., Mechanism of class 1 (glycosylhydrolase family 47) a-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control. Journal of Biological Chemistry 280:16197–16207, 2005.

3. Kim, J.-H., Hai, Y., Park, J., and Boons, G. J., A general strategy for stereoselective glycosylations. Journal of the American Chemical Society 127:12090–12097, 2005.

4. Feng, L., Orlando, R., and Prestegard, J. H., Mass spectrometry assisted assignment of NMR resonances in 15N labeled proteins. Journal of the American Chemical Society 126:14377–14379, 2004.