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Category: Highlight

Heparin Sulfate Arrays to Probe Binding Protein Specificity

jennbryant on May 12, 2014

Although hundreds of heparan sulfate binding proteins have been identified, and implicated in a myriad of physiological and pathological processes, very little information is known about ligand requirements for binding and mediating biological activities by these proteins. This difficulty results from a lack of technology for establishing structure-activity-relationships, which in turn is due to the structural complexity of natural HS and difficulties of preparing well-defined HS-oligosaccharides. To address this deficiency, we have developed a modular approach for the parallel combinatorial synthesis of HS oligosaccharides that utilizes a relatively small number of selectively protected disaccharide building blocks that can easily be converted into glycosyl donors and acceptors. These compounds can then be employed for the assembly of a wide range of HS-oligosaccharides.
The synthetic HS oligosaccharides are equipped with aminopropyl spacer, which made it possible to attach the compounds to microarray plates having reactive carboxylic acid moieties. The resulting HS oligosaccharide array is being further developed for high throughput screening to establish ligands for HS-binding proteins. In collaboration with several research groups, the synthetic HS-oligosaccharides are also employed as standards to develop mass spectrometry (MS)-based sequencing methods.

Robo1 Binds to Heparin at Two Distinct Binding Sites

on May 12, 2014

Sharp-HRPF
A team of Research Resource investigators, including members of the labs of Joshua Sharp and Kelley Moremen, have worked together to help determine the role of glycosaminoglycans (GAGs) in nervous system development. GAGs bridge a complex of two proteins, Robo1 and Slit2, to help guide the development of axons in the nervous system. Using a combination of technologies, including MS and dynamic light scattering, the team was able to discover a new, low-affinity binding site for full-length heparin that appears to be important in signaling. The discovery of this second binding site allows us to provide a structural model for GAG-mediated signaling. The model shows a high-affinity shared heparin binding site between Robo1 and Slit2 responsible for stabilizing the complex, and a second low-affinity binding site that is important in transducing a signal through the rest of the Robo1 receptor.

TSG-6 Link Module Displays Multiple modes of Glycosaminoglycan Binding

jennbryant on May 7, 2014

Link modules found in a number of cell surface proteins play important roles in stabilizing the extracellular matrix (ECM) by interacting with glycosaminoglycans (GAGs). However the specificity and geometry of these interactions is poorly defined. Driven by a collaborative project with the Tony Day lab of the University of Manchester, Younghee Park in the Prestegard group has applied forefront NMR methods to characterize binding modes of the link module of TSG-6, the secreted protein product of tumor necrosis factor-stimulated gene-6. A combination of chemical shift perturbation and paramagnetic relaxation enhancement, using defined and chemically modified oligomers of chondroitin sulfate, has identified two different binding sites (one strong and one weak). The second site appears to be formed after GAG induced dimerization of the Link module. The multiplicity of binding sites clearly can promote crosslinking and stabilization of the ECM.

Red and black ellipses on overlaid 1H-15N HSQC spectra, collected with increasing amounts of the hexameric chondroitin sulfate oligomer, show chemical shift patterns characteristic of fast and slow exchange. Fast and slow exchange residues are highlighted in red and blue respectively on the structure.

Glycosaminoglycan Builder

on May 6, 2014

glycosaminoglycan_builder
Glycosaminoglycan builder provides a point-and-click interface to build custom structures for all classes of glycosaminoglycans (GAGs). The web-tool is a simple interface where the user first chooses the desired class of GAG molecule and then enters the primary sequence by clicking through the set of constituent monosaccharides. The appropriate sulfation patterns commonly observed in each class of GAGs are provided as options. The tool generates 3D structures using the primary sequences and provides molecular structure files that can be utilized for visualization or as input for automated docking and molecular dynamic simulation programs.
This work has been done by the members of the Woods lab (Arunima Singh, Xingran Xue, Lachele B. Foley, Robert J. Woods).

Sequencing of Full Length Glycans from the Proteoglycan Bikunin Using Tandem Mass Spectrometry

on May 6, 2014

ImageForWebSite_Amster
A collaboration between UGA scientist Jonathan Amster, Robert Linhardt, at Rensselaer Polytechnic Institute, and Toshihiko Toida, at Chiba University, has led to the first ever sequencing of full length glycans from a naturally occurring proteoglycan. Although much of the structure of the protein portion of bikunin was well known, as was its homogenous N-linked glycan, the O-linked glycan was known to be a chondroitin sulfate that was heterogeneous in length and composition. The CS was released from bikunin, purified into less complex fractions by capillary gel electrophoresis, and the fractions analyzed by Fourier transform mass spectrometry. Composition analysis suggested a small number of chain lengths and degrees of sulfation (less than 20 compositions). Tandem mass spectrometry yielded the surprising result that each composition was a single sequence with a well-defined pattern of sulfation. Even more surprising was the discovery of a conserved pattern of sulfation among all of the components that were analyzed. This provides the first evidence that biosynthesis of glycosaminoglycans is not random, despite its non-template nature.

Slit Robo Interactions

on May 6, 2014

slit-robo-interactions
Co-receptor function of heparan sulfate in Slit3-Robo4 signaling: Heparan sulfate, carried by the heparan sulfate proteoglycans, such as syndecan, binds Slit3 on wildtype endothelial cell surface to form the heparan sulfate-Slit3-Robo4 ternary complexes facilitating Slit3-Robo4 signaling. Genetic ablation of N-deacetylase-N-sulfotransferase-1 (Ndst1), an dual function enzyme that initiates modification reactions during heparan sulfate biosynthesis, reduces the sulfation modifications, leading to Ndst1-/- endothelial heparan sulfate fails to bind Slit3 and disrupt heparan sulfate-Slit3-Robo4 complex formation and the subsequent angiogenic signaling.
Zhang B, Xiao WY, Qiu H, Zhang FM, Moniz HA Condac E, Gutierrez-Sanchez G, Heiss C, Clugston RD, Azadi P, Greer JJ, Bergmann C, Moremen KW, Li D, Linhardt RJ, Esko JD, Wang L (2014). Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia. J Clin Invest. 124(1):209-221. [PMC3871243]

Sequencing of Complex Mixtures of Heparan Sulfate Tetrasaccharides by Chemical Derivatization and LC-MS/MS

on May 1, 2014

GAG-sequencing
Through a collaboration between synthetic chemists investigating chemical synthesis of defined heparan sulfate oligosaccharides (Chengli Zong, Andre Venot, and Geert-Jan Boons), analytical chemists developing a derivatization, separation, and MS/MS methods for separating and sequencing complex mixtures of heparan sulfate oligosaccharides (Rongrong Huang, Dandan Zhou, and Joshua Sharp), and a bioinformaticist developing software for high-throughput analysis of heparan sulfate sequencing results (Yulun Chiu), we have developed and demonstrated a semi-automated method capable of separating and sequencing complex mixtures of heparin and heparan sulfate, including structures that are very closely related to one another. Only through the availability of both well-defined synthetic standards and advanced analytical technologies could we develop a tool that is demonstrated to be robust and reliable. Computational tools have been developed to perform automated data analysis for high-throughput heparin and heparan sulfate sequencing. A version of this software is scheduled for public release in 2014, with periodic updates made publicly available as we develop them.

Production of Recombinant Glycoproteins in Mammalian Cells for Structural and Biochemical Studies

on May 1, 2014

A platform for recombinant expression of mammalian glycoproteins has been developed in the Moremen lab for biochemical and structural studies on glycosylation enzymes and glycan binding proteins. Successful production of mammalian glycosylation enzymes in the Moremen lab led to the expanded development of a large-scale project for the expression of all mammalian glycosylation enzymes through the Repository of Glycan-related Expression Constructs as a supplement to the Resource for Integrated Glycotechnology in the last funding cycle. This project is a collaboration between the Moremen lab, and the laboratories of Joshua LaBaer (Arizona State University) and Don Jarvis (University of Wyoming). Constructs encoding the soluble catalytic domains of most human glycosyltransferases, glycoside hydrolases, sulfotransferases, and other glycan modifying enzymes have been generated for expression in both insect cells and mammalian cells. Representative enzymes are being generated in multi-milligram quantities for enzymatic characterization, structural studies, chemo-enzymatic glycan synthesis using protein tagging strategies that facilitate stable expression, purification, quantitation, and subsequent tag removal for structural studies. The technology developed for mammalian expression has also been adapted for metabolic labeling of glycoproteins with NMR-active isotopes and insertion of paramagnetic tags to facilitate NMR structural studies. This platform is also being applied for the recombinant expression of numerous glycan binding studies in the Resource Driving Biomedical Projects and Collaborations.
mammal-recomb-glycoprot-prod

Oligomeric Structure of the Chemokine CCL5/RANTES from NMR, MS, and SAXS Data

on May 1, 2014

A team of RR investigators, Xu Wang, Caroline Watson, Joshua S. Sharp, James H. Prestegard, has joined with Tracy M. Handel from UCSD, to pursue a driving biomedical project directed at understanding how glycosaminoglycans (GAGs) help regulate immune cell migration and chemokine signaling. The chemokine, CCL5 or RANTES, aids recruitment and induces endothelial transmigration of T-cells. The receptor for CCL5 also functions as a co-receptor for the aids virus adding to the interest in this project. The team employed a combination of NMR, SAXS and MS techniques to provide a structural model for CCL5 oligomerization. The model shows an extended GAG binding site and suggests a means by which simultaneous GAG binding and receptor interaction may aid cell migration.

Oligomeric Structure of the Chemokine CCL5/RANTES from NMR, MS, and SAXS Data. Xu Wang, Caroline Watson, Joshua S. Sharp, Tracy M. Handel, and James H. Prestegard.Structure (2011) 19:1138-1148DOI: 10.1038/nsmb.1853
Oligomeric Structure of the Chemokine CCL5/RANTES from NMR, MS, and SAXS Data.
Xu Wang, Caroline Watson, Joshua S. Sharp, Tracy M. Handel, and James H. Prestegard.
Structure (2011) 19:1138-1148
DOI: 10.1038/nsmb.1853

Dissemination, Contributions, & Achievements

on May 1, 2014

Resource investigators actively disseminate our technologies through presentations, publication in journals and news articles.

Representation at International Meetings

Numerous presentations at International Symposium on Glycoconjugates, Proteoglycan and Glycobiology Gordon conferences and the Society for Glycobiology meetings. More than 50 presentations annually credit resource technologies, and investigators have organized, Chaired, or Co-chaired of over 10 glycoscience related national and international meetings each year.

National Academy of Sciences Report

In 2012, Drs. Darvill and Boons served on the National Academy of Sciences committee and authored the report “Transforming Glycoscience, a Roadmap for the Future” that emphasized generation and dissemination of better tools for glycoscience and hands-on training and education.

Georgia Glycoscience Symposium

The annual international symposium hosted by the CCRC presents cutting edge carbohydrate research, including the 9thth Georgia Glycoscience Symposium (September 2015, “Proteoglycans and their Role in Heath and Disease”) focusing on tools developed by the Resource.

Awards

Recognition of research by Resource investigators include: Boons: 2012 Creative Research Inventor Award, University of Georgia Research Foundation and in 2014 was selected Roy L. Whistler International Award in Carbohydrate Chemistry by the International Carbohydrate Organization (ICO), Woods: 2014 UGA Faculty Entrepreneur of the Year, Amster: Elected a Fellow of AAAS in 2010, Moremen: 2014 Distinguished Research Professor, University of Georgia Research Foundation.

Workshops

Four annual hands-on trainings at CCRC (See Training section)
Dr. Amster co-taught a two-day short course, Mass Spectrometry of Glycans and Glycoproteins, at the ASMS Conference on Mass Spectrometry and Allied Topics, in 2012, 2013, 2014. Dr. Azadi is a co-organizer of the New and Emerging Technologies Workshop at the “Well Characterized Biotechnology Pharmaceutical in 2014.”

Consortium for Functional Glycomics (CFG)

Boons member of the steering committee of the CFG and Prestegard and Woods are CFG subgroup leaders. CFG and Resource investigators partnered in 2012 for the Georgia Glycoscience Symposium and CFG Workshop (“Paradigms for Glycan Action in Development and Disease”) Prestegard organized the CFG Participating Investigator Meeting and Symposium in 2012, co-Investigator on the CFG Workshop Grant for “Development and Application of Transformative Technologies in Glycobiology”.

Annual Society for Glycobiology

Moremen has been Secretary of the Society from 2005-present.
Azadi has been invited speaker at the satellite meeting in 2008-2012, in 2013 organized a booth, which displayed brochures and protocols on CCRC’s service and training.

Protocols and Reagents

Protocols distributed for isolation, purification and characterization of polysaccharides and glycoproteins Provide reagents (e.g. purified enzymes and synthesized oligosaccharides) generated through technology projects. Constructs produced as a supplement to the Resource as a “Repository for Glycan Related Enzymes” made available through the PSI Materials Repository at Arizona State University (dnasu.org). Proteoglycan resources include heparan sulfate oligomer arrays, mutant mouse endothelial cell lines defective in HS biosynthesis and modification, and others.

Publications and News Articles

In the last funding cycle our investigators published >100 research articles, an additional 12 publications are in press, and an additional 9 publications from Analytical Services have cited the grant number. Some of our particularly noted publications include:

  • Wang, Z., Chinoy, Z., Ambre, S., Peng, W., McBride, R., de Vries R.P., Glushka, J., Paulson, J.C., & Boons, G.J. (2013). A general strategy for the chemoenzymatic synthesis of asymmetrically branched N-glycans. Science 341, 379-383
    Highlighted in:
    • Science: Kiessling, L.L. & Kraft, M.B. (2013). A path to complex carbohydrates. Science 341(6144), 357-358
    • C&E News: Arnaud, C.H. (2013). Branching out in different ways. Carbohydrate chemistry: New synthetic strategy leads to asymmetrically branched N-glycans. C&E News 91(30), 9
    • Nature News: Johnston, R. (2013). Asymmetrical glycans synthesized in lab. Nature News (25 July 2013)
  • Ly, M., Leach, F.E. 3rd, Laremore, T.N., Toida, T., Amster, I.J., & Linhardt, R. (2011). The proteoglycan bikunin has a defined sequence. Nature Chemical Biology 7, 827-833
    Highlighted in:
    • News and Views article: Jones, C.J. and C.K. Larive. (2011). Carbohydrates: Cracking the glycan sequence code. Nature Chemical Biology 7, 758-759
    • C&E News: Highlighted as a Technology Concentrate. December 8, 2011
  • Barb, A.W. & Prestegard, J.H. (2011). NMR analysis demonstrates immunoglobulin G N-glycans are accessible and dynamic. Nature Chemical Biology 7, 147-153
    Highlighted in:
    • News and Views article in Nature Chemical Biology: Meier, S. & Duus, J. (2011). Carbohydrate dynamics: Antibody glycans wiggle and jiggle. Nature Chemical Biology 7, 131-132 (doi:10.1038/nchembio.526)
  • Chen, H., Ji, F., Olman, V., Mobley, C.K., Liu, Y., Zhou, Y., Bushweller, J.H., Prestegard, J.H., & Xu, Y. (2011). Optimal mutation sites for PRE data collection and membrane protein structure prediction. Structure 19, 484-495.
    Highlighted in:
    • Preview article in Structure: Ganguly, S., Weiner, B.E., & Meiler, J. (2011). Membrane protein structure determination using paramagnetic tags. Structure 19, 441-443 (doi:10.1016/j.str.2011.03.008)

Cover Illustrations

ligand-energies-cover
Journal of Computational Chemistry Cover-Mockup selected for publication in 2014
Nivedha, et al. (2014). The importance of ligand conformational energies in carbohydrate docking: Sorting the wheat from the chaff. J. Comput. Chem., in press.
glycobiology-cover
Glycobiology Cover Illustration
Illustrates a snapshot of ganglioside GM3 (Neu5Acα3Galβ4GlcCer) in a lipid bilayer (gray) from an all-atom explicit-solvent molecular dynamics simulation of the ganglioside using the GLYCAM force field from Glycobiology Vol. 19, 2010.
chembiochem-cover
Chembiochem Review Frontispiece in 2013
Live, et al. (2013). Dissecting the molecular basis of the role of the O-mannosylation pathway in disease: α-Dystroglycan and forms of muscular dystrophy. Chembiochem 14, 2392-402.

Faculty News Highlights

Parastoo Azadi
Highlighted in C&E News, Nov 15th 2010, “Conventional methods are inadequate when it comes to quantitative and structural analysis”.
David Live
Highlighted in Science, Jan 7th 2011, Life Sciences Technologies feature, Glycoproteomics: The sweet smell of we’re getting there”.
Joshua Sharp
Highlighted in Atlanta Business Chronicle, Sharp’s article Nov 16th 2012: “HIV research looks to copy natural immunity”.
Geert-Jan Boons
Highlighted in Athens Patch, June 11th 2013: “UGA research provides insight into a debilitating brain disease”, Coverage in Athens Banner-Herald and UGA Research Magazine, Spring/Summer 2013: “A sweet spot for sleuthing sugars”; UGA Research, June 2013: “Fighting cancer with carbohydrates”.