The ligand identification and synthesis project is led by Geert-Jan Boons, an expert in synthesis of glycoconjugates and their functional impact. He is joined by Parastoo Azadi, an expert in oligosaccharide separations and identification, and by two experts in the development of mass spectrometry (MS) methods for oligosaccharide characterization. Joshua Sharp provides expertise in the forefront MS analysis of proteoglycan structures and separations to examine protein-carbohydrate interactions. Jonathan Amster brings experience with MS-based GAG identification and is responsible for introducing the fragmentation technologies that are key to structural characterization of extended GAG oligomers. The overall approaches in this Technology Research and Development (TR&D) project involve the isolation of heparin sulfate from natural or engineered sources, fragmentation and size fractionation to produce defined oligomers for use in binding studies, and structural analysis of high affinity glycan structures. Synthetic strategies are also being developed that allow the selective synthesis of HS polymers.
The project directed at 3D structural characterization of GAG-protein complexes is led by James Prestegard, an expert in the area of NMR structure determination of biomolecules including proteins and carbohydrates. He is joined by Joshua Sharp who, as mentioned above is an expert in mass spectrometry, but more importantly for this project, is his experience with high structural resolution hydroxyl radical protein footprinting, a key method for the extension of structural characterization to larger and multimeric complexes with GAG ligands. Rob Woods is also a key contributor to this project. His expertise in computational methods and molecular modeling provides a platform for interpreting NMR and MS data as well as a means of extending structural analysis to systems for which experimental data do not exist. The overall approaches in this TR&D project are focused on collecting structural data on GAG- and PG-protein complexes using NMR and hydroxyl radical protein footprinting (HRPF) as well as computational docking and simulation to integrate structural data into complete models and provide a basis for predicting biochemical properties.
The project directed at biochemical and cellular processes is led by Kelley Moremen, an expert in protein expression and glycan biosynthesis. His skills are important to the production and characterization of various recombinant protein products used by the structural and ligand identification projects. He is joined by Lianchun Wang, an expert in cellular and organismal response to GAG variations and assessment of the effects of various GAG structures at a cellular level. The overall approaches in this TR&D project are focused on recombinant, genetic and biochemical approaches to assess PG functions. Large-scale expression strategies have been developed for glycoprotein production in mammalian cells and engineering of cellular glycosylation and leveraged for structural and functional studies in the TR&D projects. Complementary efforts employing cell lines derived from mice disrupted in numerous biosynthetic loci for the elaboration of HS polymers for use in structure-activity relationship (SAR) studies on cell-based functions. Thus, all of the TR&Ds provide complementary strategies to address the unified theme of understanding the structural and functional details of proteoglycan-protein interactions.