Many of the functions of Glycosaminoglycans (GAGs) are mediated through their interactions with proteins; which occur via contacts between the negatively charged groups of GAG oligosaccharides and positively charged amino acid side chains. As a consequence, oligosaccharides in protein-GAG complexes do not occupy hydrophobic pockets, but sit on the protein surface with only a few intermolecular contacts, making solution phase structure determination of these complexes very challenging. Despite a wealth of chemical and genetic evidence to suggest that control of the fine structure of GAGs including their detailed sulfation patterns is crucial for their function in vivo, there have been comparatively few studies of these interactions at the molecular level due to a lack of enabling chemical tools. The project aims to provide enabling synthetic methodology which will ultimately allow the application of a range of modern biophysical techniques (NMR, FRET, EPR etc.) to the study of complex protein-GAG interactions which are currently inaccessible using existing methodology. To this end, the following objectives have been identified:
Objective 1: Development of a robust methodology for the functionalisation of GAGs at the non-reducing end.
The installation of two labels into carbohydrates (e.g. as required for TR-FRET, or NMR footprinting studies) is more difficult than with proteins and nucleic acids because of a lack of unique functionality. Whilst it has been shown to be possible to introduce a fluorescent, or spin label to the reducing end of GAGs using standard techniques for oligosaccharides, there are no methods currently available for the selective dual labelling of glycosaminoglycans. We intend to utilise unique characteristics of the non-reducing end sugar produced by lyase cleavage of GAGs for installation of the second label.
Objective 2: Review of known methodologies for end labelling of GAG oligosaccharides.
A detailed assessment of methodology for the selective introduction of functional labels at either the reducing end or non-reducing end of GAG oligosaccharides, and crucially how these methods are most appropriately combined with the different procedures for GAG oligosaccharide sample preparation will be provided.
Objective 3: Dual functionalisation of heparin-derived, fully-sulfated di- and tetra-saccharides.
Methodology for the introduction of a matrix of fluorescent elements will be developed, allowing us to select the optimum pairs of fluorophores for different end-to-end distances.
Some very important processes in the human body, including blood clotting, and the body’s defence against infection, are controlled by how cells respond to signals in the gel-like material that surrounds them, or how they recognise other cells that are their neighbours. These processes can be controlled by the interaction of a particular type of sugar known as glycosaminoglycans (GAGs) with other molecules on the surface of the cell (such as proteins). So cells can recognise each other by the sugar part of one cell sticking to a protein on the surface of another cell.
GAGs are actually polymers made up of different sorts of sugars which are linked together, somewhat like a string of beads on a chain. Each “bead” has a different role to play in the overall shape of the GAG polymer and the proteins on the cell surface will stick more, or less, strongly to any particular sequence of individual beads. It is thought that GAG polymers might have many different shapes depending on exactly which sugars are linked together, e.g. a helix - as in DNA, or u-shaped - as in a horseshoe. But at the moment there is no way of determining what the shape of each complex polymer actually is, and it is not possible to predict accurately how the GAG polymer and the protein stick to each other. This kind of prediction will help in the design of new drugs.
In this project we will develop chemical methods to attach a specific label at either end of a short piece of the GAG sugar polymer. Then we will be able to use a range of new biophysical techniques to determine how far apart the ends of the GAG polymer are and if they are fixed relative to each other; or to explain how and why the protein and the GAG polymer stick to each other.
Glycosaminoglycans (GAGs) are composed of highly sulfated sugars joined together to form long polymeric chains. They are found on the surfaces of all cells and in the spaces between them. The most widely known member of this family is heparin which has an important medical application as an anticoagulant. GAGs interact with proteins displayed on the surface of cells to control signalling events. However, study of these interactions is very challenging as the GAGs sit on the surface of proteins and not in deep binding pockets, and are attracted through a series of weak interactions. In order to study protein-GAG complexes and understand more about their function in both normal and diseased states the development of new tools for the functionalisation of the ends of small portions of the GAG chain is required. This will then allow the determination of the distance between the ends of the GAG chain by fluorescence (yielding vital structural information about the GAG), and also "footprinting" of the GAG upon the surface of a protein (yielding vital information about the GAG binding site on the protein).
A number of approaches to the selective functionalisation of the non-reducing end of enzyme-cleaved glycosaminoglycan-derived oligosaccharides were explored. Of these, routes based upon oxy-mercuration, bromonium or iodonium formation, Michael addition reactions or palladium-catalysed Heck coupling reactions were found to be unsuccessful. However, efficient functionalisation of the non-reducing end of uronic acid derivatives and glycosaminoglycan-derived disaccharides was achieved using peptide coupling, mediated by the water soluble agent DMT-MM. Subsequently it was found that selective coupling of the hydrazide derivatives of a range of labels (free radical containing for spin labelling and NMR footprinting, fluorescent labels for FRET studies etc.) could be carried out at the non-reducing end of GAGs to yield diacyl hydrazides. The extension of this coupling methodology to the formation of hydrazides means that this labelling may now be carried out on a timescale that reduces competitive degradation reactions. These techniques have allowed us to look at footprinting of specific GAGs onto the surface of proteins such as factor H module 7 (fH~7) which is thought to play an important role in the development of age-related macular degeneration (one of the leading causes of blindness in the Western world).
|Effective start/end date||1/12/05 → 30/04/09|