Final Activity Report Summary - CONFBINDGAG (Conformation and binding studies of glycosaminoglycans: tagging based approaches)
Glycosaminoglycans (GAGs) are composed of highly sulphated 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).
For the development of these new techniques, samples of pure GAG species must be isolated and identified. In this project a new method for the separation of GAG species has been developed, known as ZIC-HILIC chromatography. Due to the low salt concentrations and volatile buffer used in this analysis, direct coupling of a separation technique to mass spectrometric analysis has been allowed; providing significant advantages for the ease and speed of analysis of the GAG mixture. It is likely that this technique will find widespread application in the field of GAG analysis. Taking pure samples of small portions of the GAG chain the Fellow has been able to develop new methods for the derivatisation of one end of the chain - initially through amide bond formation and more recently through diacyl hydrazide formation. In the first instance this allowed the selective coupling to one particular reactive group upon the terminal sugar found in GAG chains which have been cleaved with a lyase enzyme. With the extension of this methodology to the formation of hydrazides this may now be carried out on a timescale that reduces competitive degradation reactions. These techniques have allowed the Fellow to look at footprinting of specific GAGs onto the surface of proteins such as factor H module 7 (fHapp.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).
For the development of these new techniques, samples of pure GAG species must be isolated and identified. In this project a new method for the separation of GAG species has been developed, known as ZIC-HILIC chromatography. Due to the low salt concentrations and volatile buffer used in this analysis, direct coupling of a separation technique to mass spectrometric analysis has been allowed; providing significant advantages for the ease and speed of analysis of the GAG mixture. It is likely that this technique will find widespread application in the field of GAG analysis. Taking pure samples of small portions of the GAG chain the Fellow has been able to develop new methods for the derivatisation of one end of the chain - initially through amide bond formation and more recently through diacyl hydrazide formation. In the first instance this allowed the selective coupling to one particular reactive group upon the terminal sugar found in GAG chains which have been cleaved with a lyase enzyme. With the extension of this methodology to the formation of hydrazides this may now be carried out on a timescale that reduces competitive degradation reactions. These techniques have allowed the Fellow to look at footprinting of specific GAGs onto the surface of proteins such as factor H module 7 (fHapp.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).