Carbohydrate isoelectric focussing: A new tool for the unprecedented separation of complex glycosaminoglycan structures.

On the outside of all mammalian cells and in the spaces between them, there are a number of complex polysaccharide molecules called glycosaminoglycans (GAGs), which are known to control many important biological processes. These include cell growth and division, and are implicated in the maintenance of healthy organisms and several disease processes including new blood vessel growth in tumours, Alzheimer's, Parkinson's and prion diseases among others. Gaining a better understanding of the structure and activities of these molecules is key to understanding and intervening more effectively in these processes. GAGs bind to many different categories of proteins, including those responsible for cell communications. They have subtly different sequences when expressed by different cell types and there is currently much debate as to how sequence determines activity. Unfortunately, it is not possible to isolate sufficient quantities of many GAGs (particularly the most complex member; heparan sulfate - HS) from individual tissues to undertake useful experiments. Preparations of GAGs from natural sources are inevitably mixtures. Furthermore, having isolated crude GAG material, it is often impossible to separate the individual components (esp. HS) using current techniques. This now presents a serious obstacle to research in this area. The separation techniques which are used at present rely on two physical characteristics of GAGs; the first is their volume (approximating to their length) is expolited by gel permeation chromatography (GPC), but is relatively crude. The second property is their negative charge, which can vary. This is the basis for separation of a crude GAG mixture or of its fragments) into pools of molecules with similar structures by either high performance strong anion exchange chromatography (HPAEC) or conventional gel electrophoresis (PAGE). However, many GAGs, or their fragments, have both similar size and charge and these techniques therefore represent the current limit of separation. Here, a method is proposed, which aims to exploit an additional feature that has, until now, been overlooked. GAGs contain other chemical groups (amines), most of which are covered by one of two other (N-sulfate or N-acetyl) groups. Our hypothesis is that, if these covering groups were removed, it would confer the molecules with both positive (from the amine) and negative charges (from acid and O-sulfate). This would enable a powerful technique called isoelectric focusing (IEF) to be used. IEF is widely used in the protein field , where it has revolutionised separation. The dual positive and negative charges allow the molecules to be focussed on an electrophoresis gel in which a pH gradient has been established; each subtly different sugar arriving at a different point on the gel. Following (routine) recovery of GAG from the gel and replacement of the covering groups (there are two types of groups covering the amine-both be selectively removed or replaced by well-established methods). Hence, purified samples of the GAGs, or their fragments would be produced. The properties of the GAGs are distinct from those of most proteins and commercially available IEF gels are not suitable. Development of suitable gels (using appropriate gel materials and pH gradients) and establishment of optimal running conditions will also be undertaken. This project will aim to establish that IEF is possible on the GAGs providing samples of unprecedented purity, assisting both research and the further development of pharmaceutical and biotechnological agents, ultimately allowing biological activity to be better understood and bringing effective intervention in a host of medical situations nearer.

The ability to separate fragments (oligosaccharides) derived from the biologically important glycosaminoglycan (GAG) anionic polysaccharide family, which includes heparan sulfate (HS), heparin, chondroitin and dermatan sulfates (CS-A, CS-C and CS-B) and others, currently limits further progress in many areas; structure-function studies, development of surface-based microarrays or nano-scale probes and development of pharmaceutical agents against, for example, Alzheimer's-related BACE-1, FGF's involved in cancer-related angiogenesis or as disruptors of rosetting in Plasmodium falciparum cerebral malaria. Until now, separation GAG fragments has relied on a few techniques; GPC, which separates on volume (equating approximately to size), anion exchange chromatography (HPAEC), which distinguishes principally on the basis of different charges and gel electrophoresis, which relies on a combination of both. The problem with GAG oligosaccharides, which is especially clearly seen in the more heterogeneous GAG member, HS, is that many oligosaccharides can have essentially the same size and charge, while their sequences are distinct. Moreover, biological activity is related to subtle sequence-derived properties such as conformation, flexibility and cation binding characteristics not simply to charge density. This proposal will exploit the presence of amino functions of GAGs for the first time as an additional handle on their separation. Selective de N-sulfation or de-N-acetylation will produce zwitterionic oligosaccharide pools (conferring positively charged amine groups), which will then be focussed on bespoke elecrophoresis gels (with appropriate pH gradients) by isoelectric focussing (IEF). Recovery of the purified oligosaccharides from the gel followed by either re-N-sulfation or re-N-acetylation will provide saccharides of unprecedented purity. The principal aim of this project is to demonstrate the feasilbility of this approach.