Immobilized GAG derived ampholytes for enhanced low pH isoelectric focusing

On the outside of all mammalian cells and in the spaces between them, complex polysaccharide molecules called glycosaminoglycans (GAGs) 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. Obtaining a better insight into the structure and biological 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 always possible to isolate sufficient quantities of many GAGs (particularly the most complex member, heparan sulfate - HS) from individual tissues for useful experimentation. Preparations of GAGs from natural sources are inevitably mixtures. Furthermore, having isolated crude GAG material, it was until recently, difficult to separate the individual components (esp. HS) using previously available techniques. This now presents a serious obstacle to research in this area.

Historically, GAG separation techniques relied on two physical characteristics of GAGs; the first is their hydrodynamic volume (approximating to length) is exploited by gel filtration chromatography (GFC), although this is relatively crude. The second property is their negative charge, which varies. This strategy separates crude GAG mixtures, or fragments thereof, into pools with similar structures. However, many GAGs, or their fragments, have both similar size and charge and these techniques therefore represented the previous limit of separation.

Recently, the applicant and collaborator demonstrated successfully a method that exploited 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. Uncovering these groups confers the molecules with both positive (from the resultant amine) and negative charges (from carboxylic acid and O-sulfate groups). This has enabled a powerful technique called isoelectric focusing (IEF) to be used for their separation (Holman & Skidmore et al. (2010) 2:1550).

The dual positive and negative charges (ampholytes) 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 can be selectively removed or replaced by well-established methods). Hence, purified samples of the GAGs, or their fragments many now be produced. IEF is widely used in the protein field, where it has revolutionised separations although this has mainly been due to the development of commercially available immobilised pH gradient gels which have the added benefit of a stable, reproducible pH gradient.

The properties of the GAGs are distinct from those of most proteins and commercially available IEF gels are unsuitable. The development of suitable immobilised pH gradient (IPG) gels and the establishment of optimal run conditions for the isoelectric separation of GAGs is the main aim of this proposal. The potential to transfer GAG based IEF methodologies to current protein based IEF systems will also be investigated, which will undoubtedly facilitate widespread use. This will enable the high resolution IEF of GAGs from tissue and other biological samples. This will ultimately allow biological activities to be better understood and bring 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 HS, heparin, CS, DS and others, has historically limited progress in many areas; structure-function studies, development of surface-based microarrays or nano-scale probes and development of pharmaceuticals for Alzheimer's-related BACE-1, FGF's involved in cancer-related angiogenesis or as disruptors of rosetting in P. falciparum cerebral malaria.

The separation of GAG saccharides has until recently depended on only a few techniques; GFC separation by hydrodynamic volume (size), high performance anion exchange chromatography (HPAEC) distinguishes on the basis of charge and PAGE, which separates based on both. The problem with GAGs, especially evident in the more heterogeneous GAG member, HS, is that many oligosaccharides can have the same size and charge, with distinct sequences. Moreover, biological activity is related to subtle sequence-derived properties e.g. conformation, flexibility and cation binding characteristics, not simply to charge density.

The applicant has exploited the amino functions of GAGs as a new separation mode. Selective de-N-sulfation/de-N-acetylation yields ampholytic oligosaccharide pools (with +ve amino groups), which can be focused on bespoke gels (with appropriate pH gradients) by IEF. Physicochemical properties of the GAG are distinct from proteins and commercial IEF gels are unsuitable. Current GAG IEF gels rely on natural pH gradients, which have drawbacks to optimal resolution. Protein IEF has overcome this by using immobilised pH gradients although these gel are of an inappropriate pH range for GAG IEF. This proposal seeks to ratify this and apply the same strategy, albeit with different immobilised molecules to GAG IEF.

This will revolutionise the analytical study of GAGs in a manner akin to IPG IEF of proteins in the parallel field of proteomics.

The biologically and medically important family of carbohydrates, the glycosaminoglycans (GAGs), are present on the cell surface of almost all mammalian cells and are implicated in many crucial biological mechanisms such as cell growth, division, homeostasis and pathogen invasion. However, analysis of their structure:function relationships is compounded by the difficulty in obtaining sufficient purified material from biological samples. Historically, the separation of GAG saccharides has depended on only a few techniques; GFC separation by hydrodynamic volume (correlates approximately with size), high performance anion exchange chromatography (HPAEC) distinguishes on the basis of charge and PAGE, which separates based on both. This project will build on earlier work demonstrating proof-of principle for the IEF of GAGs, enabling immobilised pH gradient methodologies to be utilised for the first time and hence represent a new tool for the glycomics arsenal. This will revolutionise the analytical study of GAGs in a manner akin to proteins in the parallel field of proteomics.

The successful development of low pH immobilised pH gradients for GAG IEF will permit rapid progress to be made on investigations into the structure:function relationships of GAG saccharides and will ultimately contribute significantly to our understanding of GAG polysaccharides as major components of the glycome. This is a crucial facet of post-genome science, and will open up hitherto inaccessible domains within the field of glycomics.