Can GAGs be gagged?

William Booth discusses a new approach for high-throughput analysis of GAG interactions with growth factors and other bioregulatory proteins Glycosaminoglycans (GAGs) are a distinctive class of negatively-charged, linear polysaccharides composed of repeating disaccharide units. They are located mainly on cell surfaces and in the extracellular matrix (ECM), including basement membranes, where they are strategically-positioned to regulate the flow of information between cells and their local milieu. The disaccharide units of GAGs are composed of an uronic acid and an amino sugar and, with the exception of hyaluronic acid; the disaccharides are modified by sulphation.

The content and disposition of sulphate groups varies considerably amongst the members of the GAG family. Diversity of polymer sulphation dictates many of the protein binding properties of GAGs that underpin their specific biological functions. GAGs play fundamental roles in the regulation of cell growth and development, the biogenesis of the ECM and the maintenance of non-thrombogenic surfaces on vascular endothelium. The critical importance of GAGs such as heparan sulphate and the chemically-related heparin in the binding and activation of peptide growth and migration factors (eg the fibroblast growth factors, hepatocyte growth factor/scatter factor, vascular endothelial growth factor and many others) has led to increasing interest in understanding the molecular basis of protein-GAG interactions and to the search for small molecule inhibitors that may be effective “GAG-inhibitors”. Such inhibitors could have applications in suppression of cell growth in human cancer and in other diseases of aberrant cell proliferation such as retinopathy and liver fibrosis.

In this context it is also worth noting that the HS family of GAGs on endothelial surfaces of blood vessels bind in a selective manner to most members of the extensive family of chemokine migration factors (eg IL-8, MCP-1, MIP1 alpha). Chemokines are strongly implicated in the extravasation of inflammatory and immune cells and are thus critical targets in chronic inflammation, autoimmune diseases and tissue rejection.

Despite the widespread involvement of GAGs in human disease the search for inhibitors of GAG-protein interactions has been hampered by the lack of a simple high throughput screening method that is applicable to all types of GAG. The high negative charge density and hydrophilic character of GAGs prevents them from binding to conventional plastic or polystyrene surfaces of multiwell microplates.

GAGs can be chemically-modified by biotinylation and adsorbed onto streptavidin-coated plates but these procedures are time-consuming and consistently high yields of biotinylated GAG are difficult to achieve.

The 96-well Heparin/GAG binding plates overcome many of the problems normally-encountered in investigation of GAG-binding to proteins. The GAGs can be adsorbed onto the uniquely- fabricated plate surface in physiological buffers in their native states (no chemical change needed and no need to pre-coat the plate surface) with retention of protein-binding properties. Bound proteins can then be detected using specific antibodies in high-throughput formats.

In addition to their value in screening the Heparin/GAG plates can be used to investigate relative affinities of different GAGs for a particular protein and to elucidate optimum binding qualities in a GAG such as the nature of the polymer backbone and the requirement for unique sulphation patterns in favoured binding motifs. One additional intriguing possibility is that the plates may offer an attractive system for investigating receptor interactions with the growth factors bound to immobilized GAGs. Growth factors such as the FGFs mentioned above have very weak or no signalling activity unless they are bound to heparan sulphate (or heparin) – by providing a suitable surface for binding FGFs a heparin or HS coated plate may represent a realistic biochemical setting for assembly of FGF-FGF-receptor complexes. This arrangement could offer a new approach for discovery of inhibitors ligand-receptor interactions which could then be tested in bioassays of growth and migration factors that act through a so-called heparan sulphate co-receptor.

References

1. Kreuger, J. Spillmann, D., Li, J.P. Lindahl, U.  (2006) Interactions between heparan sulphate and proteins: the concept of specificity.  J. Cell Biol. 174: 323-327
2. Gallagher, J.T. (2001) Heparan sulphate: growth control with a restricted sequence menu. J. Clin. Invest. 108: 357-361
3. Capila, I and Linhardt,R.J (2002) Heparin-protein interactions, Angew Chem Int Ed Engl 41: 391–412.
4. Laremore,T.N., Zhang,F., Dordick,J.S. and Lihardt, R.J. (2009) Recent progress and applications in glycosaminoglycan and heparin research. Current Opinion in Chemical Biology. 13: 633-640

Contact e: williamb@amsbio.com t: +44-1235-828200.