Chemokine Interaction with Heparan Sulphate

The interaction of chemokines with glycosaminoglycans (GAGs) such as heparan sulphate (HS) is thought to be responsible for selectively recruiting leukocytes only at tissue sites where they are needed. The role of GAGs is to present chemokines to their target cells at the site of inflammation by multimerising the chemokines thereby increasing their local concentration. In this project, we would like to investigate the biophysics of the chemokine/HS interaction using, as a prototype, recombinant interleukin-8 (IL-8). For this purpose we will initially prepare HS(-Iike) oligosaccharides which are uniform in length and in their chemical modification (sulphation/acetylation) pattern. Although an IL-8-specific HS domain has been identified recently, we will investigate the binding affinities and kinetics dependent upon the HS chain length and chemical modification pattern since in vivo, polymerisation of chemokines on GAGs must require differential binding sites on the polysaccharide. Methods used to investigate binding will include fluorescence and CD spectroscopy, isothermal titration calorimetry, and stopped-flow kinetics. Determining the 3-D structure of a specific IL-8/HS(-Iike) oligosaccharide complex will enable us to further explore the mechanism of the interaction as well as to plan, on the long run, chemical strategies for interference. In order to obtain a more general picture of the chemokine/GAG interaction, we will extend our studies to another member of the C-X-C family and to two members of the C-C chemokine families, namely GRO-alpha/MGSA, MCP-1, and RANTES. By applying the same binding experiments as to IL-8 using identical HS oligosaccharide ligands we expect to be able to put forward a comparative, and thus more reliable, biophysical and structural model for chemokine/HS interactions. In addition to the molecular biophysical studies, we aim at cellular interaction studies, depending upon the progress of the project, by fluorescently labelling cell surface HS and by measuring the fluorescence intensity change upon chemokine binding. This will enable us to compare molecular and cellular binding affinities and should, moreover, lead to the establishment of bioassays for screening anti-inflammatory compounds in different cell types.