The Kinetic Hyperspace of Chemokines

Chemokine signalling in inflammation is mainly determined by the interaction with heparan sulfate (HS), which mediates chemokine oligomerisation as well as the activation of specific G-protein coupled receptors. Despite of intensive investigations at equilibrium, the crucial mechanisms generating the specific states dictating chemokine action, still remain unclear. For this reason, we hypothesise that the clue towards chemokine function lies in the appearance of kinetic intermediates (e.g. folding intermediates) in pre-steady state processes, which have so far been neglected in chemokine research. To bypass the limitations of equilibrium, we propose a novel kinetic approach for the underlying project, which envisages the characterisation of the multifunctional interactions of chemokines by investigating rate constants (ks) of the various molecular encounters. These are i) chemokine unfol- >chemokine fol (folding), ii) chemokine+ chemokine (oligomerisation), iii) chemokine+HS (HS- binding) iiii) chemokine+R (receptor- binding). For detection various biophysical methods (including stopped flow, fluorescence anisotropy and light scattering) will be used. The intramolecular or bi-molecular interactions, however, are biased in vivo by cross-molecular reactions (e.g. [chemokine+chemokine]+HS, [chemokineunfol- >chemokine fol ]+R, etc.) due to the simultaneous occurrence of (partly)unfolded chemokines and their interacting partners within the cell and on the cell surfaces. Our preliminary in-vitro folding studies give the indication for the chemokines existing in different folds at the same time. The ultimate purpose of this project is to unravel the kinetic relationships for all reactions (rate constant matrix) occurring during the chemokines lifetime (excl. degradation) in order to identify rate limiting steps (referring to kinetic intermediates) which may prove to be suitable for therapeutic targeting in a better way than when interactions are monitored in the equilibrium. To endorse our hypothesis of "intermediates determining chemokine function", we would like to "freeze" folding intermediates and HS-bound states by chemical cross-linking to further biophysically characterise their fold at equilibrium (by applying techniques such as circular dichroism spectroscopy and fluorescence spectroscopy). In addition, their functionality with respect to HS-/ receptor binding and oligomerisation has to be verified performing fluorescence isothermal titration experiments, isothermal titration calorimetry, in situ cell assays (Boyden chamber), etc.

Understanding the biological function of chemokines is still of utmost importance since the mebers of these protein class (currently more than 40) are involved in a plethora of diseases such as acute and chronic inflammation, cancer, infection, as well as cardiovascular and neurodegenerative diseases. In addition, despite their discovery in the late 70-ies of the last century, only two drugs targeting chemokines or their receptors have reached the market. It was therefore the `mission` of this project to shed light on a somehow under-explored aspect of chemokines, namely their interaction kinetics with their natural ligands, i.e. their receptors and co-receptors. The latter molecules are represented by a class of complex carbohydrates, the so-called glycosaminoglycans (GAGs), the influence of which on the pharmacology of chemokines has so far been underestimated. In order to investigate the kinetic interaction hyperspace of chemokines, we have established protocols to immobilise GAG ligands on surface plasmon resonance (SPR) chips which were used to study on and off rates as well as equilibrium binding of a number of chemokines and chemokine mutants. By this means we discovered that the studied proteins differed significantly in their kinetic interaction pattern with GAGs, for example RANTES exhibited a very complex interaction kinetics whereas IL-8 showed a more simple kinetic path. All interaction kinetic patterns of chemokines with their natural GAG ligands indicated at least a partial cooperative character of this molecular encounter. This means that the stoichiometry of this interaction is still an unkown parameter since binding sites are created at the protein and/or the ligand during the interaction event. By protein engineering we were able to increase the resident time of chemokines on their GAG ligands which enables us to undertake in vivo localisation studies of chemokines in the near future. Some of the project results have been published already, much is still in the process of being written up. Overall we think that the project has led our research in an important direction which needs to be explored in more detail in the future.