Endothelial heparan sulphate - a member of the glycosaminoglycan (GAG) familys - plays an important role in chemokine presentation to chemokine receptors on leukocytes and in transportation processes across the endothelial barrier. Binding to GAGs was shown to be essentially required for leukocyte transmigration in vivo [1]. We have developed GAG-binding decoy chemokines which are engineered towards higher GAG-binding affinity and which exhibit inactivated GPC receptor activity [2]. Our engineering technology is therefore strictly dependent upon the ability to characterise the chemokine-GAG interaction in sufficient detail, with high sensitivity and biological relevance.
We will present results of several in vitro assays, namely isothermal fluorescence titrations, isothermal titration calorimetry, surface plasmon resonance, and a novel type of competition assay called ELISA-like competition (ELICO) assay. For our studies, wild-type chemokines and chemokine mutants with increased GAG-binding affinity and impaired receptor binding properties were used. All the assays that were applied clearly showed a several fold increase in GAG- binding affinity for the chemokine mutants compared to the respective wild-types. The different methods will be discussed with respect to their applicability and developmental requirements.
References
- Proudfoot AE, Handel TM, Johnson Z, Lau EK, LiWang P, Clark-Lewis I, Borlat F, Wells TN, Kosco-Vilbois MH. (2003) Proc Natl Acad Sci U S A. 100:1885-90.
- Adage T, Piccinini AM, Falsone A, Trinker M, Robinson J, Gesslbauer B, Kungl AJ. (2012) Br J Pharmacol. 167:1195-205.
