Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Cientificas, Campus de Cantoblanco, Madrid, Spain.
J Leukoc Biol. 2011 Aug;90(2):399-408. doi: 10.1189/jlb.1010565. Epub 2011 May 18.
Use of SPR-based biosensors is an established method for measuring molecular interactions. Their application to the study of GPCRs is nonetheless limited to detergent-solubilized receptors that can then be reconstituted into a lipid environment. Using the chemokine receptor CXCR4 and its specific ligand CXCL12, we outline here a highly reproducible biosensor method based on receptor presentation on the surface of lentiviral particles; the approach is simple and does not require the use of antibodies to achieve correct receptor orientation on the sensorchip surface. We measured the kinetic parameters of CXCR4/CXCL12 binding in a single step and in real time and evaluated the effect of GAG presentation of chemokines on this interaction. The data indicate that at low concentrations, soluble heparin modulates CXCR4/CXCL12 interaction and at high concentrations, abrogates binding. These observations suggest that in addition to their known role in modulating local chemokine availability, GAG affect the receptor/ligand interaction, although their influence on affinity parameters is very limited. The method will also be useful for quantifying these biomarkers in biological fluids and for the development of high-throughput screening for their antagonists.
基于 SPR 的生物传感器的使用是测量分子相互作用的一种既定方法。然而，它们在 GPCR 研究中的应用仅限于可以重新构成脂质环境的去污剂溶解的受体。本文使用趋化因子受体 CXCR4 及其特异性配体 CXCL12，概述了一种基于慢病毒颗粒表面受体呈现的高度可重复的生物传感器方法；该方法简单，不需要使用抗体即可在传感器芯片表面实现正确的受体取向。我们在一步和实时测量了 CXCR4/CXCL12 结合的动力学参数，并评估了趋化因子 GAG 呈现对这种相互作用的影响。数据表明，在低浓度下，可溶性肝素调节 CXCR4/CXCL12 相互作用，而在高浓度下，肝素则会破坏结合。这些观察结果表明，除了它们在调节局部趋化因子可用性方面的已知作用外，GAG 还会影响受体/配体相互作用，尽管它们对亲和力参数的影响非常有限。该方法还将有助于定量检测生物液中的这些生物标志物，并开发高通量筛选其拮抗剂的方法。
