Technical Advance: Surface plasmon resonance-based analysis of CXCL12 binding using immobilized lentiviral particles

Abstract

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. J. Leukoc. Biol. 90: 000 -000; 2011.

Epithelial chemokine CXCL14 synergizes with CXCL12 via allosteric modulation of CXCR4

“…In brief, CXCR4-expressing LVPs are immobilized on an SPR biosensor chip, and SPR signals are recorded after injection of chemokine. In agreement with previous results (44), we determined the on-rate ( k on = 1.22 × 10 5 M −1 · s −1 ) and off-rate ( k off = 4.28 × 10 −3 s −1 ) of CXCL12 interaction with CXCR4, which resulted in a dissociation constant ( K D ) of 3.5 × 10 −8 M. Substituting CXCL12 with CXCL14 revealed strong binding of CXCL14 for immobilized CXCR4 with on-rate ( k on = 3.96 × 10 5 M −1 · s −1 ) and off-rate ( k off = 3.17 x 10 −3 s −1 ) that resulted in a K D of 8.0 × 10 −9 M (Supplemental Fig. 1 A ), which is similar to the published K D value of 1.47 × 10 −8 M obtained by Tanegashima's group using [ 125 I]-CXCL14 (32).…”

“…We have developed an SPR-based binding assay that can be applied to measuring the on- and off-rates, in real-time, of chemokines interacting with their receptors (44). In brief, CXCR4-expressing LVPs are immobilized on an SPR biosensor chip, and SPR signals are recorded after injection of chemokine.…”

“…LVPs were analyzed for CXCR4 expression by flow cytometry (44). Several batches of LVPs were standardized for CXCR4 expression, aliquoted, and stored at −80°C.…”

“…Flow cells of a CM5 sensorchip were functionalized with 8000–8500 resonance units of LVP as described in Vega et al . (44). CXCL12 or CXCL14 (12.5–200 nM) diluted in HBS buffer (100 mM HEPES, 150 mM NaCl, 0.005% polyoxyethylene sorbitan P20, pH 7.4) were injected over immobilized LVPs (30 μl/min, 2 min, 25°C; association phase), followed by a 4-min injection period of HBS buffer alone over the surface (dissociation phase).…”

Chemokine Receptor Oligomerization and Allostery

“…Oligomerization and interactions with GAGs are important for locally sequestering chemokines on cell surfaces to prevent diffusion and facilitate the formation of chemokine gradients that help guide cell movement. GAG interactions have also been shown to facilitate transcytosis of chemokines across cells, chemokine mediated signaling, and they can act as cofactors in promoting receptor interactions [21–23]. Nevertheless, as demonstrated using monomeric variants in bare filter transwell migration assays, the reversibility of chemokine oligomerization is necessary because it is the monomeric form that binds to the receptor with highest affinity and promotes cell migration [24, 25].…”