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PLoS One
2014 Jan 17;91:e87394. doi: 10.1371/journal.pone.0087394.
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C-terminal engineering of CXCL12 and CCL5 chemokines: functional characterization by electrophysiological recordings.
Picciocchi A
,
Siaučiūnaiteė-Gaubard L
,
Petit-Hartlein I
,
Sadir R
,
Revilloud J
,
Caro L
,
Vivaudou M
,
Fieschi F
,
Moreau C
,
Vivès C
.
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Chemokines are chemotactic cytokines comprised of 70-100 amino acids. The chemokines CXCL12 and CCL5 are the endogenous ligands of the CXCR4 and CCR5 G protein-coupled receptors that are also HIV co-receptors. Biochemical, structural and functional studies of receptors are ligand-consuming and the cost of commercial chemokines hinders their use in such studies. Here, we describe methods for the expression, refolding, purification, and functional characterization of CXCL12 and CCL5 constructs incorporating C-terminal epitope tags. The model tags used were hexahistidines and Strep-Tag for affinity purification, and the double lanthanoid binding tag for fluorescence imaging and crystal structure resolution. The ability of modified and purified chemokines to bind and activate CXCR4 and CCR5 receptors was tested in Xenopus oocytes expressing the receptors, together with a Kir3 G-protein activated K(+) channel that served as a reporter of receptor activation. Results demonstrate that tags greatly influence the biochemical properties of the recombinant chemokines. Besides, despite the absence of any evidence for CXCL12 or CCL5 C-terminus involvement in receptor binding and activation, we demonstrated unpredictable effects of tag insertion on the ligand apparent affinity and efficacy or on the ligand dissociation. These tagged chemokines should constitute useful tools for the selective purification of properly-folded chemokines receptors and the study of their native quaternary structures.
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24498095
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Figure 2. Scheme of expression pattern and predicted properties of the different chemokines used in this work.A. MBP-chemokine constructs. B. Chemokines expressed in E. coli inclusion bodies. Names and sequences of each construct are shown on the left. Numbers represent amino acid positions. The theoretical molecular weight and isoelectric point were determined with the Expasy ProtParam tool (http://web.expasy.org/protparam/). Chemokine expression level and purification yield are indicated. The highest chemokine levels are highlighted in bold. MBP: Maltose Binding Protein; PP: Prescission protease site; S: Strep-tag; HIS: HIS-tag; LT: Lanthanoid binding tag; (+): good expression level; (++): strong expression level. C. Purified inclusion bodies (15 µL per lane) prepared from E. coli cells over-expressing CXCL12-HIS and CXCL12-LT-HIS proteins were analysed by SDS/PAGE and stained with a Coomassie solution. PageRuler Unstained Protein Ladder and PageRuler PreStained Protein Ladder were used for CXCL12-HIS and CXCL12-LT-HIS gels, respectively.
Figure 3. SDS-PAGE analysis of purified chemokines and CXCL12-LT-HIS terbium titration.A. SDS-PAGE analysis of the purified recombinant chemokines. Proteins were loaded onto a 15% SDS-PAGE polyacrylamide gel in denaturing buffer, subjected to electrophoresis at 220 V for 1 hour and stained with a Coomassie staining solution or treated with 50 µM of TbCl3. Luminescent bands associated with the chemokine-LT constructs were visualized on a UV-transilluminator with contrast enhancement. M: Protein mass ladder. B. Tryptophan-sensitized luminescence spectra of a terbium solution with (in red) or without (in green) the purified CXCL12-LT-HIS protein (75 µM in 20 mM Hepes, pH 8.0; 100 mM NaCl) were recorded between 450 and 550 nm (left panel) as indicated in the materials and methods section. As illustrated, significant luminescence amplification is observed at 487 and 542 nm (black arrows) when the terbium ion is trapped by the lanthanoid-binding tag engineered on the purified recombinant chemokine. The CXCL12-LT-HIS terbium titration (right panel) is performed with the same purified CXCL12-LT-HIS protein sample (75 µM) using increasing terbium concentration (15 to 150 µM). After each addition, the solution was mixed and the luminescence emission spectrum was recorded between 450 and 550 nm. For each spectrum, the absorbance values at 487 (blue curve) and 542 nm (red curve) were selected and the CXCL12-LT-HIS terbium binding was visualized following the increase of the luminescence emission versus terbium concentration (expressed in equivalent per protein).
Figure 4. Functional characterization of tagged-CXCL12 chemokines.A. Representative TEVC recordings performed on Xenopus oocytes co-expressing CXCR4 and Kir3.1* or Kir3.4*. Different oocytes are used for each recording. The colored arrows represent the channel activation induced by chemokine-binding on CXCR4 and subsequent G proteins activation. The chemokines concentration is 1 µM while the Barium concentration is 3 mM. B. Dose-response curves of the indicated CXCL12 chemokines. Chemokine concentrations are applied gradually on the same oocyte and each point of the curves is the mean +/â s.e.m. of different recordings from different oocytes. C. Sequential application of CXCL12-HIS +/â LT tag during the same recording on the same oocyte but in different order shows the rapid âreversibilityâ of the activation induced by the LT-tagged CXCL12 chemokine. Each point is an average of 4 to 13 recordings.
Figure 5. Control of the activities of purified and refolded chemokines by a standard chemotaxis assay.Dose-dependent chemokine-induced migration of Jurkat cells was evaluated using a transwell system. Varying concentrations (0 to 300 nM) of WT CXCL12 or tagged-CXCL12 (A) and WT CCL5 or tagged-CCL5 (B) were added to the lower chamber. The results are expressed as percentage of input cells that migrated to the lower chamber over 3 hours from 3 replicate wells per condition (data are means ± SD). For CCL5-LT-Strep averaged values are slightly lower than the control at 0 nM clearly indicating an absence of chemotactic activity induced by this chemokine.
Figure 6. Functional characterization of the tagged-CCL5 chemokines.A. Representative TEVC recordings performed on Xenopus oocytes expressing Kir3.1* or co-expressing CCR5 and Kir3.1* and subjected to applications of the indicated CCL5 chemokines at 1 µM. B. Dose-response curves of the tagged and non-tagged CCL5 chemokines. Due to the low concentration of the mother solutions, the highest concentration cannot be extended. Each point is an average of 3 to 25 recordings.
Figure 1. Principle of the electrophysiological characterization of chemokine receptors.A. Receptors and channels are expressed in Xenopus laevis oocytes by mRNA micro-injection. After 2 day-incubation, purified chemokines are electrophysiologically characterized. B. Schematic representation of a Xenopus oocyte plasma membrane containing a heterologously-expressed chemokine receptor and the G protein-activated Kir3.1* or Kir3.4* channels. The Kir3.x* channel is mutated to function as a homomeric channel. Binding of chemokines to the receptor induces activation and release of the G protein subunits. Gβγ subunits activate the Kir3.1* channels by direct binding, resulting in an increase in ion current carried by K+. C. Schematic representation of the Two-Electrode Voltage Clamp set-up. The oocyte is impaled by 2 glass pipettes containing 3 M KCl and an Ag/AgCl electrode. Electrical current recording is performed under continuous flow of buffer +/− ligands or channel blockers. Change of solutions is controlled by a semi-automatic perfusion system. The used TEVC bath has a potassium concentration similar to the intracellular K+ concentration. D. Representative TEVC recording of Kir3* generated current at −50 mV. The basal current generated by the channels in basal state is determined in the first minute and represented in blue line and bar. This basal current is the reference (100%) for the normalization of ligand-induced effect represented in red line and bar. Barium (Ba2+) is a generic blocker of K+ channels and positions the barium-sensitive baseline.
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