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Bioluminescent CXCL12 fusion protein for cellular studies of CXCR4 and CXCR7
 
Kathryn E. Luker1, Mudit Gupta1, and Gary D. Luker1,2
1Center for Molecular Imaging, Department of Radiology, University of Michigan Medical School, Ann Arbor, MI, USA
2Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA
BioTechniques, Vol. 47, No. 1, July 2009, pp. 625–632
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Abstract

Chemokine CXCL12 and its two known receptors, CXCR4 and CXCR7, may play a role in diseases including tumor growth and metastasis, atherosclerosis, and HIV infection. Therefore, these molecules may be promising targets for drug development. While studies of cell signaling and high-throughput screening for drug discovery increasingly are based on luminescent assays because of their high sensitivity and signal-to-background ratio, there currently is no bioluminescent assay for chemokine–chemokine receptor binding. To develop a bioluminescent probe for chemokine binding and cellular uptake, we fused CXCL12 to Gaussia luciferase (GL), an ATP-independent enzyme that is the smallest known luciferase. Fusing CXCL12 to Gaussia luciferase (CXCL12-GL) did not alter the bioluminescence emission spectrum and only minimally affected enzyme function under varying conditions of pH, temperature, and NaCl concentration. CXCL12-GL also activated CXCR4-dependent signaling to a comparable extent as unfused CXCL12. Using multiwell plate assays, we established that CXCR7 increases cell-associated CXCL12 to a significantly greater extent than CXCR4. We also showed that CXCL12-GL can be used to quantify inhibition of chemokine receptor binding by compounds that specifically target CXCR7. These data validate CXCL12-GL as a bioluminescent probe to investigate molecular functions of CXCR4 and CXCR7 and screen for compounds that modulate ligand-receptor binding.

Introduction

Chemokines and chemokine receptors originally were identified as regulators of immune cell trafficking in normal physiology and inflammation. More recently, this family of proteins has been shown to regulate diseases including cancer, atherosclerosis, autoimmune diseases, and neuro-degenerative processes (1,2,3,4). As a result, chemokine receptors have emerged as new therapeutic targets, emphasizing the need to develop reagents for quantitative, high-throughput screening assays of chemokine binding and inhibition.

Conventional assays use radiolabeled chemokines to quantify binding to specific receptors and identify inhibitors of ligand-receptor interactions. While radioligand binding studies have been used successfully for these purposes, such assays generate radioactive waste that is cumbersome to dispose. As alternatives to radioactivity, investigators increasingly are using fluorescence or bioluminescence for cell-based assays in basic research and high-throughput screening. CXCL12 and other chemokines have been labeled with fluorescent dyes or proteins to detect ligand-receptor binding in intact cells (5). For example, Hatse et al. fused CXCL12 to Alexa Fluor 647 and used this reagent to detect binding and inhibition of CXCL12-CXCR4 interactions by flow cytometry (6). Similarly, binding of CXCL12 to receptor CXCR7 has been analyzed with a genetic fusion of CXCL12 to green fluorescent protein (GFP) (7).

While these studies show that fluorescent chemokines provide a viable replacement for radiolabeled chemokine binding assays in some experimental settings, bioluminescence assays have not been developed for chemokine-chemokine receptor binding studies. Relative to fluorescence-based techniques, bioluminescence assays with luciferase enzymes offer several potential advantages for quantifying accumulation of chemokines in intact cells (8). First, bioluminescence assays have substantially lower background signal than fluorescence, enhancing signal-to-noise ratios for chemokine binding. Improved sensitivity is particularly important for measuring ligand binding to chemokine and other seven-transmembrane receptors because these proteins may be expressed at modest levels in cells (9). Second, bioluminescent end points have a greater dynamic range of linear signal response than corresponding fluorescent probes. Finally, bioluminescence assays are affected less than fluorescence by colors and dyes in compounds and cell culture media. These advantages account for improved performance of bioluminescence relative to fluorescence in multiwell cell culture formats and highlight the need for a bioluminescent assay of chemokinechemokine receptor binding (10).

To develop a bioluminescent assay for binding of chemokines to receptors, we focused on chemokine CXCL12 and its two known receptors, CXCR4 and CXCR7. There is particular interest in developing specific inhibitors of CXCL12 binding to these two receptors both as chemical probes for investigating specific mechanisms of action and as potential therapeutic agents for treating diseases including cancer and HIV (11,12,13). We fused CXCL12 to the humanized form of luciferase from Gaussia princeps. Gaussia luciferase (GL) is the smallest known luciferase, containing only 185 amino acids. In cell-based assays, GL is ~1000× brighter than firefly and Renilla luciferases (two other enzymes used commonly for bioluminescent assays), which increases the dynamic range for detection and quantification (14). Bioluminescence from GL is also independent of ATP, allowing the enzyme to function in both intracellular and extracellular compartments (15). While our studies focus on CXCL12, we expect this technique can be generalized to develop bioluminescent probes for other chemokines and other peptide signaling molecules.

Materials and methods

Cells

293T cells (Open Biosystems, Huntsville, AL, USA), and human breast cancer cell lines MDA-MB-231 and MCF-7 (ATCC, Manassas, VA, USA) were cultured at 37°C and 5% CO2 in DMEM (Invitrogen, Carlsbad, CA, USA), 10% fetal bovine serum, 1% glutamine, and 0.1% penicillin/streptomycin/gentamicin (Invitrogen).

DNA constructs

Mouse CXCL12-α was amplified by PCR using plasmid CXCL12-degrakine (courtesy of Lishan Su, University of North Carolina, Chapel Hill, NC, USA) as a template. PCR primers were 5′-ATGCCTCGAGGCCACCATGGACGCCAAGGTCGTCG-3′ and 5′-GCATGAATTCCCCTTGTTTAAAGCTTTCTCCAGGTA-3′; restriction sites for XhoI and EcoRI, respectively, are underlined. The PCR product was ligated to the corresponding sites in plasmid EGFP-N1 (BD Biosciences, San Jose, CA, USA). GL was amplified using PCR primers 5′-ATGCGAATTCCGGCGGAGGTGGGTCCGGAGGCGGTGGGAGCGCCAAGCCCACCGAGAACAACGAAGACTTC-3′ and 5′-GCATGCGGCCGCTTAGCCTATGCCGCCCTGTGCGG-3′ with EcoRI and NotI restriction sites underlined (pGLuc-Basic, New England Biolabs, Ipswich, MA, USA). This strategy removes the 15–amino acid secretion signal from GL and inserts a (G4S)2 amino acid linker between CXCL12 and Gaussia (designated as CXCL12-GL). CXCL12-GL and the cytomegalovirus (CMV) promoter from EGFP-N1 were removed with restriction enzymes AseI and NotI and ligated to the blunted PacI site of FUPW (16). Unfused CXCL12 was inserted into the blunted EcoRI site of EGFP-N1 and then transferred to FUPW as described for CXCL12-GL. GL was excised from pGLuc-Basic with EcoRI and NotI and inserted into FUPW as described for CXCL12-GL. PCR products were verified by DNA sequencing.

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