A Fluorescent Ligand-Binding Alternative Using Tag-lite® Technology

Zwier, Jurriaan M.; Roux, Thomas; Cottet, Martin; Durroux, Thierry; Douzon, Stephanie; Bdioui, Sara; Gregor, Nathalie; Bourrier, Emmanuel; Oueslati, Nadia; Nicolas, L.; Tinel, Norbert; Boisseau, Chloé; Yverneau, Paul; Charrier‐Savournin, Fabienne; Fink, Michel; Trinquet, Eric

doi:10.1177/1087057110384611

Cited by 139 publications

(165 citation statements)

References 32 publications

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“…23 Similar dissociation constants (Kd) for fluorescently labeled CCL3 of 4.1 Ϯ 0.1nM, 5.9 Ϯ 0.2nM, and 7.8 Ϯ 3.7nM were obtained in cells expressing CCR5 alone or together with UL33 or UL78, respectively ( Figure 4A). Similarly, no modification of Kd values for fluorescently labeled CXCL12 were observed in cells expressing CXCR4 alone (Kd ϭ 37.8 Ϯ 3.9nM) or together with UL33 (43.1 Ϯ 9.7nM) or UL78 (28.3 Ϯ 10.1nM; For personal use only.…”

Section: Effect Of Ul33 and Ul78 On Ccr5 And Cxcr4 Ligand Binding Andsupporting

confidence: 69%

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Human cytomegalovirus-encoded UL33 and UL78 heteromerize with host CCR5 and CXCR4 impairing their HIV coreceptor activity

Tadagaki

Tudor

Gbahou

et al. 2012

Blood

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AbstractHuman cytomegalovirus (HCMV) encodes four 7-transmembrane-spanning (7TM) proteins, US28, US27, UL33, and UL78, which present important sequence homology with human chemokine receptors. Whereas US28 binds a large range of chemokines and disturbs host cell signaling at different levels, the others are orphans with largely unknown functions. Assembly of 2 different 7TM proteins into hetero-oligomeric complexes may profoundly change their respective functional properties. We show that HCMV-encoded UL33 and UL78 form heteromers with CCR5 and CXCR4 chemokine receptors in transfected human embryonic kidney 293T cells and monocytic THP-1 cells. Expression of UL33 and UL78 had pleiotropic, predominantly negative, effects on CCR5 and CXCR4 cell surface expression, ligand-induced internalization, signal transduction, and migration without modifying the chemokine binding properties of CCR5 and CXCR4. Importantly, the coreceptor activity of CCR5 and CXCR4 for HIV was largely impaired in the presence of UL33 and UL78 without affecting expression of the primary HIV entry receptor CD4 and its interaction with CCR5 and CXCR4. Collectively, we identified the first molecular function for the HCMV-encoded orphan UL33 and UL78 7TM proteins, namely the regulation of cellular chemokine receptors through receptor heteromerization.

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Section: Effect Of Ul33 and Ul78 On Ccr5 And Cxcr4 Ligand Binding Andsupporting

confidence: 69%

“…Kd values of the fluorescent ligands were obtained from saturation curves of the specific binding. 23 …”

Section: Fluorescent Ligand-binding Assaymentioning

confidence: 99%

Human cytomegalovirus-encoded UL33 and UL78 heteromerize with host CCR5 and CXCR4 impairing their HIV coreceptor activity

Tadagaki

Tudor

Gbahou

et al. 2012

Blood

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“…Tag-lite cells expressing SNAP-tagged histamine receptor 1 (H1R) and labeled with Lumi4-Tb cryptate were obtained from Cisio. Details on the generation of these cells have been described elsewhere [12]. Vials containing 1 Â 10 6 cells/ml were thawed, diluted 1:20 in ice-cold Tag-lite buffer (Cisbio), and used immediately.…”

Section: Preparation Of Tb-labeled Proteins and Cellsmentioning

confidence: 99%

A universal homogeneous assay for high-throughput determination of binding kinetics

Schiele¹,

Ayaz²,

Fernández-Montalván³

2015

Analytical Biochemistry

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“…Using the instrumental enzymes of the gene engineering mentioned above, the DNA products were reconstructed separately in plasmid pTaglite-SNAP, which is a commercialized vector expressing the receptor of interest at the cell surface, and in plasmid pABhFc, which is used to express secretory IL-6 coupling with the human Fc fragment at the C-terminus [24,25] . The recombinants, pTaglite-SNAP-IL6R and pABhFc-IL6, were analyzed and identified by restriction enzyme digestion and a sequencing analysis.…”

Section: Constructions Of the Recombinant Eukaryotic Expression Plasmidsmentioning

confidence: 99%

Establishment of a novel cell-based assay for screening small molecule antagonists of human interleukin-6 receptor

Zhang

et al. 2014

Acta Pharmacol Sin

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Aim: Blockade of interleukin-6 (IL-6) or its receptor (IL-6R) is effective in preventing the progression of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. In the present study, we established a novel cell-based assay for identifying small molecule IL-6R antagonists. Methods: HEK293A cells were transfected with recombinant plasmids pTaglite-SNAP-IL6R and pABhFc-IL6 to obtain membranebound IL-6R and recombinant human IL-6 coupled with human Fc fragment (rhIL-6), respectively. A novel screening assay based on the interaction between IL-6R and rhIL-6 was established, optimized and validated. The stability of the assay was also assessed by calculating the Z′-factor. Results: RhIL-6 dose-dependently bound to IL-6R expressed at HEK293A cell surface. The IC 50 value of the known antagonist ab47215 was 0.38±0.08 μg/mL, which was consistent with that obtained using the traditional method (0.36±0.14 μg/mL). The value of Z′-factor was 0.68, suggesting that the novel assay was stable for high throughput screening. A total of 474 compounds were screened using the novel screening assay, and 3 compounds exhibited antagonistic activities (IC 50 =8.73±0.28, 32.32±9.08, 57.83±4.24 μg/mL). Furthermore, the active compounds dose-dependently inhibited IL-6-induced proliferation of 7TD1 cells, and reduced IL-6-induced STAT3 phosphorylation in U937 cells. Conclusion: A novel cell-based screening assay for identifying small molecule IL-6R antagonists was established, which simplifies the procedures in traditional cellular ELISA screening and profiling and reduces the costs.

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A Fluorescent Ligand-Binding Alternative Using Tag-lite® Technology

Cited by 139 publications

References 32 publications

Human cytomegalovirus-encoded UL33 and UL78 heteromerize with host CCR5 and CXCR4 impairing their HIV coreceptor activity

Human cytomegalovirus-encoded UL33 and UL78 heteromerize with host CCR5 and CXCR4 impairing their HIV coreceptor activity

A universal homogeneous assay for high-throughput determination of binding kinetics

Establishment of a novel cell-based assay for screening small molecule antagonists of human interleukin-6 receptor

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