Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Dec 28;18(1):54.
doi: 10.1186/s12865-017-0237-5.

Functional expression of CCL8 and its interaction with chemokine receptor CCR3

Baosheng Ge  1 , Jiqiang Li  2 , Zhijin Wei  2 , Tingting Sun  2 , Yanzhuo Song  2 , Naseer Ullah Khan  2
Affiliations

Functional expression of CCL8 and its interaction with chemokine receptor CCR3

Baosheng Ge et al. BMC Immunol. .

Abstract

Background: Chemokines and their cognate receptors play important role in the control of leukocyte chemotaxis, HIV entry and other inflammatory diseases. Developing an effcient method to investigate the functional expression of chemokines and its interactions with specific receptors will be helpful to asses the structural and functional characteristics as well as the design of new approach to therapeutic intervention.

Results: By making systematic optimization study of expression conditions, soluble and functional production of chemokine C-C motif ligand 8 (CCL8) in Escherichia coli (E. coli) has been achieved with approx. 1.5 mg protein/l culture. Quartz crystal microbalance (QCM) analysis exhibited that the purified CCL8 could bind with C-C chemokine receptor type 3 (CCR3) with dissociation equilibrium constant (K D) as 1.2 × 10-7 M in vitro. Obvious internalization of CCR3 in vivo could be detected in 1 h when exposed to 100 nM of CCL8. Compared with chemokine C-C motif ligand 11 (CCL11) and chemokine C-C motif ligand 24 (CCL24), a weaker chemotactic effect of CCR3 expressing cells was observed when induced by CCL8 with same concentration.

Conclusion: This study delivers a simple and applicable way to produce functional chemokines in E. coli. The results clearly confirms that CCL8 can interact with chemokine receptor CCR3, therefore, it is promising area to develop drugs for the treatment of related diseases.

Keywords: Agonist; CCL8; Chemokine; Chemokine receptor CCR3; Expression; Interaction.

PubMed Disclaimer

Conflict of interest statement

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Optimization of induction phase, inducer concentration and induction temerature. Production of CCL8 was characterized using dot-blot. The intensities of dot-blot were averaged and error bars were calculated based on three times experiments. a Effect of induction phase on the expression of pET28a-CCL8. b Effect of inducer concentration on the expression of pET28a-CCL8. c Effect of expression temperature on the expression of pET28a-CCL8
Fig. 2
Fig. 2
SDS-PAGE and circular dichroim analysis of CCL8 after digestion. a SDS-PAGE analysis of CCL8. Lane M represents protein standard markers. Lane 1 is His-tagged CCL8 fusion protein; lane 2, digested mixture of His-tagged CCL8 fusion protein; lane 3, purified CCL8 protein after the second Ni affinity chromatography; and lane 4 represents the mixture of undigested fusion protein, fusion part of CCL8 and His6-TEV enzymes captured on column, respectively. b Mass spectrometry analysis of the purified CCL8. c Circular dichroism analysis of recombinant CCL8
Fig. 3
Fig. 3
QCM sensorgrams for binding of different concentrations of CCL8 with CCR3. The experimental curves are shown in black, while the fitted curves are in red. The sensorgrams were fitted globally with a 1:1 binding model [22]
Fig. 4
Fig. 4
Internalization of CCR3 induced by CCL8. Cells were imaged at different time after stimulated by 100 nM CCL8 using a Nikon A1 confocal microscopy
Fig. 5
Fig. 5
Chemotactic index of HEK293 cells stably transfected with CCR3 induced with different agonists. Errors bars were taken from three times repeats
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Van Coillie E, Van Damme J, Opdenakker G. The MCP/eotaxin subfamily of CC chemokines. Cytokine Growth Factor Rev. 1999;10(1):61–86. doi: 10.1016/S1359-6101(99)00005-2. - DOI - PubMed
    1. Martine J, SAL S, Leurs R. Fundamentals of chemokines and Chemokine receptors. Weinheim: Wiley; 2011.
    1. Rodriguez-Frade JM, Martinez AC, Mellado M. Chemokine signaling defines novel targets for therapeutic intervention. Mini Rev Med Chem. 2005;5(9):781–789. doi: 10.2174/1389557054867084. - DOI - PubMed
    1. Salanga CL, Handel TM. Chemokine oligomerization and interactions with receptors and glycosaminoglycans: the role of structural dynamics in function. Exp Cell Res. 2011;317(5):590–601. doi: 10.1016/j.yexcr.2011.01.004. - DOI - PMC - PubMed
    1. Wang X, Sharp JS, Handel TM, Prestegard JH. Chemokine oligomerization in cell signaling and migration. Prog Mol Biol Transl Sci. 2013;117:531–578. doi: 10.1016/B978-0-12-386931-9.00020-9. - DOI - PMC - PubMed

Publication types

LinkOut - more resources