Establishing roles for different chemokines in conditions of health and disease is challenging due to the large size of the chemokine family and redundancy in ligand-receptor binding. By providing increased flexibility for assay design, biotinylated chemokines can streamline common research applications and facilitate novel discovery, as highlighted by several recent publications.

What are chemokines?

Chemokines are a family of chemoattractant cytokines with essential roles in inflammation and homeostasis. To date, almost 50 different chemokines have been discovered, and are grouped into four main sub-families (CC, CXC, CX3C, and XC) based on the number and arrangement of conserved cysteine residues. Chemokines exert their biological effects by binding to complementary chemokine receptors within the G protein coupled receptor (GPCR) family. While some chemokines are proinflammatory, serving to recruit immune cells to the site of tissue damage or an infection, others are homeostatic and involved in controlling cell migration during normal tissue development or maintenance.

Challenges for chemokine research

A major challenge for chemokine research stems from redundancy in ligand-receptor binding. Just 19 chemokine receptors have so far been identified (less than half the number of known chemokines), many of which can bind multiple ligands. For example, the chemokine receptor CCR1 has been reported to bind up to 12 distinct chemokines, while CXCR1 and CXCR2 share at least three ligands, including CXCL6, CXCL7, and CXCL8, all of which have different functions.

Further hurdles are encountered when expressing and purifying chemokines, since it can often be difficult to maintain bioactivity due to the inherent instability of chemokine molecules. ChemoTactics, a company started by researchers at UC San Diego, has been able to overcome this issue by optimizing purification conditions for each individual chemokine, maximizing their stability.

An additional challenge is the low-level expression of chemokine GPCRs, which make them harder to monitor than cytokine receptors. The need for highly sensitive direct detection methods is crucial for quantitation of receptor expression, ligand binding, and receptor internalization. ChemoTactics addresses this requirement with enabling methods and reagents.

Applications of biotinylated chemokines

Biotinylated chemokines are valuable tools for studying chemokine biology because the biotin tag simplifies detection of the chemokine being investigated. They are especially useful for monitoring receptor binding and internalization, which can easily be tracked by pairing a biotinylated chemokine with a fluorophore-streptavidin conjugate for flow cytometry or microscopy-based analysis. Representative flow cytometry data is shown in Figure 1, where the uptake of biotinylated CXCL12 by U937 cells is prevented by a CXCR4 inhibitor. Figure 2 shows microscopy-based detection of biotinylated SDF-1α (one of two CXCL12 isoforms) by a temperature dependent form of CXCR4.

chemokines

Figure 1. Immunofluorescent staining and flow cytometry analysis of CXCR4 expression using ChemoTactics’ Biotinylated CXCL12 (Cat.# B-CXCL12). Data show the uptake of biotinylated CXCL12 by U937 cells in the presence (red) and absence (cyan) of a CXCR4 inhibitor, AMD3100, using a streptavidin-PE conjugate for detection. The second step control sample (streptavidin-PE alone) is shown in orange.


chemokines

Figure 2. Microscopy data showing the distribution of temperature-dependent CXCR4 at 4oC (left) and 37oC (right) using biotinylated SDF-1α (Cat.# B-CXCL12) for receptor staining in combination with streptavidin-Cy3 for detection.

Other applications for biotinylated chemokines include their use for investigating calcium flux in response to changing ligand concentrations, and as tools for promoting cell migration. Biotinylated chemokines are also useful for in vivo imaging when paired with sufficiently bright streptavidin-fluorophore conjugates, and can serve as core components of ELISAs and western blot assays.

Published uses of biotinylated chemokines

Within the last few years, biotinylated chemokines have been cited in various publications. In 2020, Torretta et al. used biotinylated-CCL5 in combination with avidin-PE to characterize the biological significance of extracellular nicotinamide phosphoribosyl transferase (eNAMPT) binding to CCR5 in cancer cells. Flow cytometry analysis showed eNAMPT to compete with CCL5-PE for CCR5 binding in an over-expressing HeLa cell line, while a series of phenotypic studies (including monitoring of CCR5 internalization and measurement of CCL5-dependent calcium signaling in the presence of eNAMPT) suggested eNAMPT to be a natural CCR5 antagonist.

More recently, in 2022, Wang et al. immobilized biotinylated CXCL12 on magnetic beads, which they used to capture transglutaminase-2 (TGM2) from solution. Pulldown experiments were performed in the presence or absence of keratin-19 (KRT19), either with or without calcium (Ca2+), using beads coated with biotinylated CXCL8 as a negative control. Data showed TGM2 to only bind CXCL12 in the presence of both bound KRT19 and Ca2+, contributing to the finding that cancer cells expressing TGM2 and KRT19 can form a TGM2-dependent, filamentous coating of CXCL12–KRT19 heterodimers that restricts the intratumoral accumulation of T cells and allows cancers to escape immune attack.

chemokines

Figure 3. The interactions among CXCL12, KRT19, and TGM2. (A) Biotinylated human CXCL8 or CXCL12 was immobilized on SA beads, which were incubated with KRT19. The bead-bound proteins were eluted with SDS and subjected to SDS-PAGE and immunoblotting with SA-HRP or anti-KRT19 antibody. (B) SA beads bearing CXCL12–biotin were incubated with increasing concentrations of KRT19. Bound KRT19 was detected by SDS-PAGE and immunoblotting with anti-KRT19 antibody, and intensities of the KRT19 bands were measured. (C) CXCL12–biotin that was immobilized on SA beads was incubated at 4°C with TGM2 in the absence or presence of KRT19 and in the absence or presence of 10 mM CaCl2. The bound proteins were eluted and subjected to SDS-PAGE and immunoblotting with SA-HRP and antibodies to KRT19 and TGM2. (D) SA beads bearing preformed complexes of CXCL12–biotin and KRT19 were incubated at 20°C for 15 min with TGM2 in the absence or presence of the TGM2 inhibitor ERW1041E. The proteins were eluted from the beads and detected by SDSPAGE and immunoblotting with antibodies to CXCL12 and KRT19. Data provided by Wang et al.

Conclusion

Biotinylated chemokines have broad utility for scientific research since they provide the flexibility to design and develop custom assays. This is demonstrated by several recent publications describing the use of biotinylated chemokines for novel oncology applications.

ChemoTactics offers a growing range of biotinylated chemokines, including the products cited here, all of which can be purchased in bulk amounts. To learn more, visit our products page here.

References

1. Torretta S, Colombo G, Travelli C, Boumya S, Lim D, Genazzani AA, Grolla AA. The Cytokine Nicotinamide Phosphoribosyltransferase (eNAMPT; PBEF; Visfatin) Acts as a Natural Antagonist of C-C Chemokine Receptor Type 5 (CCR5). Cells. 2020 Feb 21;9(2):496.

2. Wang Z, Moresco P, Yan R, Li J, Gao Y, Biasci D, Yao M, Pearson J, Hechtman JF, Janowitz T, Zaidi RM, Weiss MJ, Fearon DT. Carcinomas assemble a filamentous CXCL12-keratin-19 coating that suppresses T cell-mediated immune attack. Proc Natl Acad Sci U S A. 2022 Jan 25;119(4):e2119463119.

About the Author

Emma Easthope is the founder and director of Cambridge Technical Content Ltd, based in the U.K. Since graduating with a bachelor’s degree in biology from the University of Kent at Canterbury in 2000, she has gained extensive experience developing and running immunoassays within companies including Millennium Pharmaceuticals, AstraZeneca and Cellzome. She now produces a wide range of scientific content, including regular features for Biocompare.