Production of Recombinant Chemokines and Validation of Refolding

Veldkamp, Christopher T.; Koplinski, Chad A.; Jensen, Davin R.; Peterson, Francis C.; Smits, Kaitlin; Smith, Brittney L.; Johnson, Scott K.; Lettieri, Christina K.; Buchholz, Wallace G.; Solheim, Joyce C.; Volkman, Brian F.

doi:10.1016/bs.mie.2015.09.031

Cited by 33 publications

(45 citation statements)

References 37 publications

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“…For example, in Supplemental Fig. S1 1 H spectra of CCL21 CCL19‐N‐term show chemical shift dispersion indicating a folded chemokine and reduction results in a spectrum showing degenerate chemical shifts consistent with an unfolded protein . Additionally, 15 N‐ 1 H heteronuclear single quantum coherence spectra (HSQC) of uniformly 15 N/ 13 C labeled CCL19 CCL21‐ tail , as seen in Supplementary Fig.…”

Section: Methodsmentioning

confidence: 96%

“…The chimera was further purified using reverse phase (RP) HPLC (C18 column, 0.1% aqueous trifluoroacetic acid buffer with a CH 3 CN gradient from 21 to 42% (v/v) over 30 min). Purity, identity, and folding were verified by a combination of SDS‐PAGE, RP‐HPLC, mass spectrometry, and protein nuclear magnetic resonance (NMR) similar to Veldkamp et al . For example, in Supplemental Fig.…”

Section: Methodsmentioning

confidence: 98%

“…S2, indicate this chimera is folded and that there are significant similarities between the corresponding CCL19 and CCL21 regions of this chimera and HSQC spectra of either CCL19 or residues corresponding to the tail of CCL21 . Protein NMR data was collected at the Medical College of Wisconsin's NMR facility as previously described …”

Section: Methodsmentioning

confidence: 99%

“…with an unfolded protein. 35 Additionally, 15 N-1 H heteronuclear single quantum coherence spectra (HSQC) of uniformly 15 N/ 13 C labeled CCL19 CCL21-tail , as seen in Supplementary Fig. S2, indicate this chimera is folded and that there are significant similarities between the corresponding CCL19 and CCL21 regions of this chimera and HSQC spectra of either CCL19 36 or residues corresponding to the tail of CCL21.…”

Section: Expression and Purification Of The Human Chimeric Ccl19 Ccl2mentioning

confidence: 97%

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CCL19 with CCL21-tail displays enhanced glycosaminoglycan binding with retained chemotactic potency in dendritic cells

Jørgensen

Adogamhe

Laufer

et al. 2018

Journal of Leukocyte Biology

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CCL19 is more potent than CCL21 in inducing chemotaxis of human dendritic cells (DC). This difference is attributed to 1) a stronger interaction of the basic C-terminal tail of CCL21 with acidic glycosaminoglycans (GAGs) in the environment and 2) an autoinhibitory function of this C-terminal tail. Moreover, different receptor docking modes and tissue expression patterns of CCL19 and CCL21 contribute to fine-tuned control of CCR7 signaling. Here, we investigate the effect of the tail of CCL21 on chemokine binding to GAGs and on CCR7 activation. We show that transfer of CCL21-tail to CCL19 (CCL19 ) markedly increases binding of CCL19 to human dendritic cell surfaces, without impairing CCL19-induced intracellular calcium release or DC chemotaxis, although it causes reduced CCR7 internalization. The more potent chemotaxis induced by CCL19 and CCL19 compared to CCL21 is not transferred to CCL21 by replacing its N-terminus with that of CCL19 (CCL21 ). Measurements of cAMP production in CHO cells uncover that CCL21-tail transfer (CCL19 ) negatively affects CCL19 potency, whereas removal of CCL21-tail (CCL21 ) increases signaling compared to full-length CCL21, indicating that the tail negatively affects signaling via cAMP. Similar to chemokine-driven calcium mobilization and chemotaxis, the potency of CCL21 in cAMP is not improved by transfer of the CCL19 N-terminus to CCL21 (CCL21 ). Together these results indicate that ligands containing CCL21 core and C-terminal tail (CCL21 and CCL21 ) are most restricted in their cAMP signaling; a phenotype attributed to a stronger GAG binding of CCL21 and defined structural differences between CCL19 and CCL21.

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Section: Methodsmentioning

confidence: 96%

Section: Methodsmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

Section: Expression and Purification Of The Human Chimeric Ccl19 Ccl2mentioning

confidence: 97%

See 2 more Smart Citations

CCL19 with CCL21-tail displays enhanced glycosaminoglycan binding with retained chemotactic potency in dendritic cells

Jørgensen

Adogamhe

Laufer

et al. 2018

Journal of Leukocyte Biology

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“…Human CCL21, CCL21trunc (CCL21 residues 1–79), CCL19, and the chimera (CCL19, which contains 77 residues, with residues 78–111 of CCL21) were expressed, refolded and purified as previously described [23, 24]. …”

Section: Methodsmentioning

confidence: 99%

Transferring the C-terminus of the chemokine CCL21 to CCL19 confers enhanced heparin binding

Barmore

Castex

Gouletas

et al. 2016

Biochemical and Biophysical Research Communications

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Chemokines direct the migration of cells during various immune processes and are involved in many disease states. For example, CCL19 and CCL21, through activation of the CCR7 receptor, recruit dendritic cells and naïve T-cells to the secondary lymphoid organs aiding in balancing immune response and tolerance. However, CCL19 and CCL21 can also direct the metastasis of CCR7 expressing cancers. Chemokine binding to glycosaminoglycans, such as heparin, is as important to chemokine function as receptor activation. CCL21 is unique in that it contains an extended C-terminus not found in other chemokines like CCL19. Deletion of this extended C-terminus reduces CCL21’s affinity for heparin and transferring the CCL21 C-terminus to CCL19 enhances heparin binding mainly through non-specific, electrostatic interactions.

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CCL20 is a novel ligand for the scavenging atypical chemokine receptor 4

Matti

D’Uonnolo

Artinger

et al. 2020

Journal of Leukocyte Biology

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The chemokine CCL20 is broadly produced by endothelial cells in the liver, the lung, in lymph nodes and mucosal lymphoid tissues, and recruits CCR6 expressing leukocytes, particularly dendritic cells, mature B cells, and subpopulations of T cells. How CCL20 is systemically scavenged is currently unknown. Here, we identify that fluorescently labeled human and mouse CCL20 are efficiently taken-up by the atypical chemokine receptor ACKR4. CCL20 shares ACKR4 with the homeostatic chemokines CCL19, CCL21, and CCL25, although with a lower affinity. We demonstrate that all 4 human chemokines recruit -arrestin1 and -arrestin2 to human ACKR4. Similarly, mouse CCL19, CCL21, and CCL25 equally activate the human receptor. Interestingly, at the same chemokine concentration, mouse CCL20 did not recruit -arrestins to human ACKR4. Further cross-species analysis suggests that human ACKR4 preferentially takes-up human CCL20, whereas mouse ACKR4 similarly internalizes mouse and human CCL20. Furthermore, we engineered a fluorescently labeled chimeric chemokine consisting of the N-terminus of mouse CCL25 and the body of mouse CCL19, termed CCL25_19, which interacts with and is taken-up by human and mouse ACKR4. K E Y W O R D SACKR4, atypical chemokine receptor, CCL19, CCL20, CCL21, CCL25, chemokine scavenging, -arrestin

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Production of Recombinant Chemokines and Validation of Refolding

Cited by 33 publications

References 37 publications

CCL19 with CCL21-tail displays enhanced glycosaminoglycan binding with retained chemotactic potency in dendritic cells

CCL19 with CCL21-tail displays enhanced glycosaminoglycan binding with retained chemotactic potency in dendritic cells

Transferring the C-terminus of the chemokine CCL21 to CCL19 confers enhanced heparin binding

CCL20 is a novel ligand for the scavenging atypical chemokine receptor 4

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