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

Abstract

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.

Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7

“…The CCL21 CCL19N−term chimera was expressed and purified as described in detail in Jørgensen et al (34), while the CCL19 CCL21N−term chimera was expressed and purified as described in detail for the chemokine CCL19 in Veldkamp et al (35). The CCL21 CCL19N−term chimera contains residues 1–16 of human CCL19 and residues 17–111 of human CCL21 resulting in the protein sequence below:…”

CCR7 as a therapeutic target in Cancer

“…CCL21 consists of 111 amino acids (12 kDa) (Figure 3A), whereas CCL19 has 77 residues (8.8 kDa) (Figure 3B). The longer CCL21 contains lysine rich, glycosaminoglycan (GAG) binding domains at its C terminus, suggesting this chemokine is likely to bind endothelial cell surface glycoproteins [250][251][252][253]. The structural differences between the two chemokines CCL19 and CCL21 reflects different physiological roles between them.…”

CAL-1 as Cellular Model System to Study CCR7-Guided Human Dendritic Cell Migration

“…Serum starved cells were scraped and washed once with RPMI. Aliquots of 1.5x 10 6 cells in 50μl RPMI were incubated for 5min at 37°C and then stimulated with 50μl of pre-warmed RPMI or medium containing a 2-fold chemokine concentration (final concentration: 100nM of human CCL19 or CCL21, which is known to induce maximal responses ( 37 , 38 ); Peprotech #300-29B and #300-35) for indicated time points. Cell stimulation was terminated by the addition of 25μl of 5x Laemmli sodium dodecyl sulphate (SDS) loading buffer containing 4% β-mercaptoethanol.…”