Significance of N-Terminal Proteolysis of CCL14a to Activity on the Chemokine Receptors CCR1 and CCR5 and the Human Cytomegalovirus-Encoded Chemokine Receptor US28

Abstract

The CC chemokine CCL14a is constitutively expressed in a large variety of tissues and its inactive proform CCL14a(1–74) circulates in high concentrations in plasma. CCL14a(1–74) is converted into CCL14a(9–74) by the proteases urokinase-type plasminogen activator and plasmin and is a highly active agonist for the chemokine receptors CCR1 and CCR5. In this study, a new CCL14a analog, CCL14a(12–74), was isolated from blood filtrate. To elucidate the functional role of the N terminus, a panel of N-terminally truncated CCL14a analogs were tested on the receptors CCR1 to CCR5 and on the human cytomegalovirus (HCMV)-encoded chemokine receptor US28. The rank order of binding affinity to these receptors and of the activation of CCR1 and CCR5-mediated intracellular Ca2+ concentration mobilization is CCL14a(6–74)<(7–74)<(8–74)≪(9–74) = (10–74)≫(11–74)≫(12–74). The almost identical affinities of CCL14a(7–74), CCL14a(9–74), and CCL14a(10–74) for the US28 receptor and the inhibition of US28-mediated HIV infection of 293T cells by all of the N-terminally truncated CCL14a analogs support the promiscuous nature of the viral chemokine receptor US28. In high concentrations, CCL14a(12–74) did reveal antagonistic activity on intracellular Ca2+ concentration mobilization in CCR1- and CCR5-transfected cells, which suggests that truncation of Tyr11 might be of significance for an efficient inactivation of CCL14a. A putative inactivation pathway of CCL14a(9–74) to CCL14a(12–74) may involve the dipeptidase CD26/dipeptidyl peptidase IV (DPPIV), which generates CCL14a(11–74), and the metalloprotease aminopeptidase N (CD13), which displays the capacity to generate CCL14a(12–74) from CCL14a(11–74). Our results suggest that the activity of CCL14a might be regulated by stringent proteolytic activation and inactivation steps.

CD26/dipeptidylpeptidase IV—chemokine interactions: double-edged regulation of inflammation and tumor biology

“…Upon cleavage by CD26 and the removal of this proline residue, these chemokines become efficient substrates for CD13 (unpublished results in Fig. 2A) [80,93].…”

“…Processing into CCL14(9-74) by plasmin or urokinase plasminogen activator is necessary for CCL14 to become an agonist for CCR1, CCR3, and CCR5 (with a specific activity comparable to that of CCL5 and CCL3/LD78a) [109,110]. However, further cleavage by CD26 (possibly in combination with CD13) generates an inactive variant [93,94]. Therefore, a subtle equilibrium between these enzymes determines the fate of CCL14.…”

Pharmacological modulation of chemokine receptor function

“…In the second step, the flexible N‐terminus (triggering domain) of the chemokine is positioned in such a way that it interacts with a second site (CRS2), formed by parts of the ELs and/or TM domains, resulting in receptor activation (Figure 4C) (Monteclaro and Charo, 1996; Wu et al ., 1996; Pease et al ., 1998; Gupta et al ., 2001; Blanpain et al ., 2003; Xanthou et al ., 2003; Ott et al ., 2004; Rajagopalan and Rajarathnam, 2004). This is supported by truncations or mutations in the N‐termini of chemokines, generally leading to a loss in agonist activity, while often retaining high receptor binding affinity (Gong and Clark‐Lewis, 1995; Proudfoot et al ., 1996; Clark‐Lewis et al ., 2003; Richter et al ., 2009). In the case of CCR5, several reports indicate that a TXP motif in TM2 and surrounding aromatic residues in TM2 and 3 are involved in chemokine‐mediated activation of CCR5, but not in high‐affinity binding, suggesting that in step two the N‐terminus interacts with residues in this TM region (Govaerts et al ., 2001; 2003; Blanpain et al ., 2003).…”

Biochemical Analysis of Matrix Metalloproteinase Activation of Chemokines CCL15 and CCL23 and Increased Glycosaminoglycan Binding of CCL16

“…The CCL14-(7–74) product, here shown to result from MMP-12 processing, has been shown to enhance calcium mobilization (50). However, carboxyl-terminal truncations of the chemokine, observed here by MMP activity, have not previously been identified, and thus the functional effects of this require further investigation.…”