N-terminal pyroglutamate formation in CX3CL1 is essential for its full biologic activity.

Astrid Kehlen,Hans-Ulrich Demuth,Torsten Hoffmann,Monique Haegele,Holger Cynis,Livia Böhme

doi:10.1042/bsr20170712

Abstract

CX3CL1 (fractalkine) is a unique member of the CX3C chemokine family and mediates both adhesion and cell migration in inflammatory processes. Frequently, the activity of chemokines depends on a modified N-terminus as described for the N-terminus of CCL2 modified to a pGlu- (pyroglutamate) residue by QC (glutaminyl cyclase) activity. Here, we assess the role of the pGlu-modified residue of the CX3CL1 chemokine domain in human endothelial and smooth muscle cells. For the first time, we demonstrated using MS that QC (QPCT, gene name of QC) or its isoenzyme isoQC (iso-glutaminyl cyclase) (QPCTL, gene name of isoQC) catalyse the formation of N-terminal-modified pGlu-CX3CL1. Expression of QPCT is co-regulated with its substrates CCL2 and CX3CL1 in HUVECs (human umbilical vein endothelial cells) and HCASMCs (human coronary artery smooth muscle cells) upon stimulation with TNF-α and IL-1β whereas QPCTL expression is not affected. By contrast, inhibition of the NF-κB pathway using an IKK2 inhibitor decreased the expression of the co-regulated targets QPCT, CCL2, and CX3CL1. Furthermore, RNAi-mediated inhibition of QPCT expression resulted in a reduction in CCL2 and CX3CL1 mRNA. In HCASMCs, N-terminal-modified pGlu1-CX3CL1 induced a significant stronger effect on phosphorylation of ERK (extracellular signal regulated kinase) 1/2, Akt (protein kinase B), and p38 (p38 mitogen-activated protein kinase) kinases than the immature Gln1-CX3CL1 in a time- and concentration-dependent manner. Furthermore, pGlu1-CX3CL1 affected the expression of CCL2, CX3CL1, and the adhesion molecule ICAM1/CD54 (intercellular adhesion molecule-1) inducing in higher expression level compared with its Gln1-variant in both HCASMCs and HUVECs. These results strongly suggest that QC-catalysed N-terminal pGlu formation of CX3CL1 is important for the stability or the interaction with its receptor and opens new insights into the function of QC in inflammation.

Highlights

Increased chemokine levels and monocyte activation are common components of the pathogenesis of inflammatory diseases including atherosclerosis and chronic lung diseases
To confirm whether glutaminyl cyclase (QC) and iso-glutaminyl cyclase (isoQC) can catalyse N-terminal pGlu formation, the recombinant chemokine domain of CX3CL1 was incubated with QC or isoQC and the resulting reaction products were analysed by MS
Post-translational modification of N-terminal glutamine to pGlu by QC/isoQC is used as a cellular strategy to fine-tune the activity of the chemokines CX3CL1 and CCL2

Summary

Introduction

Increased chemokine levels and monocyte activation are common components of the pathogenesis of inflammatory diseases including atherosclerosis and chronic lung diseases. The chemokines CCL2 (MCP-1, monocyte chemoattractant protein-1) and CX3CL1 (fractalkine) are described as key players for the attraction and migration of monocytes into the inflamed tissue. In contrast with the secreted CCL2, CX3CL1 is synthesized as a transmembrane protein with its chemokine domain presented on an extended highly glycosylated mucin-like stalk [1,2]. The membrane-bound CX3CL1 promotes the integrin-independent adhesion by binding with its G-protein-coupled, 7-transmembrane receptor CX3CR1 (fractalkine receptor 1) [3] and is a survival signal for circulating monocytes [4].

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