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

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.