C-Terminal Clipping of Chemokine CCL1/I-309 Enhances CCR8-Mediated Intracellular Calcium Release and Anti-Apoptotic Activity

Abstract

Carboxypeptidase M (CPM) targets the basic amino acids arginine and lysine present at the C -terminus of peptides or proteins. CPM is thought to be involved in inflammatory processes. This is corroborated by CPM-mediated trimming and modulation of inflammatory factors, and expression of the protease in inflammatory environments. Since the function of CPM in and beyond inflammation remains mainly undefined, the identification of natural substrates can aid in discovering the (patho)physiological role of CPM. CCL1/I-309, with its three C -terminal basic amino acids, forms a potential natural substrate for CPM. CCL1 plays a role not only in inflammation but also in apoptosis, angiogenesis and tumor biology. Enzymatic processing differently impacts the biological activity of chemokines thereby contributing to the complex regulation of the chemokine system. The aim of the present study was to investigate whether (i) CCL1/I-309 is prone to trimming by CPM, and (ii) the biological activity of CCL1 is altered after C -terminal proteolytic processing. CCL1 was identified as a novel substrate for CPM in vitro using mass spectrometry. C -terminal clipping of CCL1 augmented intracellular calcium release mediated by CCR8 but reduced the binding of CCL1 to CCR8. In line with the higher intracellular calcium release, a pronounced increase of the anti-apoptotic activity of CCL1 was observed in the BW5147 cellular model. CCR8 signaling, CCR8 binding and anti-apoptotic activity were unaffected when CPM was exposed to the carboxypeptidase inhibitor DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid. The results of this study suggest that CPM is a likely candidate for the regulation of biological processes relying on the CCL1-CCR8 system.

The Positively Charged COOH-terminal Glycosaminoglycan-binding CXCL9(74–103) Peptide Inhibits CXCL8-induced Neutrophil Extravasation and Monosodium Urate Crystal-induced Gout in Mice

“…Frequently, chemokines seem to be subject to NH 2 -terminal truncation (28). In contrast, COOH-terminal truncation is only occasionally observed (51)(52)(53)(54)(55)(56)(57). The consequences of COOH-terminal truncation are less clear and seem to be rather limited to modification of GAG binding properties (58).…”

International Union of Basic and Clinical Pharmacology. LXXXIX. Update on the Extended Family of Chemokine Receptors and Introducing a New Nomenclature for Atypical Chemokine Receptors

“…In addition to cell migration, studies also support a role for the CCR8 pathway in preventing apoptosis and promoting angiogenesis (Van Snick et al, 1996;Bernardini et al, 2000;Haque et al, 2001;Ruckes et al, 2001;Denis et al, 2012). Truncation of the C terminus of CCL1 by carboxypeptidase M is reported to enhance its CCR8-mediated calcium flux and antiapoptotic activity (Denis et al, 2012).…”

Structural perspectives on antimicrobial chemokines

“…The functional activation of NAP-2 in platelets represents a unique case because no other chemokine post-translational processing events have been found to increase the antimicrobial activity (Wolf et al, 2008). In other instances where proteolytic cleavage occurs at the C-terminus, positively charged residues are lost from chemokines that are already microbicidal (Davis et al, 2005; Mortier et al, 2008; Denis et al, 2012). For example, CXCL10/IP-10 (interferon γ-induced protein-10) retains its activities against E. coli and Listeria monocytogeneses in spite of the loss of two positively charged residues, in a reaction that is catalyzed by the proprotein convertase furin (Hensbergen et al, 2004).…”