title: 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.
authors.affiliation: Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: Catherine
authors.initials: C
authors.lastname: Denis
authors.forename: Kathleen
authors.initials: K
authors.lastname: Deiteren
authors.forename: Anneleen
authors.initials: A
authors.lastname: Mortier
authors.forename: Amel
authors.initials: A
authors.lastname: Tounsi
authors.forename: Erik
authors.initials: E
authors.lastname: Fransen
authors.forename: Paul
authors.initials: P
authors.lastname: Proost
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Renauld
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Lambeir
mesh_terms: D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D054402:Chemokine CCL1 / Q000737:chemistry*; D003907:Dexamethasone / Q000494:pharmacology; D058851:GPI-Linked Proteins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D007249:Inflammation; D013058:Mass Spectrometry / Q000379:methods; D008666:Metalloendopeptidases / Q000737:chemistry; D051379:Mice; D008954:Models, Biological; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054401:Receptors, CCR8 / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: C065971:CCL1 protein, human; C103330:CCR8 protein, human; C502258:Ccl1 protein, mouse; C489319:Ccr8 protein, mouse; D054402:Chemokine CCL1; D058851:GPI-Linked Proteins; D054401:Receptors, CCR8; D003907:Dexamethasone; C058479:carboxypeptidase M; D008666:Metalloendopeptidases; D002118:Calcium