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.
Highlights
Carboxypeptidase M (CPM, EC 3.4.17.12) is a member of the metallo-carboxypeptidases, which consist of metallo-peptidases cleaving C-terminal amino acids from peptides and proteins
We examined whether CCL1 was prone to processing by CPM
In this study we demonstrate for the first time that another chemokine, CCL1, is processed at its C-terminus by CPM in vitro
Summary
Carboxypeptidase M (CPM, EC 3.4.17.12) is a member of the metallo-carboxypeptidases, which consist of metallo-peptidases cleaving C-terminal amino acids from peptides and proteins. The processing and modulation of the activity of kinins, enkephalins and anaphylatoxins quickly pointed towards an inflammatory role for CPM. This was further substantiated by the expression of CPM on differentiated immune cells such as macrophages, and up- or downregulation of CPM in inflammation, tumor and/or tumor environment (reviewed in [2]). Proteolytic processing affects the activity of chemokines in vitro and in vivo in different ways [4]. Besides orchestrating leukocyte chemotaxis and host inflammatory responses, chemokines can be regulators of fundamental developmental processes. The goal of this work was to investigate whether CPM-catalyzed C-terminal trimming of CCL1 affects its biological activity. The results of this study show that the removal of three amino acids from the C-terminus of CCL1 reduces CCR8 binding, but turns CCL1 into a more potent CCR8 agonist, resulting in, for example, increased intracellular calcium (Ca2+i) release and enhanced anti-apoptotic activity
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