Invited critical reviewCarboxypeptidase M: Multiple alliances and unknown partners☆
Introduction
Metallo-carboxypeptidases (CPs) hydrolyze a single amino acid from the C-terminus of peptides and proteins using a catalytic mechanism in which a nucleophilic attack on a peptide bond is mediated by a Zn-activated water molecule. Barrett, Rawlings and Woesner classified the CPs into clan MC, family M14, subfamilies M14A and M14B (also known as the CPA/CPB and CPN/CPE subfamilies respectively) [1]. In the MEROPS data base, Clan MC, family M14 is currently subdivided into 3 subfamilies and a 4th has been proposed. In addition to the A and B subfamilies, there is peptidase subfamily M14C (gamma-d-glutamyl-(l)-meso-diaminopimelate peptidase I from Bacillus sphaericus, MEROPS Accession MER001505 peptidase unit: 101–396). The proposed M14D subfamily is for cytosolic CPs including Nna1/CCP1 and 5 additional CCPs (numbered 2–6) [2], [3]. The most studied members are human CPA1, CPA2, CPB and CPU in the M14A subfamily and CPN, CPE, CPM and CPD (with three CP domains, of which domain three does not exhibit proteolytic activity) in the M14B subfamily. A less studied member of the M14B subfamily is CPZ [4]. Although little is known about the function of CPZ, it is highly relevant to mention it here because, like CPM, it is extracellular and it cleaves the same type of substrates. Also belonging to the M14B subfamily are a group of homologous human proteins that do not encode proteolytically active CPs because one or more critical amino acids are missing (CPX-1 [5], CPX-2 [6] and adipocyte enhancer binding protein [4], [7], [8]). The CPs are alternatively classified using parameters defining function or substrate specificity. Although CPU has no digestive function, subfamily M14A is regularly referred to as the ‘digestive’ subfamily. Subfamily M14B includes a number of CPs that are important for the activation or inactivation of biological mediators and peptide hormones, hence, this subfamily is described as the ‘regulatory’ subfamily [9], [10], [11]. CPA-type enzymes preferentially remove C-terminal aliphatic and hydrophobic residues from peptide/protein substrates. CPB-type CPs, such as CPM, exhibit a strict specificity for removing the C-terminal basic amino acids Arg and Lys from peptides/proteins and are therefore referred to as ‘basic’ CPs [9].
The renewed interest in CPs originates from recent advances in the development of CPU inhibitors. Since it was recognized that this enzyme is an important attenuator of the fibrinolytic rate, the medical and pharmaceutical industry is interested in CPU inhibitors as novel fibrinolytic agents. The CPU crystal structure [12] will probably be an important aid for the drug design. Already a number of potent CPU inhibitors (nM) were published that do not interfere with the activity of CPN which is regarded as the representative for the type-B CPs in these studies [13], [14], [15], [16]. However it is important to consider inhibition of CPM and related basic CPs by CPU inhibitors. Our work and that of others elucidated important differences in P1′ [10] and P1 [17], [18], [19], [20] substrate specificity between members of both carboxypeptidase subfamilies. In the CPB-type subfamily CPN, CPD-II and CPE prefer a P1′ Lys while CPU, CPB, CPM and CPD-I prefer an Arg residue in this place [10]. In contrast to CPU, CPM is constitutively expressed and situated at the cell membrane [21], [22], [23] where a lot of processes generate Arg/Lys C-terminal substrates such as the complement activation system, coagulation/fibrinolysis and adhesion (for example plasminogen binding). A very common phenomenon observed in monoclonal antibodies and therapeutic proteins is the C-terminal Lys or Arg variant [24]. Even though the in vivo effect of this modification it is not always established, the degree of heterogeneity should be monitored for product consistency [25].
In addition, a lot of new information has emerged in the last decade from genomic and proteomic approaches on regulated expression of CPM in different conditions. A new and unexpected finding is the expression of CPM on mesenchymal stem cells and its major upregulation during adipogenesis and osteogenesis. In this review we summarize this relatively new information on the distribution, expression and regulation of CPM. Taking into account the recent advances in the CPM research we see reasons to revise previous hypotheses on the role of CPM in homeostasis and pathophysiology.
Section snippets
History
Until the early 1980s, pancreatic carboxypeptidase CPB and plasma CPN were the only two known mammalian metallo-type basic CPs. Around 1984, there were indications for the existence of a membrane-bound member of this subfamily in various tissues including kidney, placenta and lung [26]. Around the same time two monoclonal mouse antibodies (MAX.1 and MAX.11) were found to detect a macrophage differentiation antigen [27]. Skidgel et al. purified the new CP from seminal plasma in 1988 [28] and
Current insights in the function of CPM
Roles for CPM may be deduced from its endogenous substrates, distribution in the human body and expression in states of disease. Based on a limited number of inflammatory substrates such as anaphylatoxins and kinins and the presence of CPM on mature and activated macrophages a role in the immune system has been postulated. However, at present the data are far from complete. Therefore the main challenge for future research on the physiological role of CPM is the identification of relevant
Conclusions and future perspectives
Since the discovery of CPM more than twenty years ago researchers have studied the biochemical and functional properties of this protease. Still, many questions remain. A first issue is its name. CPM was denoted ‘M’ because of its membrane bound feature but soluble forms have been found in human body fluids [26], [28], [51]. Until today however it remains a mystery how the protein is shed in vivo. In addition it was reported that the release of CPM from the cell surface by phosphatidylinositol
Acknowledgements
This work was supported by the Research Foundation-Flanders (Belgium) [F.W.O.-Vlaanderen]. K.D. is a research assistant of the F.W.O.-Vlaanderen.
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Binding to carboxypeptidase M mediates protective effects of fibrinopeptide Bβ<inf>15-42</inf>
2019, Translational ResearchCitation Excerpt :We found that the key effects of Bβ15–42 in tubular cells, ie, increase in cell migratory capacity, enhanced monolayer permeability and attenuated cytotoxic stress marker expression, were CBPM dependent. CBPM is a monomeric metalloprotease which is constitutively expressed on the outer surface of a variety of cell types in many organs.25 CBPM hydrolyses the C-terminal basic arginine or lysine of various peptides, including BK, anaphylatoxins and growth factors to either inactivate them or to increase their specific binding capacity for their respective receptors.
Enkephalin related peptides are released from jejunum wall by orally ingested bromelain
2019, PeptidesCitation Excerpt :This substrate specificity of stem bromelain cleaving after a pair of basic amino acids reminds those of the central nervous system prohormone convertases 1 and 2 (PC1 and PC2) to generate enkephalin from proenkephalin (PENK) cleaving this protein and synthetic peptides derived from it specifically and exclusively after pairs of basic amino acid residues KR, KK and RR (K = lysin) [13–17]; for review [18,19]. The cleavage of PENK by PC1 and PC2 generates enkephalin containing at its C-terminal end a pair of basic amino acids that requires a carboxypeptidase to produce opioid-active enkephalins, as carboxypeptidase E [20] carboxypeptidase D [21] or carboxypeptidase M [22]. It is well demonstrated that proenkephalin (PENK) is expressed in the neuronal tissue but it is also present in the gastrointestinal tract [23–25].
A step closer in defining glycosylphosphatidylinositol anchored proteins role in health and glycosylation disorders
2018, Molecular Genetics and Metabolism ReportsActivity of human kallikrein-related peptidase 6 (KLK6) on substrates containing sequences of basic amino acids. Is it a processing protease?
2017, Biochimica et Biophysica Acta - Proteins and ProteomicsCitation Excerpt :The cleavage after the KR pair of basic amino acids were the preferential sites of cleavages in the four FRET peptides as observed following the products of hydrolysis by HPLC – mass spectrometry, and KR is the more frequent dibasic motif flanking the enkephalins in the human proenkephalin sequence. The complete release of enkephalin requires the activity of a carboxypeptidase to remove the C-terminal basic amino acid from each peptide released by KLK6 and a possible candidate is carboxypeptidase M due its preference for basic residues and also its presence in CNS [52] as reviewed in [53,54]. Therefore, similar to hog pancreatic kallikrein [26] and HPK [27], enkephalin can be released from human proenkephalin by KLK6, particularly in the CNS.
Carboxypeptidase M is a positive allosteric modulator of the kinin B1 receptor
2013, Journal of Biological ChemistryCitation Excerpt :They are distinguished from the CPA/B (M14A) subfamily by the lack of a propeptide activation sequence and the presence of an additional C-terminal domain consisting of a seven-stranded β barrel with topological similarity to the plasma protein transthyretin (2, 6, 7). CPM is an ectoenzyme anchored to the plasma membrane of cells by a glycosylphosphatidylinositol (GPI) anchor (8, 9) and is widely distributed in the body, for example in lung and placental microvilli, kidney, blood vessels, intestine, brain, and peripheral nerves and can be found in soluble form in various body fluids (1, 10, 11). CPM has a strict specificity, cleaving only C-terminal Arg or Lys residues (12), and although >60 endogenous human peptides or proteins have been identified as potential CPM substrates, only 22 have been tested and shown to have altered activity after C-terminal cleavage (10).
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This work was supported by the Research Foundation-Flanders (Belgium) [F.W.O.-Vlaanderen]. K.D. is a research assistant of the F.W.O.-Vlaanderen.