Invited critical review
Carboxypeptidase M: Multiple alliances and unknown partners

https://doi.org/10.1016/j.cca.2008.10.003Get rights and content

Abstract

Carboxypeptidase M (EC 3.4.17.12) belongs to the family of the carboxypeptidases. These enzymes remove C-terminal amino acids from peptides and proteins and exert roles in the physiological processes of blood coagulation/fibrinolysis, inflammation, food digestion and pro-hormone and neuropeptide processing. Among the carboxypeptidases CPM is of particular importance because of its constitutive expression in an active form at the surface of specialized cells and tissues in the human body. Despite the fact that the function(s) of this enzyme is not fully understood several suggestions have been made since its discovery more than two decades ago. Based on potential substrates and its presence, often on the boundary between the host and environment, a role in inflammation was proposed. This review describes how recent discoveries affected the insights in the cellular and physiological functions of CPM. A critical analysis of the potential endogenous peptide and protein substrates is provided. The distribution of CPM on different cell types and tissues and its expression in states of disease are discussed. There is evidence that CPM functions not only as a protease but also as a binding partner in cell-surface protein–protein interactions.

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.

References (240)

  • R.J. Harris

    Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture

    J Chromatogr A

    (1995)
  • R.A. Skidgel et al.

    Hydrolysis of opioid hexapeptides by carboxypeptidase-N. Presence of carboxypeptidase in cell-membranes

    Biochem Pharmacol

    (1984)
  • F. Emmrich et al.

    Monoclonal antibodies against differentiation antigens on human macrophages

    Immunol Lett

    (1985)
  • R.A. Skidgel et al.

    Isolation and characterization of a basic carboxypeptidase from human seminal plasma

    Arch Biochem Biophys

    (1988)
  • F. Tan et al.

    Molecular cloning and sequencing of the cDNA for human membrane-bound carboxypeptidase M. Comparison with carboxypeptidases A, B, H, and N

    J Biol Chem

    (1989)
  • R.A. Skidgel et al.

    Human carboxypeptidase M. Purification and characterization of a membrane-bound carboxypeptidase that cleaves peptide hormones

    J Biol Chem

    (1989)
  • M. Rehli et al.

    Carboxypeptidase M is identical to the MAX.1 antigen and its expression is associated with monocyte to macrophage differentiation

    J Biol Chem

    (1995)
  • J. Li et al.

    Structure of the human carboxypeptidase M gene. Identification of a proximal GC-rich promoter and a unique distal promoter that consists of repetitive elements

    Gene

    (2002)
  • F. Tan et al.

    Human carboxypeptidase M

    Methods Enzymol

    (1995)
  • R.A. Skidgel et al.

    Purification of a human urinary carboxypeptidase (kininase) distinct from carboxypeptidase-A, carboxypeptidase-B, or carboxypeptidase-N

    Anal Biochem

    (1984)
  • G.B. McGwire et al.

    Carboxypeptidase M, a glycosylphosphatidylinositol-anchored protein, is localized on both the apical and basolateral domains of polarized Madin–Darby canine kidney cells

    J Biol Chem

    (1999)
  • X.Y. Li et al.

    Release of glycosylphosphatidylinositol-anchored carboxypeptidase M by phosphatidylinositol-specific phospholipase C upregulates enzyme synthesis

    Biochem Biophys Res Commun

    (1999)
  • F. Elortza et al.

    Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins

    Mol Cell Proteomics

    (2003)
  • X.M. Zhang et al.

    Carboxypeptidase M and kinin B1 receptors interact to facilitate efficient B1 signaling from B2 agonists

    J Biol Chem

    (2008)
  • S. Dhanvantari et al.

    Lipid raft association of carboxypeptidase E is necessary for its function as a regulated secretory pathway sorting receptor

    J Biol Chem

    (2000)
  • G.B. McGwire et al.

    Extracellular conversion of epidermal growth factor (EGF) to des-Arg53-EGF by carboxypeptidase M

    J Biol Chem

    (1995)
  • R.A. Skidgel

    Assays for arginine/lysine carboxypeptidases: carboxypeptidase H (E; enkephalin convertase), M, and N

  • J. Willemse et al.

    Development of a fast kinetic method for the determination of carboxypeptidase U (TAFIa) using C-terminal arginine containing peptides as substrate

    Anal Biochem

    (2005)
  • S.S. Mao et al.

    Electrochemiluminescence assay for basic carboxypeptidases: inhibition of basic carboxypeptidases and activation of thrombin-activatable fibrinolysis inhibitor

    Anal Biochem

    (2003)
  • R. Mentlein et al.

    Proteases involved in the metabolism of angiotensin II, bradykinin, calcitonin gene-related peptide (CGRP), and neuropeptide Y by vascular smooth muscle cells

    Peptides

    (1996)
  • J.L. Arolas et al.

    The three-dimensional structures of tick carboxypeptidase inhibitor in complex with A/B carboxypeptidases reveal a novel double-headed binding mode

    J Mol Biol

    (2005)
  • P. Aloy et al.

    The crystal structure of the inhibitor-complexed carboxypeptidase D domain II and the modeling of regulatory carboxypeptidases

    J Biol Chem

    (2001)
  • D. Reverter et al.

    Crystal structure of human carboxypeptidase M, a membrane-bound enzyme that regulates peptide hormone activity

    J Mol Biol

    (2004)
  • C. Keil et al.

    Crystal structure of the human carboxypeptidase N (kininase I) catalytic domain

    J Mol Biol

    (2007)
  • R.A. Skidgel

    Basic carboxypeptidases: regulators of peptide hormone activity

    Trends Pharmacol Sci

    (1988)
  • A. Zhou et al.

    Proteolytic processing in the secretory pathway

    J Biol Chem

    (1999)
  • R. Day et al.

    Prodynorphin processing by proprotein convertase 2. Cleavage at single basic residues and enhanced processing in the presence of carboxypeptidase activity

    J Biol Chem

    (1998)
  • B. de Saint-Vis et al.

    Distribution of carboxypeptidase M on lymphoid and myeloid cells parallels the other zinc-dependent proteases CD10 and CD13

    Blood

    (1995)
  • J.W. Baird et al.

    Differentiating embryonal stem cells are a rich source of haemopoietic gene products and suggest erythroid preconditioning of primitive haemopoietic stem cells

    J Biol Chem

    (2001)
  • B. Scheuerer et al.

    The CXC-chemokine platelet factor 4 promotes monocyte survival and induces monocyte differentiation into macrophages

    Blood

    (2000)
  • M. Kreutz et al.

    Retinoic acid inhibits monocyte to macrophage survival and differentiation

    Blood

    (1998)
  • S.W. Krause et al.

    Activation of lymphocytes inhibits human monocyte to macrophage differentiation

    Immunobiology

    (2001)
  • N.D. Rawlings et al.

    MEROPS: the peptidase database

    Nucleic Acids Res

    (2006)
  • E. Kalinina et al.

    A novel subfamily of mouse cytosolic carboxypeptidases

    FASEB J

    (2007)
  • M. Rodriguez de la Vega et al.

    Nna1-like proteins are active metallocarboxypeptidases of a new and diverse M14 subfamily

    FASEB J

    (2007)
  • S.E. Reznik et al.

    Carboxypeptidases from A to z: implications in embryonic development and Wnt binding

    Cell Mol Life Sci

    (2001)
  • Y.H. Lei et al.

    Identification of mouse CPX-1, a novel member of the metallocarboxypeptidase gene family with highest similarity to CPX-2

    DNA Cell Biol

    (1999)
  • X.N. Xin et al.

    Identification of mouse CPX-2, a novel member of the metallocarboxypeptidase gene family: cDNA cloning, mRNA distribution, and protein expression and characterization

    DNA Cell Biol

    (1998)
  • G.P. He et al.

    A eukaryotic transcriptional repressor with carboxypeptidase activity

    Nature

    (1995)
  • Handbook of proteolytic enzymes

    (1998)
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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.

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