Crystal Structure of Human Carboxypeptidase M, A Membrane-bound Enzyme that Regulates Peptide Hormone Activity

Tan

Journal of Biological Chemistry

et al. 2008

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Kinin B1 receptor (B1R) expression is induced by injury or inflammatory mediators, and its signaling produces both beneficial and deleterious effects. Kinins cleaved from kininogen are agonists of the B2R and must be processed by a carboxypeptidase to generate B1R agonists des-Arg 9 -bradykinin or desArg 10 Carboxypeptidase M (CPM) 2 was discovered as a glycosylphosphatidylinositol (GPI)-anchored membrane protein with B-type carboxypeptidase activity (1-3) and is a member of the "regulatory" or carboxypeptidase N/E subfamily of metallocarboxypeptidases (4 -8) based on its cDNA sequence (9), genomic structure (10), and x-ray crystal structure (11). The overall structure of CPM is composed of a 295-residue N-terminal catalytic domain, followed by an 86-residue conical ␤-sandwich (transthyretin-like domain) and a unique 25-residue extension to which the GPI anchor is post-translationally attached (11).CPM cleaves only C-terminal Arg or Lys residues, and some of its endogenous substrates include bradykinin, anaphylatoxins C3a, C4a, and C5a, Arg-or Lys-enkephalins, epidermal growth factor, and hemoglobin (2, 7, 12, 13). CPM preferentially cleaves C-terminal Arg as exemplified by the kinetics with Arg 6 -Met 5 -enkephalin (K m ϭ 46 M, k cat ϭ 934 min Ϫ1 ) versus Lys 6 -Met 5 -enkephalin (K m ϭ 375 M, k cat ϭ 663 min Ϫ1 ) (2). Bradykinin exhibits the lowest K m (16 M) of any CPM substrate tested (2), a concentration that is still much higher than the typical physiological concentration of this peptide in the nanomolar range. However, peptidases in vivo typically work at substrate concentrations far below the K m as exemplified by angiotensin I-converting enzyme (ACE), which has the same relatively high K m (16 M) with angiotensin I (14), a major physiological substrate. The development of ACE inhibitors as effective agents for treating hypertension and cardiovascular diseases was based in large part on the critical role of this enzyme in converting angiotensin I to II in vivo (15).The kinin peptides bradykinin and kallidin (Lys-bradykinin) are generated by the proteolytic action of plasma or tissue kallikrein on high or low molecular weight kininogen (16,17). Removal of the C-terminal Arg from bradykinin or kallidin inactivates these peptides as agonists of the constitutively expressed B2 receptor (B2R) (16,17). However, this conversion is a required processing step to generate desArg 9 -bradykinin or des-Arg 10 -kallidin (16, 18), metabolites that have a variety of biological activities mediated by specific activation of a different B1 receptor (B1R) whose expression is induced by inflammatory mediators (19,20). Thus, CPM acts as a cell surface processing enzyme to generate des-Arg-kinin agonists for the B1R, and without this catalytic conversion, B1R signaling could not occur. This is of potential importance in inflammatory or pathological responses. For example, B1R activation stimulates extracellular signal-regulated kinase phosphorylation, prostaglandin production (20), and inducible nitric-oxide synthase-mediate...

Section: Effect Of Receptor Antagonists and Cpm Activity On B1r Signamentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Carboxypeptidase M and Kinin B1 Receptors Interact to Facilitate Efficient B1 Signaling from B2 Agonists

Tan

Journal of Biological Chemistry

et al. 2008

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International Immunopharmacology

“…Helices (α1 to α9) and β-strands (β1 to β15) are represented as golden helices and blue arrows, and the residual chain as a red rope. Also shown are the zinc ion cofactor inserted according to the CPM structure [52] (purple sphere), the two disulfide bridges (yellow stick model), the three glycosylated residues Thr380, Thr382 and Thr389 (orange stick models), and the O-linked proximal sugars (green stick models) in the TT domain. The figure was prepared with Pymol (http://pymol.sourceforge.net/).…”

Section: Hydrolysis Of Biologically Relevant Peptides In Vivomentioning

confidence: 99%

“…3). The structure of the CPN catalytic subunit is topologically similar to those of the duck CPD-2 fragment [51] and human CPM [52]. The polypeptide chain of the C-terminally truncated CPN catalytic subunit was traced in the final electron density to Ser398 except for nine residues around the Arg218-Arg219 proteasesensitive cleavage site (Fig.…”

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confidence: 97%

Structure and function of human plasma carboxypeptidase N, the anaphylatoxin inactivator

Skidgel

Erdös

2007

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Human carboxypeptidase N (CPN) was discovered in the early 1960s as a plasma enzyme that inactivates bradykinin and was identified 8 years later as the major "anaphylatoxin inactivator" of blood. CPN plays in important role in protecting the body from excessive buildup of potentially deleterious peptides that normally act as local autocrine or paracrine hormones. This review summarizes the structure, enzymatic properties and function of this important human enzyme, including insights gained by the recent elucidation of the crystal structure of the CPN catalytic subunit and structural modeling of the non-catalytic regulatory 83 kDa subunit. We also discuss its physiological role in cleaving substrates such as kinins, anaphylatoxins, creatine kinase, plasminogen receptors, hemoglobin and stromal cell-derived factor-1α (SDF-1α). HistoryCarboxypeptidases were initially isolated from pancreatic extracts and so were associated with protein and peptide degradation in the digestive tract [1][2][3][4]. However, work beginning in the early 1960's by Erdös and Sloane [5] revealed the presence of a novel carboxypeptidase in the blood that plays a regulatory role by inactivating bradykinin via removal of its C-terminal Arg residue. (Subsequent research some 2 decades later led to the discovery of a second kinin B 1 receptor activated by the carboxypeptidase metabolite that is its endogenous agonist -see below). Initially it was assumed that the enzyme represents a circulating form of pancreatic carboxypeptidase B, but this was disproved by the characterization of its enzymatic properties [5][6][7][8], which was later borne out by biochemical studies, sequence analysis [9][10][11][12][13][14][15] and ultimately X-ray crystallography [16]. To differentiate it from the pancreatic enzyme, it was named carboxypeptidase N (CPN; EC 3.4.17.3) and was the first member of a family of carboxypeptidases now known as the regulatory or CPN/E subfamily of metallocarboxypeptidases [17][18][19]. It has since been re-discovered many times as other substrates were found and so has acquired aliases such as creatine kinase conversion factor, plasma carboxypeptidase B, arginine carboxypeptidase, lysine carboxypeptidase and protaminase [8,17,20,21]. The most notable was the finding by Bokisch and Müller-Eberhard in 1969 -70 [22,23] that the human plasma "anaphylatoxin inactivator" is identical with CPN. This finding explains our interest in the work of Dr. Tony Hugli, who has made many seminal contributions to the understanding of the structure and function of anaphylatoxins [24][25][26][27]. In Send Correspondence and Proofs To: Randal A. Skidgel, PhD, Dept. of Pharmacology (M/C 868), Univ. of Illinois College of Medicine, 835 S. Wolcott, Chicago, IL 60612, Phone: 312-996-9179, FAX: 312-996-1648, EMAIL: E-mail: rskidgel@uic.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript w...

Structural and functional characterization of binding sites in metallocarboxypeptidases based on Optimal Docking Area analysis

et al. 2007

The metallocarboxypeptidases (MCPs) belonging to the clan MC were studied by the Optimal Docking Area (ODA) method to evaluate protein-protein binding sites and to provide a basis for the identification of binding partners for this class of enzymes. The ODA method identifies surface patches with optimal desolvation energy based on the selection of low-energy docking regions, generated from a set of surface points around the protein. With few exceptions, the ODA method identified surface patches with a significant low-energy docking surface for all the MCPs with known three-dimensional structure. Overall, in 14 out of 24 cases, the detected ODA patches were correctly located (i.e. more than 50% of the predicted residues were in known protein-protein binding sites), yielding a global success rate of 58%. More specifically, the success rate increased up to 80% on the ODA patches detected for the catalytic domains of the M14A subfamily, independently on the partner. Interestingly, the ODA residues on the catalytic domain were correctly located in the interface with the N-terminal pro domain in all MCPs. The spatial distribution of the ODA patches for the different members of the family is in relation to the origin and function of the particular MCP, which allowed distinguishing between them. In good agreement with the experimentally characterized protein interfaces, the total average surface area of the theoretically derived ODA patches for the catalytic domain of MCPs is around 1700 A2 and their content in hydrophobic residues is about 40%. As a particular case, the average surface area of the ODA patches in MCPs of crop insect pests is about twice that of the MCPs of vertebrates, which might be related to their particular function. We recognized two binding regions for the catalytic domain of the MCPs, one of them accounting for nearly all the known intermolecular interactions made up by the enzymes. Protein inhibitors seem to have evolved to dock on this subset of ODA patches, evoking the binding mode of the N-terminal pro domains. The second binding region detected, for which no ligands have been identified so far, seems to be related to the acquisition/maintenance of the native structure of the peptidase. Overall, the ODA method has been successful in identifying low-energy docking areas in a set of structurally and functionally related proteins, suggesting that it can be easily extended to other families in the search for protein-protein binding sites and for their functional significance.