Carboxypeptidase N Research Articles

Two carboxypeptidase counterparts from rock bream (Oplegnathus fasciatus): Molecular characterization, genomic arrangement and immune responses upon pathogenic stresses

Carboxypeptidases (CPs) are proteases that hydrolyze C-terminal peptide bonds. They are involved in regulating the complement system of the immune system. Here, we report the molecular characterization and immune response of two carboxypeptidases, named carboxypeptidase A (Rb-CPA) and carboxypeptidase N1 (Rb-CPN1), from rock bream. The genomic sequence of Rb-CPA contains 12 exons interrupted by 11 introns, while the genomic sequence of Rb-CPN1 has 9 exons and 8 introns. The cDNA sequence of Rb-CPA encodes a 421-amino-acid (AA) polypeptide (48kDa), and the cDNA of Rb-CPN1 encodes a 448-AA polypeptide (51kDa). The amino acid sequences of Rb-CPA and Rb-CPN1 were found to harbor two characteristic Zn-binding signature domains and a peptidase-M14 Zn carboxypeptidase site. Pairwise analysis revealed that Rb-CPA and Rb-CPN1 had the highest identity with the corresponding proteins from Anoplopoma fimbria (87.6%) and Dicentrarchus labrax (96.9%), respectively. qPCR results indicated that Rb-CPA and Rb-CPN1 were constitutively expressed mainly in the kidney, heart, liver, and head kidney. Both genes were transcriptionally regulated in the liver upon challenge with pathogenic bacteria (Streptococcus iniae, Edwardsiella tarda), rock bream iridovirus (RBIV), and the immune modulators polyinosinic:polycytidylic acid and lipopolysaccharide. Taken together, our findings suggest that Rb-CPA and Rb-CPN1 have immune-related functions in rock bream.

Abstract 2507: Circulating proteolytic products of carboxypeptidase N for early detection of breast cancer

Abstract Background: Carboxypeptidase N (CPN) plays an important role in regulating vasoactive peptide hormones, growth factors, and cytokines by specifically cleaving their C-terminal basic residues. Herein we demonstrated that the circulating peptides specifically cleaved by CPN in the tumor microenvironment can indeed be stage-specific indicators of breast cancer. Methods: The activity of CPN was evaluated using an ex-vivo peptide cleavage assay, in which synthesized C3f peptide (His6-C3f_S1304-R1320-His6) is incubated in interstitial fluids of breast tumor and adjacent normal breast tissues in mice with orthotopic implantation of the human cell line MDA-MB-231. Fragment profiling, by an approach combining nanopore fractionation and mass spectrometry, revealed the nature and extent of cleavage by CPN. These results correlated with the level of CPN-catalyzed peptides in blood specimens taken from the tumor-bearing mice, healthy women and breast cancer patients. CPN expression in the same set of samples was further examined by immunohistochemistry and immunoblotting. Results: We showed that generation of C3f_R1310-L1319 specifically correlated with the CPN expression level. In both the mouse and clinical patient samples, the amount of CPN was clearly increased in tumor tissues compared to that seen in normal breast tissue, while its counterpart in blood remained constant. The amount of 6 CPN-catalyzed peptides predominantly increased in sera taken from the mice (n=8) at 2 weeks after orthotropic implantation. Six homologous peptides displayed the significantly higher expression in the patients' plasma as early as the first pathologic stage of breast cancer. Conclusions: The amount of circulating CPN-catalyzed peptides reported here reflects the extent of this enzyme's activity in tumors, and our results clearly indicate their strong potential as biomarkers for non-invasive and early diagnosis of breast cancer. It would certainly be naïve to think that CPN-catalyzed peptides and the enzyme itself are the only biomarkers specific for breast cancer diagnosis. We do however argue that they can be used as a complementary measure for very early diagnosis of tumor growth, particularly because CPN activity within the tumor at this point is already detectable by blood sampling. Such sensitivity together with all of the features mentioned above lend well to their translation to the clinic, for breast cancer and perhaps other pathologies. Citation Format: Ye Hu. Circulating proteolytic products of carboxypeptidase N for early detection of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2507. doi:10.1158/1538-7445.AM2014-2507

Abstract LB-247: Circulating proteolytic products of tumor-resident enzyme as potential biomarkers for early detection of breast cancer

Abstract In theory, any or all of tumor-secreted proteins can serve as cancer biomarkers. However, the reality is challenging to monitor because of the large degree of fluctuation in abundance and localization of these tumor-secreted proteins, especially in the early stage of tumor development and/or metastasis. As such, it seems feasible that we might take advantage of the fact that secreted proteases/peptidases in the tumor microenvironment generate proteolytic products, also referred to as “circulating peptides”, which are detectable in bloodstream and provide ample information about the body, “coded” in the patterns and quantity of these peptides. Herein we clearly link the catalytic activity of Carboxypeptidase N (CPN) to its proteolytic products during breast tumor progression in mouse model and clinical samples. CPN plays important roles in regulating vasoactive peptide hormones, growth factors, and cytokines by specifically cleaving their C-terminal basic residues. Circulating fragment profiling, by an approach combining nanopore fractionation and mass spectrometry, revealed the nature and extent of cleavage by CPN. These results correlated with the level of CPN-catalyzed peptides in blood taken from the tumor-bearing mice, healthy women and breast cancer patients. We showed that generation of C3f_R1310-L1319 specifically correlated with the CPN expression level. In both mouse and clinical patient samples, the amount of CPN was increased in tumor tissues compared to that seen in normal breast tissue, while its counterpart in blood remained constant. The amount of 6 CPN-catalyzed peptides predominantly increased in sera taken from both the mice at 2 weeks after orthotropic implantation and the patients’ plasma as early as the first pathologic stage of breast cancer. In conclusion, the circulating level of the selected 6 CPN-catalyzed proteolytic products reflects the extent of this enzyme's activity in tumors, and our results clearly indicate their strong potential as biomarkers for non-invasive early diagnosis of breast cancer. Citation Format: Ye Hu. Circulating proteolytic products of tumor-resident enzyme as potential biomarkers for early detection of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-247. doi:10.1158/1538-7445.AM2014-LB-247

Proteolytic activity of fetoplacental complex in norm and pathology

The activity of angiotensin converting enzyme (ACE), carboxypeptidase N (CPN), and leucine aminopeptidase (LAP) has been investigated in the fetoplacental complex (FPC) in normal and placental insufficiency (FPI). ACE and LAP activities were significantly higher in the placental tissue than in maternal serum and umbilical vein serum. CPN activity was significantly lower in umbilical vein serum as compared to that of women in childbirth. Probably, the studied enzymes are involved in formation of reduced sensitivity of FPC of blood vessels during physiological pregnancy. In cases of placental insufficiency a significant increase of LAP activity was found in the placental tissue and umbilical vein serum. In addition, the pathological course of pregnancy caused a significant increase of CPN activity in serum of pregnant women in comparison to the norm. The obtained data suggest that during FPI proteolytic enzymes participate in the formation of compensatoty-adaptive reactions in the FPC. Results of this study are interesting in context of development of methods for prevention and correction of metabolic disorders in pathologies of pregnancy.

Carboxypeptidase N-deficient mice present with polymorphic disease phenotypes on induction of experimental autoimmune encephalomyelitis

Carboxypeptidase N (CPN) is a member of the carboxypeptidase family of enzymes that cleave carboxy-terminal lysine and arginine residues from a large number of biologically active peptides and proteins. These enzymes are best known for their roles in modulating the activity of kinins, complement anaphylatoxins and coagulation proteins. Although CPN makes important contributions to acute inflammatory events, little is known about its role in autoimmune disease. In this study we used CPN−/− mice in experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. Unexpectedly, we observed several EAE disease phenotypes in CPN−/− mice compared to wild type mice. The majority of CPN−/− mice died within five to seven days after disease induction, before displaying clinical signs of disease. The remaining mice presented with either mild EAE or did not develop EAE. In addition, CPN−/− mice injected with complete or incomplete Freund's adjuvant died within the same time frame and in similar numbers as those induced for EAE. Overall, the course of EAE in CPN−/− mice was significantly delayed and attenuated compared to wild type mice. Spinal cord histopathology in CPN−/− mice revealed meningeal, but not parenchymal leukocyte infiltration, and minimal demyelination. Our results indicate that CPN plays an important role in EAE development and progression and suggests that multiple CPN ligands contribute to the disease phenotypes we observed.

Peptidase activity of the fetoplacental complex in the norm and pathology

The activity of angiotensin converting enzyme (ACE), carboxypeptidase N (CPN), and leucine aminopeptidase (LAP) has been investigated in the fetoplacental complex (FPC) in norm and fetoplacental insufficiency (FPI). ACE and LAP activities were significantly higher in the placental tissue than in maternal serum and umbilical vein serum. CPN activity was significantly lower in umbilical vein serum as compared to that of women in childbirth. Probably, the studied enzymes are involved in formation of reduced vascular sensitivity of FPC during physiological pregnancy. In cases of placental insufficiency a significant increase of LAP activity was found in the placental tissue and umbilical vein serum. In addition, the pathological course of pregnancy caused a significant increase of CPN activity in serum of pregnant women in comparison to the norm. The obtained data suggest that during FPI proteolytic enzymes participate in the formation of compensatory-adaptive reactions in the FPC. Results of this study are interesting in the context of development of methods for prevention and correction of metabolic disorders in pathologies of pregnancy.

Binding of carboxypeptidase N to fibrinogen and fibrin

The ultimate step in the blood coagulation cascade is the formation of fibrin. Several proteins are known to bind to fibrin and may thereby change clot properties or clot function. Our previous studies identified carboxypeptidase N (CPN) as a novel plasma clot component. CPN cleaves C-terminal lysine and arginine residues from several proteins. The activity of CPN is increased upon its proteolysis by several proteases. The aim of this study is to investigate the presence of CPN in a plasma clot in more detail. Plasma clots were formed by adding thrombin, CaCl2 and aprotinin to citrated plasma. Unbound proteins were washed away and non-covalently bound proteins were extracted and analyzed with 2D gel electrophoresis and mass spectrometry. The identification of CPN as a fibrin clot-bound protein was verified using Western blotting. Clot-bound CPN consisted of the same molecular forms as CPN in plasma and its content was approximately 30ng/ml plasma clot. Using surface plasmon resonance we showed that CPN can bind to fibrinogen as well as to fibrin. In conclusion, CPN binds to fibrinogen and is present in a fibrin clot prepared from plasma. Because CPN binds to a fibrin clot, there could be a possible role for CPN as a fibrinolysis inhibitor.

Extraction of Peptidase Substrates by the Isolated Perfused Rat Lung

Peptidases in the lung are well placed to have an important role in regulating levels of circulating endogenous and therapeutic peptides. They also present a first-pass metabolic barrier for peptides delivered to the lung for systemic absorption. The activities of five peptidases were surveyed in the pulmonary circulation of the asanguinous isolated perfused rat lung (IPRL) using synthetic substrates and selective inhibitors. Extraction ratios (ER) were calculated for the substrates of: aminopeptidase N (AMN), ER 0·37 ± 0·04; dipeptidyl peptidase IV (DPP), ER 0·69 ± 0·05; and angiotensin-converting enzyme (ACE), ER 0·40 ± 0·02. These activities were inhibited (> 95%) by the selective inhibitors bestatin, diprotin A, and captopril, respectively. Substrates for neutral endopeptidase 24.11 (NEP) and carboxypeptidase M (CPM) were minimally degraded with ER of 0·02 and 0·00, respectively. The low activity of NEP, a major membrane bound endopeptidase, indicates that the lungs may not contribute greatly to degradation of either systemic NEP substrates, or exopeptidase-resistant peptides absorbed from the peripheral lung. In contrast peptides susceptible to the exopeptidases AMN, DPP, and ACE, will be substantially degraded during passage through the lung. The absence of CPM activity in the pulmonary circulation of the asanguinous IPRL implies that basic carboxypeptidase activity in blood perfused lungs reported in the literature is more likely to be the result of plasma carboxypeptidase N activity.

Measurement of carboxypeptidase U (active thrombin-activatable fibrinolysis inhibitor) in plasma: Challenges overcome by a novel selective assay

This article introduces a novel assay for the measurement of carboxypeptidase U (CPU) in plasma using the selective CPU substrate Bz-o-cyano-Phe-Arg (N-benzoyl-ortho-cyano-phenylalanyl-arginine), thereby limiting the interference of plasma carboxypeptidase N (CPN) as well as the intrinsic activity of procarboxypeptidase U (proCPU). A limit of detection of 0.05U/L (10pM) was reached. In addition, the current assay has the advantage of being easy to perform and shows excellent linearity and variability, rendering it a useful tool in the screening of samples for the presence of CPU in several patient populations and encouraging in-depth exploration of the pathophysiological role of the proCPU/CPU system.

Prochemerin Is a New Substrate for Thrombin.

Abstract 3591Poster Board III-528 IntroductionProchemerin is a 163 amino acid precursor protein with a C-terminal domain highly susceptible to proteolysis. Its chemotactic activity is unmasked upon C-terminal cleavage by proteases of the coagulation, fibrinolytic and inflammatory systems. Here, we studied whether thrombin is able to cleave prochemerin to generate active forms of chemerin. MethodsThe 15mer peptide prochemerin C-terminal sequence (YFPGQFAFSKALPRS) or prochemerin was incubated with thrombin at different concentrations and times. The reactions were stopped by addition of PPACK before determining their chemotactic activity in a transwell assay using CMKLR1-transfected cells and their mass by mass-spectrometry. ResultsThrombin (0-100 nM) dose-dependently cleaved 15mer to 14mer (YFPGQFAFSKALPR). Over a longer reaction time, the 10mer (YFPGQFAFSK) was also detected. The 15mer was almost inert in the chemotaxis assay but thrombin-cleaved 15mer caused migration of CMKLR1 transfectants. The 14mer and 10mer at 1 μM induced CMKLR1 cell migration at a rate of 3200 and 2800 cells/ml, but 1 μM 15mer did not induce any chemotaxis. Thrombin can also cleave the 14mer to a 10mer as determined by mass spectrometry. Using selected thrombin mutants for the Na+ binding site (E229K) and the active site YPPW-insertion loop (W50A), we found that thrombin hydrolysis of the 15mer was dependent on the Na+ bound “fast” form of thrombin and active site topology. The 10mer could be further activated by carboxypeptidase N (CPN) by removing the C-terminal lysine, whereas the C-terminal arginine of 14mer could not be cleaved by CPN, which did not affect its activity. Full-length prochemerin was also activated by thrombin (100nM) and the chemotactic activity further increased by CPN (50nM) about 6 fold. ConclusionsProchemerin is a new substrate for thrombin. Thrombin-cleaved chemerins are active chemoattractants in chemotaxis. This extends the molecular link between blood coagulation and CMKLR1-mediated immune responses. Disclosures:No relevant conflicts of interest to declare.

Development of a sensitive and selective assay for the determination of procarboxypeptidase U (thrombin-activatable fibrinolysis inhibitor) in plasma

To date, several assays for procarboxypeptidase U (proCPU) determination exist, all having their own inherent disadvantages and advantages. A drawback of activity-based assays is the interference of the constitutively active carboxypeptidase N (CPN) in plasma. Recent screening of Bz- Xaa-Arg peptides with modified aromatic amino acids at the P1 position revealed a selective CPU substrate, N-benzoyl- ortho-cyano-phenylalanyl-arginine (Bz- o-cyano-Phe-Arg), which will allow straightforward determination of proCPU in plasma. Our assay shows an excellent linearity in the concentration range of 20–2600 U/L, with within- and between-run precision values of 2.7% and 4.6%, respectively. A good correlation with our high-performance liquid chromatography (HPLC)-assisted proCPU activity assay using hippuryl- l-arginine (HipArg) as substrate was found. Besides the major improvement regarding the selectivity, the assay is much easier to perform and far less time-consuming compared with the proCPU activity assay using HipArg as substrate.

Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (Activated Thrombin-activable Fibrinolysis Inhibitor), and Platelets

Chemerin is a potent chemoattractant for cells expressing the serpentine receptor CMKLR1 (chemokine-like receptor 1), such as plasmacytoid dendritic cells and tissue macrophages. The bioactivity of chemerin is post-translationally regulated; the attractant circulates in blood in a relatively inactive form (prochemerin) and is activated by carboxyl-terminal proteolytic cleavage. We discovered that plasma carboxypeptidase N (CPN) and B (CPB or activated thrombin-activable fibrinolysis inhibitor, TAFIa) enhanced the bioactivity of 10-mer chemerin peptide NH2-YFPGQFAFSK-COOH by removing the carboxyl-terminal lysine (K). Sequential cleavages of either a prochemerin peptide (NH2-YFPGQFAFSKALPRS-COOH) or recombinant full-length prochemerin by plasmin and CPN/CPB substantially increased their chemotactic activities. Endogenous CPN present in circulating plasma enhanced the activity of plasmin-cleaved prochemerin. In addition, we discovered that platelets store chemerin protein and release it upon stimulation. Thus circulating CPN/CPB and platelets may potentially contribute to regulating the bioactivity of leukocyte chemoattractant chemerin, and further extend the molecular link between blood coagulation/fibrinolysis and CMKLR1-mediated immune responses.

Proteolytic cleavage of carboxypeptidase N markedly increases its antifibrinolytic activity.

Carboxypeptidase N (CPN) is a constitutively active basic carboxypeptidase sharing specificity with activated thrombin-activable fibrinolysis inhibitor (TAFIa). Generally, CPN is regarded as being non-antifibrinolytic. However, this assumption has not been thoroughly investigated, particularly with respect to long-term antifibrinolysis. In addition, a recent report has shown that plasmin cleavage increases the catalytic activity of CPN. Therefore, we investigated the antifibrinolytic properties of CPN and plasmin-cleaved CPN (CPNc). CPN was incubated with plasmin for various periods of time and the prolongation of clot lysis at various concentrations of CPN/CPNc mixture was investigated in TAFI-depleted plasma. CPN cleavage was analyzed by electrophoresis and catalytic activity was determined by monitoring cleavage of the small substrate, FA-Ala-Lys. CPN exhibited antifibrinolytic properties in plasma clot lysis assays when present at supraphysiological concentrations. Depletion of CPN from plasma decreased the lysis time of clots formed from minimally diluted plasma at low tissue-type plasminogen activator (t-PA) concentrations. Plasmin cleavage of CPN markedly increased the antifibrinolytic properties. CPN and CPNc prolonged lysis in a non-saturable, dose-dependent, and t-PA-dependent manner. At sufficient concentration, CPN and CPNc prolonged lysis at least forty-fivefold. CPNc was 700% more antifibrinolytic than CPN but only 7% more active toward FA-Ala-Lys. The active site inhibitor GEMSA eliminated the antifibrinolytic effects of CPN and CPNc. Antifibrinolytic activity correlated with cleavage of active and/or regulatory subunits, presumably generating heterodimeric CPNc. Limited proteolysis of CPN by plasmin generates an enzyme with greatly increased antifibrinolytic properties. We speculate that (patho)physiological proteolysis of CPN may generate a long-term antifibrinolytic enzyme.

Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (TAFIa) and Platelets.

Chemerin is a potent chemoattractant for cells that express the serpentine receptor CMKLR1 (chemokine-like receptor 1), such as plasmacytoid dendritic cells and tissue macrophages. Chemerin circulates in the blood in a relatively inactive form (prochemerin). Its chemotactic activity is unleashed following proteolytic cleavage of carboxyl-terminal amino acids by serine proteases including plasmin, factor XIIa and VIIa. Recruitment of plasmacytoid dendritic cells and macrophages by chemerin may play a role in local tissue immune and inflammatory responses. The shortest bioactive chemerin peptide NH2-YFPGQFAFS-COOH (9mer) is present in the carboxyl-terminal domain. In this work, we show that plasma carboxypeptidase N (CPN) and B (CPB or activated thrombin-activatable fibrinolysis inhibitor (TAFIa)) (30 nM) remove the C-terminal lysine (K) from YFPGQFAFSK (10mer) (200nM) and enhance the migration of CMKLR1-transfected cells by ∼16-fold. To investigate if sequential proteolysis by plasmin and carboxypeptidases can modulate the activity of chemerin peptides, we generated the carboxyl-terminal 15mer of prochemerin, NH2-YFPGQFAFSKALPRS-COOH. Plasmin cleavage (1 μM) generated a 10mer, which was further processed to 9mer by CPN or CPB (30 nM) cleavage. These sequential cleavages were paralleled by corresponding increases in chemotactic activity. At concentrations as high as 1 μM the 15 mer did not induce chemotaxis; after plasmin cleavage and conversion to 10mer, however, significant chemotactic activity was demonstrated. Treatment with CPN or CPB further enhanced this chemotactic activity. We observed a similar enhancement in bioactivity following sequential plasmin/CPN-or-CPB cleavage of full-length prochemerin. Endogenous plasma CPN supports the activation (∼2.5 fold increase in bioactivity) of plasmin-cleaved prochemerin (0.2 nM). This activation was blocked by incubating plasma with MGTA (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid), a specific CPN inhibitor. In addition, we show that platelets are a rich source of chemerin (20–30 ng of chemerin/5x108 platelets). Chemerin released from activated platelets triggers CMKLR1-transfectant chemotaxis, which was blocked by anti-chemerin antibodies. Thus, the circulating humoral factors reported here (platelets, serine proteases, and carboxypeptidases) may contribute to the regulation of chemerin bioactivity in vivo and therefore play a critical role in CMKLR1-mediated immune responses.

Discovery of Potent & Selective Inhibitors of Activated Thrombin-Activatable Fibrinolysis Inhibitor for the Treatment of Thrombosis

Thrombin-activatable fibrinolysis inhibitor (TAFI) has emerged as a key link between the coagulation and fibrinolysis cascades and represents a promising new target for the treatment of thrombosis. A novel series of imidazolepropionic acids has been designed that exhibit high potency against activated TAFI (TAFIa) and excellent selectivity over plasma carboxypeptidase N (CPN). Structure activity relationships suggest that the imidazole moiety plays a key role in binding to the catalytic zinc of TAFIa, and this has been supported by crystallographic studies using porcine pancreatic carboxypeptidase B as a surrogate for TAFIa. The SAR program led to the identification of 21 (TAFIa Ki = 10 nM, selectivity TAFIa/CPN > 1000) as a candidate for clinical development. Compound 21 exhibited antithrombotic efficacy in a rabbit model of venous thrombosis, yet had no effect on surgical bleeding in the rabbit. In addition, 21 exhibited an excellent preclinical and clinical pharmacokinetic profile, characterized by paracellular absorption, low clearance, and a low volume of distribution, fully consistent with its physicochemical properties of low molecular weight (MW = 239) and high hydrophilicity (log D = -2.8). These data indicate 21 (UK-396,082) has potential as a novel TAFIa inhibitor for the treatment of thrombosis and other fibrin-dependent diseases in humans.

Comparative substrate specificity study of carboxypeptidase U (TAFIa) and carboxypeptidase N: Development of highly selective CPU substrates as useful tools for assay development

BackgroundMeasurement of procarboxypeptidase U (TAFI) in plasma by activity-based assays is complicated by the presence of plasma carboxypeptidase N (CPN). Accurate blank measurements, correcting for this interfering CPN activity, should therefore be performed. A selective CPU substrate will make proCPU determination much less time-consuming. MethodsWe searched for selective and sensitive CPU substrates by kinetic screening of different Bz-Xaa-Arg (Xaa=a naturally occurring amino acid) substrates using a novel kinetic assay. ResultsThe presence of an aromatic amino acid (Phe, Tyr, Trp) resulted in a fairly high selectivity for CPU which was most pronounced with Bz-Trp-Arg showing a 56-fold higher kcat/Km value for CPU compared to CPN. Next we performed chemical modifications on the structure of those aromatic amino acids. This approach resulted in a fully selective CPU substrate with a 2.5-fold increase in kcat value compared to the commonly used Hip-Arg (Bz-Gly-Arg). DiscussionWe demonstrated significant differences in substrate specificity between CPU and CPN that were previously not fully appreciated. The selective CPU substrate presented in this paper will allow straightforward determination of proCPU in plasma in the future.

Crystal Structure of the Human Carboxypeptidase N (Kininase I) Catalytic Domain

Human carboxypeptidase N (CPN), a member of the CPN/E subfamily of “regulatory” metallo-carboxypeptidases, is an extracellular glycoprotein synthesized in the liver and secreted into the blood, where it controls the activity of vasoactive peptide hormones, growth factors and cytokines by specifically removing C-terminal basic residues. Normally, CPN circulates in blood plasma as a hetero-tetramer consisting of two 83 kDa (CPN2) domains each flanked by a 48 to 55 kDa catalytic (CPN1) domain. We have prepared and crystallized the recombinant C-terminally truncated catalytic domain of human CPN1, and have determined and refined its 2.1 Å crystal structure. The structural analysis reveals that CPN1 has a pear-like shape, consisting of a 319 residue N-terminal catalytic domain and an abutting, cylindrically shaped 79 residue C-terminal β-sandwich transthyretin (TT) domain, more resembling CPD-2 than CPM. Like these other CPN/E members, two surface loops surrounding the active-site groove restrict access to the catalytic center, offering an explanation for why some larger protein carboxypeptidase inhibitors do not inhibit CPN. Modeling of the Pro-Phe-Arg C-terminal end of the natural substrate bradykinin into the active site shows that the S1′ pocket of CPN1 might better accommodate P1′-Lys than Arg residues, in agreement with CPN's preference for cleaving off C-terminal Lys residues. Three Thr residues at the distal TT edge of CPN1 are O-linked to N-acetyl glucosamine sugars; equivalent sites in the membrane-anchored CPM are occupied by basic residues probably involved in membrane interaction. In tetrameric CPN, each CPN1 subunit might interact with the central leucine-rich repeat tandem of the cognate CPN2 subunit via a unique hydrophobic surface patch wrapping around the catalytic domain–TT interface, exposing the two active centers.

Plasmin alters the activity and quaternary structure of human plasma carboxypeptidase N

Human CPN (carboxypeptidase N) is a tetrameric plasma enzyme containing two glycosylated 83 kDa non-catalytic/regulatory subunits that carry and protect two active catalytic subunits. Because CPN can regulate the level of plasminogen binding to cell surface proteins, we investigated how plasmin cleaves CPN and the consequences. The products of hydrolysis were analysed by activity assays, Western blotting, gel filtration and sequencing. When incubated with intact CPN tetramer, plasmin rapidly cleaved the 83 kDa subunit at the Arg457-Ser458 bond near the C-terminus to produce fragments of 72 and 13 kDa, thereby releasing an active 142 kDa heterodimer, and also cleaved the active subunit, decreasing its size from 55 kDa to 48 kDa. Further evidence for the heterodimeric form of CPN was obtained by re-complexing the non-catalytic 72 kDa fragment with recombinant catalytic subunit or by immunoprecipitation of the catalytic subunit after plasmin treatment of CPN using an antibody specific for the 83 kDa subunit. Upon longer incubation, plasmin cleaved the catalytic subunit at Arg218-Arg219 to generate fragments of 27 kDa and 21 kDa, held together by non-covalent bonds, that were more active than the native enzyme. These data show that plasmin can alter CPN structure and activity, and that the C-terminal 13 kDa fragment of the CPN 83 kDa subunit is a docking peptide that is necessary to maintain the stable active tetrameric form of human CPN in plasma.

Carboxypeptidase N: a pleiotropic regulator of inflammation

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits (CPN1) and two large subunits (CPN2) that protect the protein from degradation. CPN cleaves carboxy-terminal arginines and lysines from peptides found in the bloodstream such as complement anaphylatoxins, kinins, and creatine kinase MM (CK-MM). By removing only one amino acid, CPN has the ability to change peptide activity and receptor binding. CPN is a member of a larger family of carboxypeptidases, many of which also cleave arginine and lysine. Because of the highly conserved active sites and the possible redundant functions of carboxypeptidases, it has been difficult to elucidate the role of CPN in disease processes. The future use of gene ablation technology may be the most appropriate way to understand the function of CPN in vivo.

Modulation of procarboxypeptidase R (ProCPR) activation by complementary peptides to thrombomodulin.

We designed complementary peptides (C-peptides) using a novel computer program (MIMETIC), which generates a series of peptides designed to interact with a target peptide sequence. Carboxypeptidase R (CPR) is an unstable basic carboxypeptidase found in fresh serum in addition to carboxypeptidase N (CPN) which is stable. CPR is generated from its precursor form (proCPR) by trypsin-like enzymes, and its activation is mediated by thrombin generated in the coagulation cascade. The efficiency of activation is enhanced approximately 1,200-fold when thrombin (T) is bound to thrombomodulin (TM). We attempted to generate C-peptides which recognize the T-binding site within TM assuming that some of these might interfere with the generation of T and TM complexes (T-TM). Among three peptides designed, two inhibited the enhancement in activation of proCPR by T in the presence of TM. One of the peptides at 16 microM reduced the activation of proCPR to the level obtained by T alone.