Serum Carboxypeptidase N1 Serves as a Potential Biomarker Complementing CA15-3 for Breast Cancer.

BACKGROUND: The incidence and mortality of breast cancer are increasing annually. Breast cancer seriously threatens women's health and quality of life. We aimed to measure the clinical value of CPN1, a new serum marker of breast cancer and to evaluate the efficacy of CPN1 in combination with CA15-3. METHODS: Seventy samples of breast cancer with lymph node metastasis, seventy-three samples of nonmetastatic breast cancer and twenty-five samples of healthy human serum were collected. Serum CA15-3 concentration was determined by Roche Elecsys, and serum CPN1 concentration was determined by ELISA. RESULTS: In breast cancer patients, serum CPN1 concentration was positively correlated with tumour size, clinical stage and CA15-3 concentration (r = 0.376, P<0.0001). ROC curve analysis showed that the optimal critical concentration of CPN1 for breast cancer diagnosis was 32.8pg/ml. The optimal critical concentration of CPN1 in the diagnosis of metastatic breast cancer was 66.121pg/ml. CPN1 has a greater diagnostic ability for breast cancer (AUCCA15-3=0.702 vs. AUCCPN1=0.886, P<0.0001) and metastatic breast cancer (AUCCA15-3=0.629 vs. AUCCPN1=0.887, P<0.0001) than CA15-3, and the combined detection of CA15-3 and CPN1 can improve the diagnostic efficiency for breast cancer (AUCCA15-3+CPN1=0.916) and for distinguishing between metastatic and non-metastatic breast cancer (AUCCA15-3+CPN1=0.895). CONCLUSION: CPN1 can be used as a new tumour marker to diagnose and evaluate the invasion and metastasis of breast cancer. The combined detection of CPN1 and CA15-3 is more accurate and has a certain value in clinical application.

Fine-tune regulation of carboxypeptidase N1 controls vascular patterning during zebrafish development.

Vascular development is regulated by complicated signals and molecules in vertebrates. In this study, we characterized a novel function of carboxypeptidase N1 (Cpn1) in the vasculature. We show that cpn1 mRNA is expressed in developing vessels. The knockdown of cpn1 by morpholino injection impairs the growth of intersegmental vessels (ISV) and caudal vein plexus (CVP), suggesting the role of cpn1 in vascular development. We showed that vascular defects are not caused by cell death but are due to the impairment of migration and proliferation. Consistent with vascular growth defects, loss of cpn1 affects the expression of the vascular markers flt4, mrc1, flk, stabilin, and ephrinb2. Furthermore, the overexpression of cpn1 impaired the growth of ISV and CVP, but the remodeling expression of vascular markers was different from the knockdown of cpn1, indicating the differential regulation mechanisms in cpn1-overexpressing embryos. We examine the interaction between cpn1 and multiple signals and observed that cpn1 is regulated by Notch/VEGF signals for ISV growth and likely regulates BMP signals for CVP patterning. In conclusion, we demonstrate that cpn1 has a critical role in the vascular development of zebrafish. We also reveal a fine-tune regulation of cpn1 that controls vascular patterning mediated by multiple signals.

[Basic carboxypeptidases of blood: significance for coagulology].

This review considers the basic metallocarboxypeptidases of human blood and their role in coagulologic disorders. In includes information on the history of the discovery and biological characteristics of potential enzymes-regulators of the fibrinolytic process: carboxypeptidase U and carboxypeptidase N. Certain attention is paid to the biochemical mechanisms and the main modern concepts of the antifibrinolytic effects of these enzymes.

Distinct conditions support a novel classification for bradykinin-mediated angio-oedema.

BACKGROUND: Angio-oedema (AO) can be attributable to bradykinin (BK) accumulation, as is the case for prototypical hereditary AO (HAO) due to C1 inhibitor (C1-INH) deficiency. However, our clinical experience in a reference centre has shown that some patients display a clinical history suggestive of HAO, but exhibit normal C1-INH function, have no mutation in the causative genes associated with HAO (SERPING1, F12), and report no intake of drugs known to promote AO. OBJECTIVE: We sought to determine the frequency and distribution of different AO subtypes suspected to be BK-mediated AO (BK-AO) and defined by clinical, history and biological criteria (enzyme activities implicated in BK formation and catabolism). METHODS: The files of all patients referred to our centre for suspected BK-AO were retrospectively analysed. RESULTS: The distribution of patients (n = 162) was 16 and 4% with a hereditary deficiency of C1-INH or a gain of factor XII function, respectively, 29% with iatrogenic BK-AO, 21% with non-iatrogenic defective kininase activity and 30% with idiopathic increased kinin formation. CONCLUSION: BK-AO may be caused by multiple inherited or acquired factors triggering BK accumulation. Therefore, we propose a novel typology for BK-AO based on the imbalance of production/catabolism of BK.

[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.

Circulating proteolytic products of carboxypeptidase N for early detection of breast cancer.

BACKGROUND: Carboxypeptidase N (CPN) is important in regulating vasoactive peptide hormones, growth factors, and cytokines by specifically cleaving their C-terminal basic residues. We investigated whether circulating peptides specifically cleaved by CPN in the tumor microenvironment can be stage-specific indicators of breast cancer. METHODS: CPN activity was measured using an ex vivo peptide cleavage assay by incubating synthesized C3f peptide (His6-C3f_S1304-R1320-His6) in interstitial fluids of breast tumors and adjacent normal breast tissues in mice with orthotopic implantation of the human cell line MDA-MB-231. The nature and extent of peptide cleavage by CPN was investigated by fragment profiling using nanopore fractionation and mass spectrometry. The fragment profiles in interstitial fluid correlated with concentrations of CPN-catalyzed peptides in blood samples 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, CPN was clearly increased in tumor tissues compared with normal breast tissue, whereas corresponding CPN abundance in blood remained constant. Concentrations of 6 CPN-catalyzed peptides predominantly increased in sera taken from the mice (n = 8) at 2 weeks after orthotopic implantation. Six homologous peptides displayed significantly higher expression in the patients' plasma as early as the first pathologic stage of breast cancer. CONCLUSIONS: Circulating CPN-catalyzed peptide concentrations reflect the CPN activity in tumors. These biomarkers show strong potential for the noninvasive and early diagnosis of breast cancer.

Dietary intake increases serum levels of carboxymethil-lysine (CML) in diabetic patients / La ingesta dietaria de carboximetil-lisina (CML) aumenta los niveles plasmáticos de este compuesto en pacientes diabéticos

Introduction: Advanced glycation end products are produced endogenously, in association with hyperglycemia and oxidative stress. They can also be generated during cooking or food processing and, once absorbed, alter protein function and promote inflammation. Methods: We selected 40 healthy male subjects, 17 patients with type 2 diabetes of both sexes and 15 patients with type 1 diabetes of both sexes. Each participant underwent both a food frequency questionnaire (FFQ) and 24-hour dietary recall specially adapted for measuring CML intake, anthropometry, measurement of blood pressure and biochemical parameters in blood and urine. Results: Serum CML levels were significantly higher in patients with diabetes compared to healthy subjects (p 0.04), showing a direct relationship between dietary intake and serum levels of CML in T2D patients (r 0.53 p 0.03). sCML levels correlated positively with length of diabetes mellitus, and inversely with body mass index (BMI). The most important dietary factor contributing to raise CML levels in these patients with diabetes was the consumption of milk powder. Conclusion: Serum levels of CML were found to be higher among diabetic subjects, associated to length of diabetes as expected, but also with the ingestion of foods containing higher amounts of ML. The consumption of milk powder in this group is a major determinant of increased serum levels (AU)

Introducción: Los productos avanzados de la glicación se producen de forma endógena en relación con la hiperglucemia y el estrés oxidativo. También pueden generarse durante el cocinado o el procesamiento de los alimentos; una vez absorbidos, alteran la función proteica y favorecen la inflamación. Métodos: Seleccionamos a 40 hombres sanos, 17 pacientes con diabetes tipo 2 de ambos sexos y 15 pacientes con diabetes tipo 1 de ambos sexos. A cada participante se le realizó un cuestionario de frecuencia de consumo de alimentos (CFA) y un recordatorio de 24 horas especialmente adaptado para medir el consumo de CML, antropometría, medición de la presión sanguínea y parámetros bioquímicos en la sangre y la orina. Resultados: Las concentraciones séricas de CML fueron significativamente mayores en pacientes con diabetes en comparación con los individuos sanos (p = 0,04). Se encontró una relación directa entre el consumo dietético y las concentraciones séricas de CML en los pacientes con diabetes tipo 2 (r = 0,53; p = 0,03). Las concentraciones séricas de CML se correlacionan positivamente con la duración de la diabetes mellitus e inversamente con el índice de masa corporal (IMC). El alimento que más contribuye al aumento de las concentraciones plasmáticas de CML en estos pacientes fue el consumo de leche en polvo. Conclusión: Se encontró que las concentraciones séricas de CML eran mayores en los sujetos diabéticos, asociado con la duración de la diabetes, como era de esperar, pero también con la ingestión de alimentos que contienen mayores cantidades de CML. El consumo de leche en polvo en este grupo es un factor determinante en el aumento de las concentraciones séricas de CML (AU)

Deletion of carboxypeptidase N delays onset of experimental cerebral malaria.

Complement contributes to inflammation during pathogen infections; however, less is known regarding its role during malaria and in the severest form of the disease, cerebral malaria. Recent studies have shown that deletion of the complement anaphylatoxins receptors, C3aR and C5aR, does not alter disease susceptibility in experimental cerebral malaria (ECM). This does not, however, preclude C3a- and C5a-mediated contributions to inflammation in ECM and raises the possibility that carboxypeptidase regulation of anaphylatoxin activity rapidly over rides their functions. To address this question, we performed ECM using carboxypeptidase N-deficient (CPN(-/-)) mice. Unexpectedly, we found that CPN(-/-) mice survived longer than wild-type mice, but they were fully susceptible to ECM. CD4(+) and CD8(+) T cell infiltration was not reduced at the peak of disease in CPN(-/-) mice, and there was no corresponding reduction in pro-inflammatory cytokine production. Our results indicate that carboxypeptidases contribute to the pathogenesis of ECM and that studies examining the contribution of other carboxypeptidase families and family members may provide greater insight into the role these enzymes play in malaria.

Both B1R and B2R act as intermediate signaling molecules in high glucose-induced stimulation of glutamate uptake in ARPE cells.

Bradykinin (BK) is a potent modulator of biological processes in the retina, and retinal pigment epithelial cells (RPE) and the regulation of glutamate are believed to be important in the pathogenesis of diabetic retinopathy. However, the mechanism by which BK regulates glutamate uptake in RPE cells in diabetic retinopathy is unknown. Here, we examined the involvement of BK receptors in high glucose-induced dysfunction of glutamate uptake in human ARPE cells. High glucose stimulated glutamate uptake and the expression of excitatory amino acid transporter-4 (EAAT4) mRNA, and these were blocked by treatment with small interfering RNA (siRNA) for BK1 receptor (B1R) and BK2 receptor (B2R), but not scrambled siRNA, supporting an involvement of B1R and B2R in this process. High glucose-stimulated glutamate uptake was also blocked by the B1R antagonist [des-Arg(10)]-HOE 140 and the B2R antagonist HOE 140. High glucose increased B1R and B2R mRNA and protein expression in a time-dependent manner, increased B1R and B2R translocation from the cytosol to the nucleus, and stimulated kininogen, kallikrein, and kininase I mRNA expression. We examined whether BK receptors were involved in high glucose-induced signaling pathways. High glucose stimulated arachidonic acid release, cytosolic phospholipase A(2) and cyclooxygenase-2 proteins, nuclear factor-kappaB activation, and inhibitor-kappaB activation; these events were blocked by treatment with B1R and B2R siRNAs, but not scrambled siRNA. In addition, high glucose-induced stimulation of glutamate uptake was blocked by the cyclooxygenase-2 inhibitors arachidonyl trifluoromethyl ketone, mepacrine, 5-bromo-2-(4-fluorophenyl)-3-[4-(methyl-sulfonyl)phenyl]-thiophene, and N-[2-cyclohexyloxy-4-nitrophenyl] methane-sulfonamide, and by the nuclear factor-kappaB inhibitors pyrrolidine dithiocarbamate and SN-50.

A scrutiny of the biochemical pathways from Ang II to Ang-(3-4) in renal basolateral membranes.

In a previous paper we demonstrated that Ang-(3-4) counteracts inhibition of the Ca(2+)-ATPase by Ang II in the basolateral membranes of kidney proximal tubules cells (BLM). We have now investigated the enzymatic routs by which Ang II is converted to Ang-(3-4). Membrane-bound angiotensin converting enzyme, aminopeptidases and neprilysin were identified using fluorescent substrates. HPLC showed that Plummer's inhibitor but not Z-pro-prolinal blocks Ang II metabolism, suggesting that carboxypeptidase N catalyzes the conversion Ang II--> Ang-(1-7). Different combinations of bestatin, thiorphan, Plummer's inhibitor, Ang II and Ang-(1-5), and use of short proteolysis times, indicate that Ang-(1-7)--> Ang-(1-5)--> Ang-(1-4)--> Ang-(3-4) is a major route. When Ang III was combined with the same inhibitors, the following pathway was demonstrated: Ang III--> Ang IV--> Ang-(3-4). Ca(2+)-ATPase assays with different Ang II concentrations and different peptidase inhibitors confirm the existence of these pathways in BLM and show that a prolyl-carboxypeptidase may be an alternative catalyst for converting Ang II to Ang-(1-7). Overall, we demonstrated that BLM have all the peptidase machinery required to produce Ang-(3-4) in the vicinity of the Ca(2+)-ATPase, enabling a local RAS axis to effect rapid modulation of active Ca(2+) fluxes.

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 NH(2)-YFPGQFAFSK-COOH by removing the carboxyl-terminal lysine (K). Sequential cleavages of either a prochemerin peptide (NH(2)-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.

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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

BACKGROUND: Measurement 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. METHODS: We 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. RESULTS: The 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 k(cat)/K(m) 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 k(cat) value compared to the commonly used Hip-Arg (Bz-Gly-Arg). DISCUSSION: We 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.

Acute adverse reactions associated with angiotensin-converting enzyme inhibitors: genetic factors and therapeutic implications.

Angiotensin-converting enzyme inhibitors (ACEIs) have been used in the treatment of various cardiovascular diseases. Despite the therapeutic benefits of ACEIs, there are several reported side effects, including chronic cough, angioedema and anaphylactoid reactions. These adverse events cannot be explained by the vasodilatory effects of this group of medications. Preliminary studies have shown that patients with a history of developing these side effects have a lower activity of an enzyme called aminopeptidase-P. This enzyme has an important role in degrading bradykinin. This defect in enzymatic activity can be partially explained by genetic variation. Using genome-wide screening strategies, the locus (loci), gene(s) and untimely polymorphisms that explain the low enzymatic activity and side effects can be identified.

Identification of carboxypeptidase N as an enzyme responsible for C-terminal cleavage of stromal cell-derived factor-1alpha in the circulation.

The chemokine stromal-derived factor-1alpha (SDF-1alpha) is an essential regulator of hematopoiesis, lymphocyte homing, pre-B-cell growth, and angiogenesis. As SDF-1alpha is constitutively expressed in many tissues, chemokine function is mostly regulated by proteolytic degradation. Human serum cleaves the 68-amino acid chemokine, SDF-1alpha, at both termini. The enzyme or enzymes responsible for the removal of the carboxy-terminal lysine from SDF-1alpha, leading to significant reduction in biologic activity, have not been identified. Using a new biochemical assay for measuring the carboxy-terminal cleavage activity, we purified from serum and plasma a peptidase that specifically removes the carboxy-terminal lysine from SDF-1alpha and identified it as carboxypeptidase N (CPN, also known as kininase I, arginine carboxypeptidase, and anaphylotoxin inactivator). We demonstrate that SDF-1alpha in serum and plasma lacks the carboxy terminal lysine, and depletion of CPN from serum and plasma significantly reduces the SDF-1alpha carboxypeptidase activity. Purified CPN effectively and specifically removes the carboxy-terminal lysine from SDF-1alpha and significantly reduces the chemokine's biologic activity as a pre-B-cell growth factor and chemoattractant. Thus, in addition to its role as a regulator of the biologic activity of kinins and anaphylatoxins, CPN is an important regulator of the biologic activity of SDF-1alpha by reducing the chemokine-specific activity.

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.

Hemi-orolingual angioedema and ACE inhibition after alteplase treatment of stroke.

One hundred seventy-six consecutive patients treated with IV tissue plasminogen activator (tPA) for acute ischemic stroke were examined prospectively, and orolingual angioedema was found in nine (5.1%; 95% CI 2.3 to 9.5). The reaction was typically mild, transient, and contralateral to the ischemic hemisphere. Risk of angioedema was associated with angiotensin-converting enzyme inhibitors (relative risk [RR] 13.6; 95% CI 3.0 to 62.7) and signs on initial CT of ischemia in the insular and frontal cortex (RR 9.1; 95% CI 1.4 to 30.0).

DNA polymorphism and mutations in CPN1, including the genomic basis of carboxypeptidase N deficiency.

Carboxypeptidase N (EC 3.4.17.3) regulates the activity of peptides such as kinins and anaphylatoxins. Although deficiency of carboxypeptidase N (MIM 212070) produces a severe allergic syndrome, no human mutations have ever been described. Therefore, using archival genomic DNA from a subject with documented carboxypeptidase N deficiency, we sequenced CPN1 (MIM 603103), which encodes the catalytic subunit of carboxypeptidase N. In the genomic DNA of the proband, we discovered three CPN1 variants: (1) 385fsInsG, a frameshift mutation in exon 1 due to a single G insertion at nucleotide 385; (2) 746G>A single-nucleotide polymorphism (SNP), a missense mutation in exon 3 that predicted substitution of aspartic acid for the wild-type conserved glycine at amino acid 178 (G178D); and (3) IVS1 +6C>T, an SNP in intron 1. Among 128 normal Caucasians, the 385fsInsG mutation was absent and the G178D mutation had a frequency of 0.0078, suggesting that these were rare molecular events that likely contributed to the carboxypeptidase N deficiency phenotype. The frequency of the IVS1 +6C>T polymorphism was 0.051. The reagents described here provide tools for further study of association with clinical and biochemical phenotypes related to allergy and immunity.