Carboxypeptidase N Research Articles

Genetic Deletion of Carboxypeptidase N Attenuates Angiotensin II-Induced Hypertension and Target Organ Damage

Objectives: Kininase I transforms bradykinin and kallidin, into kinin B1 receptor (B1R) agonists des-Arg9-BK and des-Arg10-kallidin, respectively. Carboxypeptidase M (CPM) is the membrane bound isoform of kininase I and carboxypeptidase N (CPN) is the isoform located in the plasma. Activation of the B1R has been implicated in the development of hypertension, diabetes and atherosclerosis. Pharmacologic inhibition of kininase I is associated with reductions in B1R expression, inflammation, oxidative stress, and improvements in insulin resistance. B1R can interact with aminopeptidase N (CD13) or high-mobility group box 1 (HMGB1) to mediate an inflammatory response. However, the exact role of kininase I in the development of hypertension and the regulation of these B1R-mediated signaling mechanisms remains unknown. In this study, we hypothesize that gene deletion of CPN will prevent B1R activation, reduce inflammation via interfering with CD13 and HMGB1 signaling, and ultimately attenuate the progression of hypertension and target organ damage. Methods and Results: To test our hypothesis we utilized 12-week-old male and female C57BL/6NJ wild-type (WT) and CPN gene knockout (CPNKO) mice. These mice were infused angiotensin II (Ang II, 600 ng/kg/min) or saline (vehicle) via osmotic minipumps for 2 weeks. Vehicle treated CPNKO and WT mice showed no significant difference in mean arterial blood pressure (MAP) (104 ±7 vs 102 ±5 mmHg, n=5, p≤0.05). Ang II-induced elevations in MAP were significantly reduced in CPNKO mice compared to WT mice infused with Ang II (129 ±8 vs 150 ±6 mmHg, n=5, p≤0.05). Interestingly, we did not find any significant sex differences in MAP in CPNKO groups. Additionally, Ang II treatment significantly increased cardiomyocyte hypertrophy in male and female WT mice, but this effect was blunted in CPNKO mice (n=5, p≤0.05; wheat germ agglutinin staining). Ang II infusion promoted collagen deposition in the aorta of WT male and female mice, but not in CPNKO mice (n=5, p≤0.05; picrosirius red/Masson’s trichrome). In male WT mice, Ang II significantly increased the immunoreactivity of CPM in the heart, but this was abrogated in CPNKO mice infused with Ang II (n=4, p≤0.05). Similarly, B1R was also upregulated in male WT mice hearts when compared to CPNKO mice after Ang II infusions (n=3, p≤0.05). We also found that Ang II significantly increased HMGB1 and CD13 in male WT hearts and brains, but not in CPNKO mice (n=4, p≤0.05). In addition, oxidative stress levels in the brain were significantly increased by Ang II in WT mice and was attenuated in CPNKO mice (n=4, p≤0.05; dihydroethidium staining). Conclusions: Genetic deletion of CPN attenuates Ang II-induced hypertension by reducing B1R mediated reductions in CD13 and HMGB1-mediated inflammatory responses and oxidative stress. Our data suggests that targeting kininase I may be a novel therapeutic target for hypertension and target organ damage. Funding: This study was supported by NHLBI/NIH 5R01HL153115 (S. Sriramula). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hereditary angioedema with normal C1 inhibitor associated with carboxypeptidase N deficiency

BackgroundHereditary angioedema (HAE) is a potentially life-threatening disorder characterized by recurrent episodes of subcutaneous or submucosal swelling. HAE with normal C1 Inhibitor (HAE-nC1-INH) is an under-diagnosed condition. Although the association with genetic variants has been identified for some families, the genetic causes in many patients with HAE-nC1-INH remain unknown. The role of genes associated with bradykinin catabolism is not fully understood. ObjectiveWe investigated the biological parameters and the genes related to kallikrein-kinin system (KKS) in families with a clinical phenotype of HAE-nC1-INH and presenting with a carboxypeptidase N (CPN) deficiency. MethodsThis study includes four families presenting with HAE-nC1-INH and CPN deficiency. Patients’ clinical records were examined, biological parameters of KKS measured, genetics was analyzed by next-generation sequencing and Sanger sequencing. Predictive algorithms (HSF®, SIFT®, Polyphen-2®, MutationTaster®, ClinPred®) were used to classify variants as affecting splicing, as benign to deleterious, or as disease-causing. ResultsPatients presented with angioedema and urticaria, mainly on face/lips, but also with abdominal pain or laryngeal symptoms. Affected patients displayed low CPN activity –30 to 50% of median value in plasma. We identified three variants of the CPN1 gene encoding the catalytic 55-kDa subunit of CPN at: c.533G>A, c.582A>G and c.734C>T. CPN deficiency associated with genetic variants segregated with HAE-nC1-INH symptoms in affected family members. ConclusionsCPN1 gene variants are associated with CPN deficiency and HAE-nC1-INH symptoms in four unrelated families. Genetic CPN deficiency may contribute to bradykinin and anaphylatoxins accumulation, with synergistic effects in angioedema and urticarial symptoms.

Impact of Vitamin D Status Correction on Serum Lipid Profile, Carboxypeptidase N and Nitric Oxide Levels in Saudi Adults.

This study aimed to determine the impact on the lipid profile, carboxypeptidase N (CPN) and nitric oxide (NOx) associated with vitamin D (VD) status correction among Saudi adults with VD deficiency. A total 111 VD deficient (25(OH)D < 50 nmol/L)) adult Saudis aged 18-50 years old (57 females and 54 males) were enrolled in this 6-month interventional study. They were given 50,000 IU VD weekly for the first 2 months and then twice a month for the next 2 months, followed by 1000 IU daily for the last 2 months. The fasting lipid profile and the blood glucose, VD, NOx and CPN concentrations were measured at baseline and after intervention. Post-supplementation, the median VD was significantly higher (p < 0.001) in females [58.3 (50.6-71.2)] and males [57.8 (51.0-71.8)]. HDL cholesterol significantly increased (p = 0.05) and NOx significantly decreased (p = 0.02) in males post-supplementation. Triglycerides were positively associated with NOx in all subjects before (r = 0.44, p = 0.01) and after (r = 0.37, p = 0.01) VD status correction. There was a significant increase in serum levels of CPN2 (p = 0.02) in all subjects. Furthermore, CPN was inversely correlated with NOx (r = -0.35, p = 0.05) in males post-supplementation. In conclusion, VD status correction reduced serum NOx, particularly in males. The inhibition of NOx synthesis may be responsible for the anti-inflammatory effects of VD supplementation. An inverse association was found between NOx and CPN2.

High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses.

Objective: The purpose of current research is to explore the function of retinoic acid-induced protein 14 (RAI14), being a reciprocal protein of carboxypeptidase N1 (CPN1), and as a biomarker for prognosis and immunoregulatory effects in breast cancers. Methods: Interacting proteins of CPN1 were characterized by co-immunoprecipitation (CO-IP) and mass spectrometry. We evaluated RAI14 expression and related clinical prognosis based on bioinformatics methods. The level of relevance between RAI14 and infiltrating immune cells biomarkers was investigated by using TIMER and certificated by immunohistochemical staining and cytology experiments. Results: RAI14 is an interacting protein of CPN1. Higher RAI14 expression in TNBC was significantly correlated with poor prognosis in TNBC, especially (RFS: HR = 1.32, p = 0.015; DFS: HR = 1.18, p = 0.035). The estrogen receptor (ER), P53 status, and histological types and triple-negative status were observed and correlated with RAI14 expression. Moreover, the level of RAI14 was positive in relation with the expression of CD163 (M2 macrophages marker, r = 0.393, p = 1.89e-06) and PD-1 (T-cell exhaustion marker, r = 0.626, p = 4.82e-03), indicating RAI14 levels were mainly related to M2 macrophages and T-cell exhaustion infiltration in TNBC. Furthermore, CPN1 overexpression was accompanied by RAI14 and PD-L1 upregulation, and a correlation was found among them. Conclusions: RAI14 is a potential downstream molecule of CPN1, which may be a potential prognostic biomarker and identification of an immunosuppressive tumor microenvironment in TNBC.

High Angiotensin-Converting Enzyme and Low Carboxypeptidase N Serum Activity Correlate with Disease Severity in COVID-19 Patients.

(1) Background: Angiotensin-converting enzyme 2 (ACE2) is a functional receptor of SARS-CoV-2 and counter-balances ACE in the renin–angiotensin system (RAS). An imbalance of the RAS could be associated with severe COVID-19 progression. (2) Methods: Activities of serum proteases angiotensin-converting enzyme (ACE) and carboxypeptidase N (CPN) for 45 hospitalized and 26 convalescent COVID-19 patients were investigated vs. healthy controls using labeled bradykinin (DBK) degradation with and without inhibition by captopril as a read-out. Data were correlated to clinical parameters. (3) Results: DBK degradation and CPN activity were significantly reduced gender-independently in COVID-19 and returned to normal during convalescence. ACE activity was over-active in severe disease progression; product DBK1-5 was significantly increased in critically ill patients and strongly correlated with clinical heart and liver parameters. ACE inhibitors seemed to be protective, as DBK1-5 levels were normal in moderately ill patients in contrast to critically ill patients. (4) Conclusions: CPN and ACE serum activity correlated with disease severity. The RAS is affected in COVID-19, and ACE could be a therapeutic target. Further proof from dedicated studies is needed.

Carboxypeptidase N1 is anticipated to be a synergy metrics for chemotherapy effectiveness and prognostic significance in invasive breast cancer

BackgroundThe incidence and mortality of invasive breast cancer (IBC) are increasing annually. Hence, it is urgently needed to determine reliable biomarkers for not only monitoring curative effects, but evaluating prognosis. In present study, we aim to determine the potential role of Carboxypeptidase N1 (CPN1) in IBC tissues on chemotherapeutic efficacy and poor prognosis.MethodsThe expression level of CPN1 in IBC tissue samples (n = 123) was quantified by tissue microarray technique and immunohistochemical staining. Moreover, sera of IBC patients (n = 34) that underwent three to five consecutive chemotherapy sessions were collected. The patients were randomly stratified into a training (n = 15) as well as a validation group (n = 19). The expression of serum CA153 and CPN1 was quantified by electrochemiluminescence and ELISA assay, respectively.ResultsBy univariate and multivariate Cox regression analysis, we show that CPN1 expression in IBC tissues, as an independent risk factor, is related to a poor overall survival (OS) and progression-free survival (PFS) (P < 0.05). Analysis of the data revealed that CPN1 over-expression could be consistently linked to adverse clinicopathological features such as lymph node metastasis and the pathological stage (pTNM) (P < 0.05). The serum CPN1 level trajectory of individual patients generally decreased during chemotherapy. In line with these findings were changes in the follow-up ultrasonography and a consistent decrease in serum CPN1 levels. The comparison of the area under the receiver operating curves (ROC) revealed that CPN1 has a better surveillance value than CA153 in the training (AUCCPN1 = 0.834 vs. AUCCA153 = 0.724) as well as the validation set (AUCCPN1 = 0.860 vs. AUCCA153 = 0.720) when comparing cycle2 versus cycle3.ConclusionsCPN1 is a suitable potential biomarker for chemotherapeutic surveillance purposes as well as being an appropriate prognostic indicator which would support an improved chemotherapy regimen.

Reduced serum protease activity in Complex Regional Pain Syndrome: The impact of angiotensin-converting enzyme and carboxypeptidases

Complex Regional Pain Syndrome (CRPS) occurs in about 2% of patients after fracture of the limbs. In an earlier clinical study with 102 probands we have shown that the serum protease network in CRPS might be less effective. Based on these results we hypothesized that angiotensin-converting enzyme (ACE) and carboxypeptidase N (CPN) activity contribute to the differences of labeled bradykinin (DBK) degradation by patients’ sera. Details of the enzymatic processes remained however unclear. The contributions of ACE and CPN in the serum degradation of DBK were studied using specific inhibitors. CPN1-ELISA was performed in serum. It was confirmed that the majority of DBK was degraded by ACE and CPN. The data delivered proof that the ACE serum activity was lowered in CRPS. High-resolution mass spectrometry was additionally used for protein expression analysis of sera of above study cohort (CRPS vs. healthy probands). According to principal component analysis of these data, significant differences between CRPS and control samples only occurred in sera of females younger than 46 years. In these CRPS patients, a number of defence / immunity-related proteins and members of the renin-angiotensin system (RAS) protein network were regulated. The impact of CPN in CRPS pathophysiology is subject to further investigation. The data support the hypothesis that both the RAS and the innate immune system might be affected in CRPS. A database of regulated serum proteins was established for future research.

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

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. 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. 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). 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.

Analysis of different proteins in primary open angle glaucoma based on aqueous proteome

Objective To analyze the changes of protein expression in aqueous humor of patients with primary open angle glaucoma (POAG), and to explore the biological markers and potential therapeutic targets associated with disease occurrence. Methods A retrospective case-control study was adopted.Ten patients with age-related cataract and 10 patients with POAG combined with cataract.were collected at the Tianjin Medical University Eye Hospital from October 2016 to December 2017.In the process of surgery, 100 μl of aqueous humor were collected with 1 ml syringe and No.1 needle through the surgical access.Those proteins extracted from aqueous humor were analyzed by quantitative proteomic mass spectrometry.The differential significance test was performed by Maxquant significances A. The differential proteins of the two groups were screened and determined with the conditions of P 2.The biological big data was annotated by the function of GO and KEGG.The study was approved by the Ethics Committee of the Tianjin Medical University Eye Hospital (No.2013KY[L]-10). Patients and their guardians all signed the informed consent. Results Ninty-seven differential proteins were detected in this proteomic analysis, including 48 up-regulated proteins and 49 down-regulated proteins.GO analysis significantly differs in protein function involved in many aspects, some different proteins including lipopolysaccharide-binding protein (LBP), cluster of Differentiation 163(CD163), C-reactive protein (CRP), annexin A1 (ANXA1) were involved in inflammation; redox-related proteins were glutathione S-transferase P (GSTP1), thioredoxin (TXN), some different proteins related with cell adhesion movement were cartilage oligomeric matrix protein (COMP), desmocollin-2 (DSC2) and laminin subunit beta-2 (LAMB2); procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1), solid protein (tenascin, TNC) are associated with fibrosis; some different proteins related to nerve growth were reelin (RELN), semaphorin-3F(SEMA3F), semaphorin-4B(SEMA4B). Metabolism-related proteins included pyruvate kinase (PKM), carb oxypeptidase N subunit 2 (CPN2) and so on.KEGG analysis indicated that the expression of NrF2/ERK signaling pathway and TGF-β/NF-κB signaling pathway were different between the two groups. Conclusions The expression of GSTP1 and TXN in the aqueous humor of POAG is significantly decreased, which may regulate cell adhesion and activity and expression of extracellular matrix by regulating NrF2/ERK1/2 and TGF-β/NF-κB signaling pathways.GSTP1 and TXN may serve as a potential biomarker and therapeutic target of POAG. Key words: Primary open angle glaucoma; Aqueous humor; Label free quantitative proteome; Glutathione S-transferase P; Thioredoxin; Transforming growth factor-β; nuclear factor-kappa B; Nuclear factor erythroid 2-related factor 2; extracellular regulated protein kinases

Identification and characterization of a carboxypeptidase N1 from red lip mullet (Liza haematocheila); revealing its immune relevance

Complement system orchestrates the innate and adaptive immunity via the activation, recruitment, and regulation of immune molecules to destroy pathogens. However, regulation of the complement is essential to avoid injuries to the autologous tissues. The present study unveils the characteristic features of an important complement component, anaphylatoxin inactivator from red lip mullet at its molecular and functional level. Mullet carboxypeptidase N1 (MuCPN1) cDNA sequence possessed an open reading frame of 1347 bp, which encoded a protein of 449 amino acids with a predicted molecular weight of 51 kDa. In silico analysis discovered two domains of PM14-Zn carboxypeptidase and a C-terminal domain of M14 N/E carboxypeptidase, two zinc-binding signature motifs, and an N-glycosylation site in the MuCPN1 sequence. Homology analysis revealed that most of the residues in the sequence are conserved among the other selected homologs. Phylogeny analysis showed that MuCPN1 closely cladded with the Maylandia zebra CPN1 and clustered together with the teleostean counterparts. A challenge experiment showed modulated expression of MuCPN1 upon polyinosinic:polycytidylic acid and Lactococcus garviae in head kidney, spleen, gill, and liver tissues. The highest upregulation of MuCPN1 was observed 24 h post infection against poly I:C in each tissue. Moreover, the highest relative expressions upon L. garviae challenge were observed at 24 h post infection in head kidney tissue and 48 h post infection in spleen, gill, and liver tissues. MuCPN1 transfected cells triggered a 2.2-fold increase of nitric oxide (NO) production upon LPS stimulation compared to the un-transfected controls suggesting that MuCPN1 is an active protease which releases arginine from complement C3a, C4a, and C5a. These results have driven certain way towards enhancing the understanding of immune role of MuCPN1 in the complement defense mechanism of red lip mullet.

Fibrinolytic potential of DS-1040, a novel orally available inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa)

An activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis by removing C-terminal lysine/arginine residues from partially degraded fibrin. We have identified a novel low-molecular-weight inhibitor of TAFIa, DS-1040, to be potentially useful for treating thrombotic diseases.In this study, we investigated its in vitro pharmacological profile and in vivo effects in animal models of microthrombosis and bleeding.DS-1040 inhibited human TAFIa and carboxypeptidase N (CPN) in vitro with IC50 values of 5.92 and 3.02 × 106 nmol/L, respectively, suggesting that DS-1040 is highly selective for TAFIa over CPN. DS-1040 did not affect platelet aggregation and coagulation time. In a tissue factor-induced rat microthrombosis model, intravenously administered DS-1040 reduced existing fibrin clots in the lung, whereas post-treatment with enoxaparin had limited effect. Both intravenously and orally administered DS-1040 elevated plasma D-dimer levels with similar plasma exposures of DS-1040. DS-1040 significantly augmented plasma D-dimer level on top of silent dose of recombinant tissue-plasminogen activator (t-PA), suggesting DS-1040 enhances fibrinolytic activity of t-PA. In addition, DS-1040 did not prolong the tail bleeding time beyond its efficacy dose.These results indicate that DS-1040 is a potent, selective, intravenously/orally available inhibitor of TAFIa with minimum risk of bleeding. DS-1040 is a potential novel fibrinolysis enhancer useful in treating thrombotic diseases.

Structural basis for the selective inhibition of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) by a selenium-containing inhibitor with chloro-aminopyridine as a basic group

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) is a target molecule for treating thromboembolic disorders. We previously reported that design and synthesis of compound 1 containing a selenol group and chloloaminopyridine. Compound 1 showed high inhibitory activity towards TAFIa, with a high degree of selectivity for TAFIa over carboxypeptidase N (CPN). Here we report investigation of this selectivity. To obtain co-crystal of 1/pp-CPB (a surrogate of TAFIa), we synthesized protected compound 5 as a stabilized precursor of 1. The X-ray crystal structure and docking study indicated that the Cl substituent is accommodated in the pp-CPB specific pocket whereas CPN has no identical pocket. This is important information for the design of drugs targeting TAFIa with high selectivity.

Carboxypeptidase B2 and N play different roles in regulation of activated complements C3a and C5a in mice

Background There are two basic carboxypeptidases in plasma. Carboxypeptidase B2 (CPB2) is activated from a circulating zymogen, proCPB2, and carboxypeptidase N (CPN) is constitutively active with both inactivating complement C3a and C5a. Aims To test the roles of CPB2 and CPN in complement-driven mouse models of cobra venom factor (CVF) challenge and hemolytic-uremic syndrome (HUS). Methods Cpb2-/- , Cpn-/- and wild-type (WT) mice were compared in an HUS model induced by Shiga toxin and lipopolysaccharide administration and following CVF administration. Results HUS was exacerbated in Cpb2-/- mice more than in Cpn-/- mice, compared with WT mice. Cpb2-/- mice developed the HUS clinical triad of microangiopathic hemolytic anemia, uremia and thrombocytopenia. Treatment with anti-C5 antibody improved survival of both Cpb2-/- and Cpn-/- mice. In contrast, when challenged acutely with CVF, the reverse phenotype was observed. Cpn-/- mice had markedly worse disease than Cpb2-/- mice, whereas the WT mice were resistant. Conclusions CPN and CPB2 play overlapping but non-redundant roles in regulating complement activation in vivo. The constitutively active CPN is key for inactivation of systemic C5a, whereas CPB2 functions as an on-demand supplementary anaphylatoxin inhibitor in inactivating excessive C5a formed locally.

Kinin system of blood plasma as pathogenesis factor in brain tumors.

e13542 Background: The kallikrein-kinin system (KKS) does not only perform its adaptive-regulatory function; it is confirmed to be involved into pathology, neoplastic processes in particular. Our purpose was to study the kallikrein family (KLKs), pro-KLKs and carboxypeptidase N (CPN) in the blood plasma of patients with benign and malignant brain tumors. Methods: We studied citrated blood plasma of 164 patients with brain neoplasms: 37 meningiomas (M), 74 primary glioblastomas (G) and 53 brain metastases (MTS) from breast cancer. The kinetics of KKS enzymatic reactions was determined in the blood plasma before treatment using the U-2900 UV Solutions double-beam spectrophotometer (USA). The results were compared to the values in 39 healthy donors (N). The data were processed using the Statistica 10 program. The significance of differences was determined by Student's t-test. Results: Activity of KLKs in the blood plasma in patients with M exceeded N by 1.3 times (p≤0.05), in G – by 3.9 times, in MTS – by 5.1 times. Levels of pro-KLKs in M were lower than in N by 1.6 times, and in G and MTS they were similar to N. CPN activity in M was decreased compared to N by 3.1 times; in G and MTS it did not differ significantly from N. The pro-KLKs/KLKs coefficient in patients with M was lower than in N by 2 times, in G – by 3.7 times, and in MTS – by 5.1 times. Activity of KLKs in G and MTS was 3 and 4 times higher, respectively, than in M (p &lt; 0.001). It supposed realization of effects of KLKs and produced bradykinin on cells, blood enzymes and microvessel walls in malignant brain tumors to a greater extent than in benign ones. Conclusions: Activation of KKS in the blood plasma in malignant brain tumors is more intense than in benign tumors, it directly and indirectly influences cell migration, increases capillary permeability and causes an imbalance in the related enzyme systems becoming a pathogenesis factor.

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.

Deficiency of Either Carboxypeptidase B2 or Carboxypeptidase N Exacerbated Disease Activity in a Mouse Model of Hemolytic Uremic Syndrome

Two different basic carboxypeptidases circulate in blood - carboxypeptidase N (CPN) and proCPB2. CPN is constitutively active, while proCPB2 is a zymogen, (also termed thrombin activatable fibrinolysis inhibitor, TAFI), and is activated by the endothelial thrombin/thrombomodulin complex to CPB2. Their kinetics of substrate cleavage are distinct but both can efficiently inactivate the complement anaphylatoxins, C3a and C5a. Hemolytic uremic syndrome (HUS) is caused by Shiga toxin (stx) producing strains of E. coli and is characterized by the triad of hemolysis, thrombocytopenia and uremia. We hypothesized that in a mouse model of HUS, Cpb2-/- and Cpn-/- mice would have prolonged C5a anaphylatoxin activity thus causing disease exacerbation.HUS was induced by stx2 and LPS administration in WT, Cpn-/- and Cpb2-/- mice. In Cpb2-/- mice, median time to death was earlier (60 hours, n=15) than in WT mice (96 hours: n=42, p<0.0001), and had greater kidney and liver damage shown by increases in ALT, AST, BUN and creatinine levels at 48 hours (creatinine Cpb2-/-: 1.01 mg/dL, WT: 0.25 mg/dL; Cpb2-/- control: 0.20 mg/dL and WT control: 0.19 mg/dL; Cpb2-/- p<0.0001 vs. all other groups, n>9). An increase in hemolysis was demonstrated by reduced RBC count and hemoglobin level plus an increase in total bilirubin and LDH. Profound thrombocytopenia (Cpb2-/-: 121,000/μL vs. WT: 217,000/μL; p=ns) developed in both genotypes (control Cpb2-/-: 1,001,000/μL vs. control WT: 1,141,000/μL; p=ns but vs. either Cpb2-/- or WT with HUS, p<0.0001) and thus the HUS clinical triad was present. Histology showed tubular epithelial necrosis in the kidney ante-mortem. Administration of either toxin separately caused milder disease without the characteristics of HUS and with no observed mortality. Induction of the disease depended on co-administration of both toxins. Treatment with anti-murine C5 antibody (0.75 mg every 24 hours from 3 hours before disease initiation) improved survival of both WT and Cpb2-/- mice with a median survival time of 168 hours for both genotypes (n=11, p=0.003 and <0.0001 respectively) and normalized the outcomes between the genotypes.Cpn-/- mice also died sooner (median time to survival 81.5 hours, n=28) than WT mice (96 hours, n=42, p=0.0002). The median survival time between Cpb2-/- and Cpn-/- mice was also significantly different (60 vs. 81.5 hours, p=0.0083). This is a first direct comparison of the role of CPN vs. CPB2 in regulating C5a activity in a disease relevant mouse model. Our study suggests that both CPB2 and CPN protect against HUS by inactivation of C5a with CPB2 having a greater effect than CPN. When Cpb2+/-/Cpn+/- mice are crossed, all expected genotypes are recovered in the expected Mendelian ratios including double deficient Cpb2-/-/Cpn-/- mice. Thus absence of both plasma basic carboxypeptidases is not essential for murine life. We are currently evaluating the Cpb2-/-/Cpn-/- mice in our HUS model. DisclosuresNo relevant conflicts of interest to declare.

Prochemerin Cleavage By Factor XIa Links Adipogenesis, Inflammation and Coagulation

Chemerin is a chemoattractant and adipokine that circulates in blood as inactive prochemerin (chem163S). Chem163S is activated by a series of C-terminal proteolytic cleavages resulting in diverse chemerin forms with different levels of activity. Cleavage of chem163S at Lys158 generates chem158K, which has low bioactivity, with subsequent cleavage by the basic carboxypeptidases in plasma, carboxypeptidase N (CPN) or thrombin-activatable CPB2 (also known as thrombin-activatable fibrinolysis inhibitor or TAFI), to produce chem157S, the most active form of chemerin. Inactivation of chem157S to chem155A and smaller forms of chemerin occurs by further proteolytic cleavages.We have developed specific ELISAs for chem163S, 158K, 157S and 155A. Using these chemerin-specific ELISAs, we found that most of the chemerin in normal human plasma is inactive chem163S, while chem158K is the dominant form in synovial fluids of arthritis patients. To identify the physiological activator of chemerin in plasma, we screened proteases from the coagulation, fibrinolytic and complement pathways. The proteases were incubated with purified chem163S protein in buffer and the resultant products were analyzed by MALDI-TOF mass spectrometry. The results showed that Factor XIa (FXIa) can cleave chem163S, generating a novel chemerin form, chem162R, as an intermediate product, and chem158K, as the final product. We compared the kinetics of FXIa cleavage of chem163S and FIX, the established physiological FXIa substrate. Peptides from chem163S and FIX containing the FXIa cleavage site were subjected to FXIa cleavage followed by HPLC analysis. The catalytic efficiency (kcat/KM) of FXIa cleavage of FIX was ~3-fold more efficient than chemerin cleavage, but the hydrolysis of chem163S still occurred at a physiologically relevant rate. By substituting Arg162 with Ala in chemerin protein/peptides, we found that processing at Arg162 was not required for cleavage at Lys158 or regulation of chemerin bioactivity.We further investigated FXIa cleavage of chem163 in plasma triggered by kaolin to activate FXI. FXI activity was measured by inclusion of a specific chromogenic substrate, and chemerin levels were measured by the chemerin-specific ELISAs. Contact phase activation of human platelet-poor plasma led to generation of FXIa and cleavage of chem163S, which was abolished in FXI-depleted plasma. Platelets markedly enhanced FXI activation and chem163S cleavage in platelet-rich plasma comparing to platelet-poor plasma. Following contact activation, plasma levels of the most potent form of chemerin, chem157S, as well as inactive chem155A, increased. Plasma levels of chem163S in FXI-deficient patients were significantly higher compared to a matched control group (90.69±9.6 ng/mL vs. 58.11±2.51 ng/mL, n=8; P<0.01) and inversely correlated with the plasma FXI levels. FXIa, generated upon contact phase activation, cleaves chem163S to generate chem158K, which can be further processed to the most active chemerin form, thus providing a molecular link between coagulation, inflammation, innate immunity and adipogenesis. DisclosuresNo relevant conflicts of interest to declare.

Role of Carboxypeptidase N Invasion and Migration in Breast Cancer.

Carboxypeptidase N (CPN) is highly expressed in breast cancer and plays an important role in cleaving specific polypeptide fragments within the tumor microenvironment, so here we studied the important role of its invasion and migration in breast cancer. MDA-MB-231, MDA-MB-468, and MCF-7 cells were selected for cell culture. We used real-time polymerase chain reaction (PCR) and western blotting to determine CPN gene and protein expression. If CPN was obviously expressed, we designed and synthesized a molecular sequence using an RNA interference approach to remove the CPN and observed its proliferation, migration, and invasion within tumor cells. Real-time PCR and western blotting show the following CPN expression: minimal in MDA-MB-468 cells but obvious in MDA-MB-231 and MCF7 cells. MDA-MB-231 breast cancer cell lines were selected for the control group and CPN was knocked out for the experimental group. Compared to the control group, the experimental group had significantly less migration and invasion. CPN may play an important biological function in breast cancer and will provide a new target for the effective diagnosis and treatment of breast cancer.

Profiling of Cross-Functional Peptidases Regulated Circulating Peptides in BRCA1 Mutant Breast Cancer.

Women with inherited BRCA1 mutations are more likely to develop breast cancer (BC); however, not every carrier will progress to BC. The aim of this study was to identify and characterize circulating peptides that correlate with BC patients carrying BRCA1 mutations. Circulating peptides were enriched using our well-designed nanoporous silica thin films (NanoTraps) and profiled by mass spectrometry to identify among four clinical groups. To determine the corresponding proteolytic processes and their sites of activity, purified candidate peptidases and synthesized substrates were assayed to verify the processes predicted by the MERPOS database. Proteolytic processes were validated using patient serum samples. The peptides, KNG1K438-R457 and C 3fS1304-R1320, were identified as putative peptide candidates to differentiate BRCA1 mutant BC from sporadic BC and cancer-free BRCA1 mutant carriers. Kallikrein-2 (KLK2) is the major peptidase that cleaves KNG1K438-R457 from kininogen-1, and its expressions and activities were also found to be dependent on BRCA1 status. We further determined that KNG1K438-R457 is cleaved at its C-terminal arginine by carboxypeptidase N1 (CPN1). Increased KLK2 activity, with decreased CPN1 activity, results in the accumulation of KNG1K438-R457 in BRCA1-associated BC. Our work outlined a useful strategy for determining the peptide-petidase relationship and thus establishing a biological mechanism for changes in the peptidome in BRCA1-associated BC.

Fine‐tune Regulation of Cpn1 (Carboxypeptidase N1) to Control Vascular Patterning during Zebrafish Development

Vascular development is important for vertebrates and regulated by regulators via complicated signaling pathways. In our previous study, transcription factor islet2 regulated vein and ISV formation mediated by notch signaling. Genome‐wide transcriptional analysis showed that cpn1 is regulated by islet2/coupTFIb, suggested its function in vasculature. However, no direct evidence shown the role of Cpn1 in vascular function so far. Thus, we hypothesize that cpn1 encode enzymatic active subunits to perform CPN activity and function in vascular developmentIn‐situ hybridization showed cpn1 is expressed in developing vessels. Overexpression of cpn1 impairs the growth of ISV (intersegmental vessel) and CVP (cardinal vein plexus), which is similar to the phenotype of isl2 morphant. Knockdown of cpn1 by morpholino injection also causes vascular defects, suggesting the role of cpn1 in controlling ISV and CVP growth. We further showed the cpn1 MO knockdown works efficiently by probing cpn1 expression products and knockdown specificity by using 2nd MO. We found that a loss of cpn1 results in a decreased expression of vascular markers flk, stabilin, ephrinb2. This indicates the regulatory role of cpn1 in controlling vascular development. Promoter analysis showed cpn1 is likely regulated by isl2/coupTFIb. To examine the interaction between cpn1 and isl2/coupTFIb, we performed luciferase assay and showed cpn1 is regulated by isl2, which is consistent with our microarray results. We also revealed cpn1 is regulated by notch and VEGF signalings. Together, we showed cpn1 plays an important role for vascular development in zebrafish. We also uncovered a fine‐tune regulation of cpn1 that controls vascular patterning mediated by isl2‐Notch signaling.