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

Essentials Two basic carboxypeptidases are present in plasma, B2 (CPB2) and N (CPN). Cpb2-/- and Cpn-/- mice were challenged in a hemolytic uremic syndrome (HUS) model vs. wild type. Cpb2-/- exacerbates HUS while Cpn-/- exacerbates cobra venom factor challenge vs. wild type mice. CPB2 and CPN have overlapping but non-redundant roles. SUMMARY: 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.

Iatrogenic angioedema associated with ACEi, sitagliptin, and deficiency of 3 enzymes catabolizing bradykinin.

New concepts of idiopathic and iatrogenic angioedema underline the role of bradykinin, and the importance of catabolizing enzymes. A case is described of Angiotensin converting enzyme inhibitor (ACEi) and sitagliptin induced angioedema, where AO attacks decreased after the withdrawal of lisinopril but resolved only after the withdrawal of sitagliptin, an inhibitor of dipeptylpeptidase IV. ACE, aminopeptidase P and carboxypeptidase N were decreased down to 17%, 42%, 64% of median references values, and remained low one year after the interruption of these drugs: 56%, 28% and 50%, respectively. The combined deficiency of APP and CPN might enhance the inhibiting effect of the DPP IV inhibitor. The fact that this triple deficiency remained latent before and after the treatment indicates that searching for latent enzyme deficiencies should be carried out when there is intention to treat with a combination of drugs interfering with the bradykinin metabolism.

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.

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.

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.

Decreased synthesis of serum carboxypeptidase N (SCPN) in familial SCPN deficiency.

Serum carboxypeptidase N (SCPN) is the primary inactivator of the C3a, C4a, and C5a anaphylatoxins as well as an inactivator of bradykinin. Thus SCPN deficiency potentially could result in significant pathophysiologic consequences. Previous studies identified a deficient subject afflicted with frequent episodes of angioedema, and other family members also had SCPN deficiency. To delineate this abnormality further, the fractional catabolic rate (FRC) and enzyme synthesis were determined in three members of the afflicted kindred as well as in five normal persons following the infusion of homogeneous 125I-SCPN. The mean FCR and synthesis rates for SCPN in the normal subjects were 1.3%/hr and 20,793 U/kg/hr, respectively. Reduced synthesis was concluded to be primarily responsible for the low SCPN levels in the afflicted kindred. The high FRC of SCPN discourages attempted maintenance therapy with infusions of enriched SCPN preparations.

Familial carboxypeptidase N deficiency.

Carboxypeptidase N is a serum metalloenzyme that inactivates C3a, C4a, C5a, bradykinin, kalladin, and fibrinopeptides. Of 172 sera from patients with chronic urticaria or angioedema, one had a remarkably depressed carboxypeptidase N level (21% of normal). Of sera from 103 patients with other diseases, elevated levels were observed in cases of neoplasms, and one abnormally low value was detected in a patient with cirrhosis. The patient with a remarkably low carboxypeptidase N level was a 65-year-old man with an 11-year history of episodic angioedema occurring about 40 times per year. Inactivation of C3a and lysyl-bradykinin by his serum was markedly prolonged. Plasma histamine was elevated during attacks, but serotonin and kinin activity were not. The proband's sister had an equally depressed serum carboxypeptidase N level, and studies of other family members suggested an autosomal recessive inheritance of the enzyme deficiency.