A novel rat carboxypeptidase, CPA2: characterization, molecular cloning, and evolutionary implications on substrate specificity in the carboxypeptidase gene family

A new member of the carboxypeptidase gene family, carboxypeptidase A2 (CPA2), has been identified from the predicted amino acid sequence of a rat pancreatic cDNA clone. In vivo recombination and in situ hybridization techniques employing the CPA2 cDNA resulted in the isolation of two genomic clones spanning the 25-kilobase pair rat CPA2 gene. Evolutionary trees built from the amino acid sequences of the known pancreatic carboxypeptidases show that CPA2 and carboxypeptidase A1 (CPA1) are the products of genes which duplicated before the mammalian radiation, and that bovine CPA is of the A1 type. The substrate specificities of CPA1 and CPA2 isolated from rat pancreas are similar to bovine CPA in that carboxyl-terminal amino acids with aromatic or branched aliphatic side chains are preferred. However, the substrate preference of rat CPA1 is skewed toward smaller amino acids, while that of rat CPA2 is skewed toward bulkier amino acids as compared to bovine CPA. The differences in the substrate specificities of these three carboxypeptidases are compatible with the nature of the amino acid replacements in their binding pockets for the carboxylterminal amino acid of the substrate.     

Enzyme immobilization via monoclonal antibodies I. Preparation of a highly active immobilized carboxypeptidase A

A novel method for the preparation of highly active immobilized enzymes is described. It is based on the binding of enzymes to suitable carriers via monoclonal antibodies, which bind to the enzyme with high affinity without affecting its catalytic activity. The applicability of the method forwarded has been illustrated by the preparation of two samples of highly active immobilized carboxypeptidase A (CPA) preparations as follows: A mouse monoclonal antibody (mAb 100)to CPA that binds to the enzyme with a high-affinity constant without affecting its catalytic activity was prepared, purified, and characterized. Covalent binding of this monoclonal antibody to Eupergit C (EC) or noncovalent binding to Sepharose-protein A (SPA)yielded the conjugated carriers EC-mAb and SPA.mAb, respectively, which reacted specifically with CPA to give the immobilized enzyme preparations EC-mAb.CPA and SPA.mAb.CPA displaying full catalytic activity and improved stability. At pH 7.5 and a temperature range of 4-37 degrees C an apparent binding constant of approximately 10(8)M(-1) characterizing the interaction of CPA with EC-mAb and SPA.mAb, was obtained. To compare the properties of EC-mAb.CPA and SPA.mAb.CPA with those of immobilized CPA preparations obtained by some representative techniques of covalent binding of the enzyme with a corresponding carrier, the following immobilized CPA preparations were obtained and their properties investigated: EC-CPA (I), a preparation obtained by direct binding of EC with CPA; EC-NH-GA-CPA (II), a derivative obtained by covalent binding of CPA to aminated EC via glutaraldehyde; EC-NH-Su-CPA (III), a CPA derivative obtained by binding the enzyme to aminated EC via a succinyl residue; and EC-HMD-GA-CPA (IV), obtained by binding the enzyme via glutaraldehyde to a hexamethylene diamine derivative of EC. Full enzymic activity for all of the bound enzyme, such as that recorded for the immobilized CPA preparations EC-mAb.CPA and SPA.mAb.CPA, was not detected in any of the insoluble covalently bound enzyme preparations.     

Identification of a novel polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene required for the biosynthesis of cyclopiazonic acid in Aspergillus oryzae

Cyclopiazonic acid (CPA) is a mycotoxin produced by several strains of Penicillium and Aspergillus species. Aspergillus oryzae strains used in fermented foods do not produce CPA; however, several wild-type A. oryzae strains produce CPA. Here, we identified a novel polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene involved in CPA production by comparing the telomere-adjacent region of a CPA-producing strain (A. oryzae NBRC 4177) with that of a nonproducing strain (A. oryzae RIB40). NBRC 4177 has an additional 17-18-kb sequence beyond the region corresponding to the telomere repeat in RIB40 and this additional regions contains 3' region of the PKS-NRPS gene, while RIB40 has only the 5' region of the PKS-NRPS gene. Gene disruption of the PKS-NRPS gene in NBRC 4177 resulted in elimination of CPA production. Thus, the PKS-NRPS gene is required for CPA biosynthesis, and the truncation of this gene is presumed as one of the determinants of CPA nonproductivity in A. oryzae RIB40.     

Substrate Specificity of Human Carboxypeptidase A6

Carboxypeptidase A6 (CPA6) is an extracellular matrix-bound metallocarboxypeptidase (CP) that has been implicated in Duane syndrome, a neurodevelopmental disorder in which the lateral rectus extraocular muscle is not properly innervated. Consistent with a role in Duane syndrome, CPA6 is expressed in a number of chondrocytic and nervous tissues during embryogenesis. To better characterize the enzymatic function and specificity of CPA6 and to compare this with other CPs, CPA6 was expressed in HEK293 cells and purified. Kinetic parameters were determined using a panel of synthetic carboxypeptidase substrates, indicating a preference of CPA6 for large hydrophobic C-terminal amino acids and only very weak activity toward small amino acids and histidine. A quantitative peptidomics approach using a mixture of peptides representative of the neuropeptidome allowed the characterization of CPA6 preferences at the P1 substrate position and suggested that small and acidic P1 residues significantly inhibit CPA6 cleavage. Finally, a comparison of available kinetic data for CPA enzymes shows a gradient of specificity across the subfamily, from the very restricted specificity of CPA2 to the very broad activity of CPA4. Structural data and modeling for all CPA/B subfamily members suggests the structural basis for the unique specificities observed for each member of the CPA/B subfamily of metallocarboxypeptidases.     

Production of aflatoxin and cyclopiazonic acid by various aspergilli: An ELISA analysis

An enzyme-linked Immunosorbent assay (ELISA) was used to monitor a total of 153 fungi in theAspergillus flavus group, Including 130A. flavus, 15A. parasiticus and 8A. tamarii, for their ability to produce aflatoxins (AFs) and cyclopiazonic acid (CPA) in a mycologlcal broth-sucrose-yeast extract medium. Of 15A. parasiticus isolates, ten produced AFs In a range of 12.4 to 89.3 μg/vial (average 56.9 μg/vial); two isolates produced only trace amounts of AFs and three isolates produced none at all. Production of CPA was not demonstrated in anyA. parasiticus isolate. On the other hand, all A. tamarii isolates produced only CPA with a range of 310 to 1100 gmg/vial. Fifteen percent (14.6%) of theA. flavus isolates (19/130) produced more than 500 μg CPA/vial, but yielded no or little AF (less than 0.1 μg/vial). About 22.3% ofA. flavus (29/130) that produced less than 500 μg of CPA also yielded little or no aflatoxin. MostA. flavus isolates (44.6%) produced both CPA (50 to 300 μg/vial) and AFs (10 to 40 μg/vial). About 9.2% of theA. flavus are low CPA producers (less than 100 μg/vial) but yielded higher amounts of AFs. A small percentage (12/130 or 9.2%) of A. flavus isolates produced neither CPA nor aflatoxin. Excluding the isolates that produced neither AFs nor CPA, there is a negative correlation between the production of CPA and AFs by most A.flavus isolates. Data obtained from ELISA for the production of CPA were consistent with TLC results. Thus, the ELISA method for CPA and AFB could be applied to the screening of toxigenic fungi. Data on the simultaneous production of both toxins by a large percentage of the toxigenicA. flavus isolates suggest that there is a potential health hazard for co-existence of both toxins in foods and feeds.     

Cyclopiazonic acid production by submerged cultures of Penicillium and Aspergillus strains

Abstract 62 isolates of Penicillium and Aspergillus were screened for cyclopiazonic acid (CPA) production by surface and submerged culture on different media. The production of this mycotoxin was restricted to Penicillium camembertii group II (and its domesticated form P. camembertii ), P. griseofulvum , and Aspergillus flavus (and its domesticated form A. oryzae ). The best yield of CPA was obtained by a strain of P. griseofulvum , but several strains of P. camembertii group II were also good producers. Propionic acid (500 and 1000 mg/l medium) did not enhance the production of CPA. The best yields of CPA were obtained in submerged culture, but in some cases growth and CPA production only occured in surface culture. A simplified procedure for isolation of CPA is described.     

Clustered genes involved in cyclopiazonic acid production are next to the aflatoxin biosynthesis gene cluster in Aspergillus flavus

Cyclopiazonic acid (CPA), an indole-tetramic acid mycotoxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins. Aflatoxin biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. flavus lacking aflatoxin production due to the loss of the entire aflatoxin gene cluster and portions of the subtelomeric region are often unable to produce CPA, which suggests a physical link of genes involved in CPA biosynthesis to the aflatoxin gene cluster. Examining the subtelomeric region in A. flavus isolates of different chemotypes revealed a region possibly associated with CPA production. Disruption of three of the four genes present in this region predicted to encode a monoamine oxidase, a dimethylallyl tryptophan synthase, and a hybrid polyketide non-ribosomal peptide synthase abolished CPA production in an aflatoxigenic A. flavus strain. Therefore, some of the CPA biosynthesis genes are organized in a mini-gene cluster that is next to the aflatoxin gene cluster in A. flavus.     

Characterization of carboxypeptidase A6, an extracellular matrix peptidase

Carboxypeptidase A6 (CPA6) is a member of the M14 metallocarboxypeptidase family that is highly expressed in the adult mouse olfactory bulb and broadly expressed in embryonic brain and other tissues. A disruption in the human CPA6 gene is linked to Duane syndrome, a defect in the abducens nerve/lateral rectus muscle connection. In this study the cellular distribution, processing, and substrate specificity of human CPA6 were investigated. The 50-kDa pro-CPA6 is routed through the constitutive secretory pathway, processed by furin or a furin-like enzyme into the 37-kDa active form, and secreted into the extracellular matrix. CPA6 cleaves the C-terminal residue from a range of substrates, including small synthetic substrates, larger peptides, and proteins. CPA6 has a preference for large hydrophobic C-terminal amino acids as well as histidine. Peptides with a penultimate glycine or proline are very poorly cleaved. Several neuropeptides were found to be processed by CPA6, including Met- and Leu-enkephalin, angiotensin I, and neurotensin. Whereas CPA6 converts enkephalin and neurotensin into forms known to be inactive toward their receptors, CPA6 converts inactive angiotensin I into the biologically active angiotensin II. Taken together, these data suggest a role for CPA6 in the regulation of neuropeptides in the extracellular environment within the olfactory bulb and other parts of the brain.     

Naturally Occurring Carboxypeptidase A6 Mutations: EFFECT ON ENZYME FUNCTION AND ASSOCIATION WITH EPILEPSY*

Carboxypeptidase A6 (CPA6) is a member of the A/B subfamily of M14 metallocarboxypeptidases that is expressed in brain and many other tissues during development. Recently, two mutations in human CPA6 were associated with febrile seizures and/or temporal lobe epilepsy. In this study we screened for additional CPA6 mutations in patients with febrile seizures and focal epilepsy, which encompasses the temporal lobe epilepsy subtype. Mutations found from this analysis as well as CPA6 mutations reported in databases of single nucleotide polymorphisms were further screened by analysis of the modeled proCPA6 protein structure and the functional role of the mutated amino acid. The point mutations predicted to affect activity and/or protein folding were tested by expression of the mutant in HEK293 cells and analysis of the resulting CPA6 protein. Common polymorphisms in CPA6 were also included in this analysis. Several mutations resulted in reduced enzyme activity or CPA6 protein levels in the extracellular matrix. The mutants with reduced extracellular CPA6 protein levels showed normal levels of 50-kDa proCPA6 in the cell, and this could be converted into 37-kDa CPA6 by trypsin, suggesting that protein folding was not greatly affected by the mutations. Interestingly, three of the mutations that reduced extracellular CPA6 protein levels were found in patients with epilepsy. Taken together, these results provide further evidence for the involvement of CPA6 mutations in human epilepsy and reveal additional rare mutations that inactivate CPA6 and could, therefore, also be associated with epileptic phenotypes.     

A reversed-phase high-performance liquid chromatographic method for the simultaneous determination of serum concentrations of cyproterone acetate and 15 beta-hydroxycyproterone acetate

A reversed-phase high-performance liquid chromatographic method for the simultaneous determination of cyproterone acetate (CPA), 15 beta-hydroxycyproterone acetate (15 beta-OH-CPA) and cyproterone (CP) was reported. This method was specific, sensitive, precise, easy and rapid for determination of the serum concentrations of these steroids in patients receiving CPA. Although no peak corresponding to CP was observed for serum, peaks corresponding to CPA and 15 beta-OH-CPA were detected and well separated in all subjects undergoing long-term CPA therapy. In these patients, there seemed to be a dose-dependent relationship between the amount of CPA administered and the serum concentrations of these steroids, and the serum concentrations of CPA were either similar or low compared with those of 15 beta-OH-CPA. In conclusion, this simplified method is thought to be very valuable for studies on the pharmacokinetics of CPA and 15 beta-OH-CPA, and on the relationship between the CPA dosage and the therapeutic or side effects on adrenal and gonadal steroid production.     

Use of directed mutagenesis to probe the role of tyrosine 198 in the catalytic mechanism of carboxypeptidase A

Derivatization of Tyr198 in carboxypeptidase A (CPA) results in lowered catalytic activity toward peptide substrates (Cueni, L., and Riordan, J.F. (1978) Biochemistry 17, 1834-1842). We have synthesized via directed mutagenesis a rat CPA variant [Phe198] CPA containing a Tyr198-to-Phe substitution in order to test whether the phenolic hydroxyl plays a critical role in catalysis. A double mutant [Phe193, Phe248]CPA in which both Tyr198 and Tyr248 have been replaced by phenylalanine has also been engineered. Enzymatic characterization of [Phe198]CPA indicates that the Tyr198 hydroxyl is not obligatory for the hydrolysis of peptide and ester substrates. Furthermore, parallel studies with [Phe198, Phe248]CPA show that simultaneous removal of both the Tyr198 and Tyr248 hydroxyls does not abolish catalytic activity. Analysis of the acetylated derivatives of [Phe198]CPA, [Phe248]CPA, and [Phe198, Phe248]CPA establishes that Tyr198 and Tyr248 are the active site tyrosines which are modified by N-acetylimidazole. In addition, the perturbations of enzymatic activity which accompany acetylation of native CPA can be largely assigned to derivatization of Tyr248. The changes in the kinetic constants of substrate hydrolysis due to the Tyr198-to-Phe substitution are manifested as small decreases in the kcat values, but the Km values are essentially unaffected. This exclusive effect on the kcat values suggests that the Tyr198 hydroxyl participates in catalysis by stabilizing the rate-determining transition-state complex.     

Structural evolution of an enzyme specificity. The structure of rat carboxypeptidase A2 at 1.9-A resolution.

The structure of rat carboxypeptidase A2 (CPA2), which has a unique specificity for tryptophan-containing COOH-terminal peptides, has been determined in an unliganded state at 1.9-A resolution and refined to a crystallographic R-factor of 18.3%. Comparison of the structure of CPA2 with that of bovine carboxypeptidase A (referred to here as CPA1) reveals that the specificity of the former for larger amino acids probably arises from two amino acid replacements within the binding cavity (Thr268----Ala and Leu203----Met), coupled with differences in the positions of conserved residues in a surface loop on one face of the specificity pocket. The position of the reactive-site surface loop may be affected also by a disulfide bridge between Cys210 and Cys244. In this unliganded form of the enzyme, Tyr248 takes up a position interior to the specificity pocket and is distinct from that observed in bovine CPA1. The structural differences between CPA1 and CPA2 correlate strongly with crystallographically determined temperature factors and thus appear to be largest where the enzyme is flexible.     

Sulfamide derivatives as transition state analogue inhibitors for carboxypeptidase A

3-Phenyl-2-sulfamoyloxypropionic acid (2), 2-benzyl-3-sulfamoylpropionic acid (3), and N-(N-hydroxysulfamoyl)phenylalanine (5) have been synthesized and evaluated as inhibitors for carboxypeptidase A (CPA) to find that they inhibit the enzyme competitively with the Ki values in the microM range, suggesting that their binding modes to CPA are analogous to each other, and resemble the binding mode of N-sulfamoylphenylalanine (1) that has been established by the X-ray crystallographic method to form a complex with CPA in a manner reminiscent of the binding of a transition state in the catalytic pathway. It was concluded thus that they are a new type of transition state analogue inhibitors for CPA. (R)-N-Hydroxy-N-sulfamoyl-beta-phenylalanine (8) was shown to be also a potent CPA inhibitor (Ki = 39 microM), the high potency of which may be ascribed to the involvement of the hydroxyl in the binding of CPA, most likely forming bidentate coordinative bonds to the zinc ion in CPA together with the sulfamoyl oxygen atom.     

Zinc-mediated thermal stabilization of carboxypeptidase A

In this study we investigated the contribution of Zn ions to the catalytic and structural thermostability of carboxypeptidase A (CPA). Structural studies on CPA molecule, performed in the presence of a number of ligands, demonstrated the multiple binding models around Zn ions which may affect the enzyme functions. Zinc was reported to bind at various sites in the CPA molecule at room temperature leading to inhibition of its enzymic activity. In this study we found that binding of Zn to CPA molecule followed by exposure to 50 degrees C did not inhibit the enzymic activity but activates and protects it against heat denaturation. The stabilization effect was found to be dependent on the increasing Zn/CPA ratios. The moderate changes of CPA activity as well as the UV and fluorescence spectra analyses indicate that the main function of the newly introduced zinc atoms is structural rather than catalytical.     

The molecular basis for cyclopiazonic acid inhibition of the sarcoplasmic reticulum calcium pump

The sarcoplasmic reticulum Ca(2+)-ATPase is essential for calcium reuptake in the muscle contraction-relaxation cycle. Here we present structures of a calcium-free state with bound cyclopiazonic acid (CPA) and magnesium fluoride at 2.65 A resolution and a calcium-free state with bound CPA and ADP at 3.4A resolution. In both structures, CPA occupies the calcium access channel delimited by transmembrane segments M1-M4. Inhibition of Ca(2+)-ATPase is stabilized by a polar pocket that surrounds the tetramic acid of CPA and a hydrophobic platform that cradles the inhibitor. The calcium pump residues involved include Gln(56), Leu(61), Val(62), and Asn(101). We conclude that CPA inhibits the calcium pump by blocking the calcium access channel and immobilizing a subset of transmembrane helices. In the E2(CPA) structure, ADP is bound in a distinct orientation within the nucleotide binding pocket. The adenine ring is sandwiched between Arg(489) of the nucleotide-binding domain and Arg(678) of the phosphorylation domain. This mode of binding conforms to an adenine recognition motif commonly found in ATP-dependent proteins.     

Identification and characterization of three members of the human metallocarboxypeptidase gene family

Amino acid homology searches of the human genome revealed three members of the metallocarboxypeptidase (metallo-CP) family that had not been described in the literature in addition to the 14 known genes. One of these three, named CPA5, is present in a gene cluster with CPA1, CPA2, and CPA4 on chromosome 7. The cDNA encoding a mouse homolog of human CPA5 was isolated from a testis library and sequenced. The deduced amino acid sequence of human CPA5 has highest amino acid sequence identity (60%) to CPA1. Modeling analysis shows the overall structure to be very similar to that of other members of the A/B subfamily of metallocarboxypeptidases. The active site of CPA5 is predicted to cleave substrates with C-terminal hydrophobic residues, as do CPA1, -2, and -3. Using Northern blot analysis, CPA5 mRNA is detected in testis but not in kidney, liver, brain, or lung. In situ hybridization analysis shows that CPA5 is localized to testis germ cells. Mouse pro-CPA5 protein expressed in Sf9 cells using the baculovirus system was retained in the particulate fraction of the cells and was not secreted into the media. Pro-CPA5 was not enzymatically active toward standard CPA substrates, but after incubation with prohormone convertase 4 the resulting protein was able to cleave furylacryloyl-Gly-Leu, with 3-4-fold greater activity at pH 7.4 than at 5.6. Two additional members of the human CP gene family were also studied. Modeling analysis indicates that both contain the necessary amino acids required for enzymatic activity. The CP on chromosome 8 is predicted to have a CPA-like specificity for C-terminal hydrophobic residues and was named CPA6. The CP on chromosome 2 is predicted to cleave substrates with C-terminal acidic residues and was named CPO.