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Dirk Hendriks - One of the best experts on this subject based on the ideXlab platform.
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242 – Carboxypeptidase U
Handbook of Proteolytic Enzymes, 2020Co-Authors: Dirk HendriksAbstract:Publisher Summary This chapter elaborates the structural chemistry and the biological aspects of Carboxypeptidase U (CPU). The primary structure of proCPU has been deduced from a cDNA sequence. The primary translation product consists of 423 amino acids containing a 22 amino acid signal peptide, a 92 amino acid activation peptide and a 309 amino acid catalytic domain. ProCPU has a molecular mass of 60 kDa (SDS-PAGE). Proteolytic cleavage by thrombin, plasmin or trypsin releases the 92 amino acid activation peptide, which is heavily glycosylated, results in the formation of the nonglycosylated 36 kDa catalytic unit. After its activation, CPU is very unstable. This instability is strongly temperature dependent, with a CPU half-life of 10 min at 37°C. Inactivation of CPU is based on its conformational instability rather than on further proteolytic cleavage at Arg302. Both proCPU and CPU bind to plasminogen and are substrates for trans-glutaminase catalyzed crosslinking. CPU exhibits high sequence similarity (around 50% identity) to both pancreatic Carboxypeptidases A and B and thus is presumed to have a fold similar to that of the well-known Carboxypeptidase A structure.
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Carboxypeptidase u a plasma Carboxypeptidase with high affinity for plasminogen
Journal of Biological Chemistry, 1994Co-Authors: W. Wang, Dirk Hendriks, Simon ScharpeAbstract:: A novel basic Carboxypeptidase clearly different from Carboxypeptidase N has been isolated from human plasma. It circulates as an enzymatically inactive precursor enzyme bound to plasminogen. During fibrinolysis, it can be converted to its active form, Carboxypeptidase U, through the action of plasmin. The active enzyme has an apparent molecular weight of 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It hydrolyzes the synthetic peptides hippuryl-L-arginine and hippuryl-L-lysine but, in contrast to other human basic Carboxypeptidases, has only a limited esterase activity. After its activation, Carboxypeptidase U tends to be very unstable.
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On the specificity of Carboxypeptidase N, a comparative study
Biological chemistry Hoppe-Seyler, 1993Co-Authors: Dirk Hendriks, W. Wang, Martin Vingron, Gerrit Vriend, Dominique Nalis, Simon ScharpeAbstract:: The structure of the enzymatically active subunit of human plasma Carboxypeptidase N was modeled based on the homology with bovine Carboxypeptidase A. The active site of Carboxypeptidase N is well conserved in comparison with Carboxypeptidase A. From a comparison of energetically favorable binding sites for different atomic probe groups a hypothesis for the differences in substrate specificity between Carboxypeptidases A and N was derived. Small synthetic peptide substrates were synthesized to confirm this hypothesis. This study shows that even with very low homology model building by homology can be employed to build models of sufficient quality to aid in drug design.
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purification and characterization of a new arginine Carboxypeptidase in human serum
Biochimica et Biophysica Acta, 1990Co-Authors: Dirk Hendriks, Marie-paule Lommaert, Simon Scharpe, W. Wang, Marc Van SandeAbstract:Abstract A Carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates in present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its unstability at room temperature and at 37°C, we named it unstable Carboxypeptidase (Carboxypeptidase U). Carboxypeptidase U was partially purified from fresh human serum by chromatography on DEAE-cellulose and Mono-Q sepharose and was found to be a 435 kDa protein. We compared this enzyme with Carboxypeptidase N, purified from human serum by a two-step affinity chromatography on arginine-Sepharose 4B, followed by ion-exchange chromatography on Mono-Q sepharose. Carboxypeptidase U cleaves hippuryl- l -arginine and hippuryl- l -lysine, but at a different relative rate than Carboxypeptidase N, and has no esterase activity on hippuryl- l -argininic acid. Its activity was inhibited by o-phenanthroline, dl -2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, CoCl2, 2-mercaptoethanol, dithiothreitol and 4-chloromercuribenzoic acid. These characteristics differentiate Carboxypeptidase U from Carboxypeptidase N and other known Carboxypeptidase.
Simon Scharpe - One of the best experts on this subject based on the ideXlab platform.
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Carboxypeptidase u a plasma Carboxypeptidase with high affinity for plasminogen
Journal of Biological Chemistry, 1994Co-Authors: W. Wang, Dirk Hendriks, Simon ScharpeAbstract:: A novel basic Carboxypeptidase clearly different from Carboxypeptidase N has been isolated from human plasma. It circulates as an enzymatically inactive precursor enzyme bound to plasminogen. During fibrinolysis, it can be converted to its active form, Carboxypeptidase U, through the action of plasmin. The active enzyme has an apparent molecular weight of 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It hydrolyzes the synthetic peptides hippuryl-L-arginine and hippuryl-L-lysine but, in contrast to other human basic Carboxypeptidases, has only a limited esterase activity. After its activation, Carboxypeptidase U tends to be very unstable.
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On the specificity of Carboxypeptidase N, a comparative study
Biological chemistry Hoppe-Seyler, 1993Co-Authors: Dirk Hendriks, W. Wang, Martin Vingron, Gerrit Vriend, Dominique Nalis, Simon ScharpeAbstract:: The structure of the enzymatically active subunit of human plasma Carboxypeptidase N was modeled based on the homology with bovine Carboxypeptidase A. The active site of Carboxypeptidase N is well conserved in comparison with Carboxypeptidase A. From a comparison of energetically favorable binding sites for different atomic probe groups a hypothesis for the differences in substrate specificity between Carboxypeptidases A and N was derived. Small synthetic peptide substrates were synthesized to confirm this hypothesis. This study shows that even with very low homology model building by homology can be employed to build models of sufficient quality to aid in drug design.
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purification and characterization of a new arginine Carboxypeptidase in human serum
Biochimica et Biophysica Acta, 1990Co-Authors: Dirk Hendriks, Marie-paule Lommaert, Simon Scharpe, W. Wang, Marc Van SandeAbstract:Abstract A Carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates in present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its unstability at room temperature and at 37°C, we named it unstable Carboxypeptidase (Carboxypeptidase U). Carboxypeptidase U was partially purified from fresh human serum by chromatography on DEAE-cellulose and Mono-Q sepharose and was found to be a 435 kDa protein. We compared this enzyme with Carboxypeptidase N, purified from human serum by a two-step affinity chromatography on arginine-Sepharose 4B, followed by ion-exchange chromatography on Mono-Q sepharose. Carboxypeptidase U cleaves hippuryl- l -arginine and hippuryl- l -lysine, but at a different relative rate than Carboxypeptidase N, and has no esterase activity on hippuryl- l -argininic acid. Its activity was inhibited by o-phenanthroline, dl -2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, CoCl2, 2-mercaptoethanol, dithiothreitol and 4-chloromercuribenzoic acid. These characteristics differentiate Carboxypeptidase U from Carboxypeptidase N and other known Carboxypeptidase.
Lloyd D. Fricker - One of the best experts on this subject based on the ideXlab platform.
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Carboxypeptidase o is a glycosylphosphatidylinositol anchored intestinal peptidase with acidic amino acid specificity
Journal of Biological Chemistry, 2011Co-Authors: Peter J Lyons, Lloyd D. FrickerAbstract:Abstract The first metalloCarboxypeptidase (CP) was identified in pancreatic extracts more than 80 years ago and named Carboxypeptidase A (CPA; now known as CPA1). Since that time, seven additional mammalian members of the CPA subfamily have been described, all of which are initially produced as proenzymes, are activated by endoproteases, and remove either C-terminal hydrophobic or basic amino acids from peptides. Here we describe the enzymatic and structural properties of Carboxypeptidase O (CPO), a previously uncharacterized and unique member of the CPA subfamily. Whereas all other members of the CPA subfamily contain an N-terminal prodomain necessary for folding, bioinformatics and expression of both human and zebrafish CPO orthologs revealed that CPO does not require a prodomain. CPO was purified by affinity chromatography, and the purified enzyme was able to cleave proteins and synthetic peptides with greatest activity toward acidic C-terminal amino acids unlike other CPA-like enzymes. CPO displayed a neutral pH optimum and was inhibited by common metalloCarboxypeptidase inhibitors as well as citrate. CPO was modified by attachment of a glycosylphosphatidylinositol membrane anchor to the C terminus of the protein. Immunocytochemistry of Madin-Darby canine kidney cells stably expressing CPO showed localization to vesicular membranes in subconfluent cells and to the plasma membrane in differentiated cells. CPO is highly expressed in intestinal epithelial cells in both zebrafish and human. These results suggest that CPO cleaves acidic amino acids from dietary proteins and peptides, thus complementing the actions of well known digestive Carboxypeptidases CPA and CPB.
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The Crystal Structure of the Inhibitor-complexed Carboxypeptidase D Domain II and the Modeling of Regulatory Carboxypeptidases
Journal of Biological Chemistry, 2001Co-Authors: Patrick Aloy, Josep Vendrell, Francesc X. Avilés, Verònica Companys, Lloyd D. Fricker, Miquel Coll, F. Xavier Gomis-rüthAbstract:Abstract The three-dimensional crystal structure of duck Carboxypeptidase D domain II has been solved in a complex with the peptidomimetic inhibitor, guanidinoethylmercaptosuccinic acid, occupying the specificity pocket. This structure allows a clear definition of the substrate binding sites and the substrate funnel-like access. The structure of domain II is the only one available from the regulatory Carboxypeptidase family and can be used as a general template for its members. Here, it has been used to model the structures of domains I and III from the former protein and of human Carboxypeptidase E. The models obtained show that the overall topology is similar in all cases, the main differences being local and because of insertions in non-regular loops. In both Carboxypeptidase D domain I and Carboxypeptidase E slightly different shapes of the access to the active site are predicted, implying some kind of structural selection of protein or peptide substrates. Furthermore, emplacement of the inhibitor structure in the active site of the constructed models showed that the inhibitor fits very well in all of them and that the relevant interactions observed with domain II are conserved in domain I and Carboxypeptidase E but not in the non-active domain III because of the absence of catalytically indispensable residues in the latter protein. However, in domain III some of the residues potentially involved in substrate binding are well preserved, together with others of unknown roles, which also are highly conserved among all Carboxypeptidases. These observations, taken together with others, suggest that domain III might play a role in the binding and presentation of proteins or peptide substrates, such as the pre-S domain of the large envelope protein of duck hepatitis B virus.
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cloning and expression of human Carboxypeptidase z a novel metalloCarboxypeptidase
Journal of Biological Chemistry, 1997Co-Authors: Lixin Song, Lloyd D. FrickerAbstract:Abstract A novel cDNA, designated Carboxypeptidase Z (CPZ), was identified based on its homology to known metalloCarboxypeptidases. Northern blot analysis shows bands of 2.1 and/or 2.6 kilobases in all tissues examined. The major form of CPZ mRNA in human salivary gland encodes a protein with an open reading frame of 641 amino acids. In addition, three variants were found that presumably arise due to alternative intron splicing. The 641-amino acid protein contains an 18-residue signal peptide-like sequence, a 120-residue region that shows 23–29% amino acid identity with a Cys-rich domain found in frizzled proteins and in type XVIII collagen, and then a 390-residue Carboxypeptidase domain with 49% amino acid identity to Carboxypeptidases E and N. The 641-amino acid form of CPZ expressed in the baculovirus system cleaves 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Phe-Ala-Arg, although the level of enzyme activity was approximately 10-fold lower than either Carboxypeptidase E or D expressed using the same viral system. The CPZ activity is more active at neutral pH than at pH 5.5 and is inhibited by active site-directed inhibitors of metalloCarboxypeptidases. In summary, CPZ is a novel metalloCarboxypeptidase that is active toward substrates with C-terminal basic amino acids.
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Carboxypeptidase e activity is deficient in mice with the fat mutation effect on peptide processing
Journal of Biological Chemistry, 1996Co-Authors: Lloyd D. Fricker, Yemiliya Berman, Edward H Leiter, Lakshmi A DeviAbstract:Abstract Carboxypeptidase E (CPE) is involved in the biosynthesis of many peptide hormones and neurotransmitters. Mice with the fat mutation have previously been found to have a point mutation in the cpe gene, and to have greatly reduced levels of CPE-like enzyme activity in the pituitary and pancreatic islets (Naggert, J. K., Fricker, L. D., Varlamov, O., Nishina, P. M., Rouille, Y., Steiner, D. F., Carroll, R. J., Paigen, B. J., and Leiter, E. H. (1995) Nat. Genet. 10, 135-142). In the present report, we examined CPE-like activity and peptide processing in several tissues of C57BLKS/LtJ-Cpefat/Cpefat mutant (Cpefat/Cpefat) mice. Whereas CPE-like activity is detected in homogenates of Cpefat/Cpefat mouse tissues, the majority of this activity is not due to CPE based on the sensitivity to p-chloromercuriphenyl sulfonate. In addition, the Cpefat/Cpefat activity does not bind to a substrate affinity column under conditions that bind CPE. Furthermore, the enzyme activity and immunoreactive properties of the activity purified from Cpefat/Cpefat brain are distinct from those of CPE. Taken together, these data suggest that CPE is completely inactive in the Cpefat/Cpefat mice, and that all of the CPE-like activity is due to other Carboxypeptidases such as Carboxypeptidase D. Levels of Leu-enkephalin in Cpefat/Cpefat mouse brain are approximately 5-fold lower than those in control brain. Treatment of the Cpefat/Cpefat brain extract with Carboxypeptidase B restores the level of Leu-enkephalin to the level in control brain. Interestingly, the large molecular weight enkephalin-containing peptides are elevated 2-3-fold in Cpefat/Cpefat mouse brain. These data indicate that CPE plays an important role in the processing of peptide hormones in various tissues, but that other Carboxypeptidases also contribute to peptide processing. Furthermore, the increase in levels of high molecular weight enkephalin peptides in the Cpefat/Cpefat mouse suggests that CPE is required for efficient peptide processing by the endopeptidases.
W. Wang - One of the best experts on this subject based on the ideXlab platform.
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Carboxypeptidase u a plasma Carboxypeptidase with high affinity for plasminogen
Journal of Biological Chemistry, 1994Co-Authors: W. Wang, Dirk Hendriks, Simon ScharpeAbstract:: A novel basic Carboxypeptidase clearly different from Carboxypeptidase N has been isolated from human plasma. It circulates as an enzymatically inactive precursor enzyme bound to plasminogen. During fibrinolysis, it can be converted to its active form, Carboxypeptidase U, through the action of plasmin. The active enzyme has an apparent molecular weight of 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It hydrolyzes the synthetic peptides hippuryl-L-arginine and hippuryl-L-lysine but, in contrast to other human basic Carboxypeptidases, has only a limited esterase activity. After its activation, Carboxypeptidase U tends to be very unstable.
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On the specificity of Carboxypeptidase N, a comparative study
Biological chemistry Hoppe-Seyler, 1993Co-Authors: Dirk Hendriks, W. Wang, Martin Vingron, Gerrit Vriend, Dominique Nalis, Simon ScharpeAbstract:: The structure of the enzymatically active subunit of human plasma Carboxypeptidase N was modeled based on the homology with bovine Carboxypeptidase A. The active site of Carboxypeptidase N is well conserved in comparison with Carboxypeptidase A. From a comparison of energetically favorable binding sites for different atomic probe groups a hypothesis for the differences in substrate specificity between Carboxypeptidases A and N was derived. Small synthetic peptide substrates were synthesized to confirm this hypothesis. This study shows that even with very low homology model building by homology can be employed to build models of sufficient quality to aid in drug design.
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purification and characterization of a new arginine Carboxypeptidase in human serum
Biochimica et Biophysica Acta, 1990Co-Authors: Dirk Hendriks, Marie-paule Lommaert, Simon Scharpe, W. Wang, Marc Van SandeAbstract:Abstract A Carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates in present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its unstability at room temperature and at 37°C, we named it unstable Carboxypeptidase (Carboxypeptidase U). Carboxypeptidase U was partially purified from fresh human serum by chromatography on DEAE-cellulose and Mono-Q sepharose and was found to be a 435 kDa protein. We compared this enzyme with Carboxypeptidase N, purified from human serum by a two-step affinity chromatography on arginine-Sepharose 4B, followed by ion-exchange chromatography on Mono-Q sepharose. Carboxypeptidase U cleaves hippuryl- l -arginine and hippuryl- l -lysine, but at a different relative rate than Carboxypeptidase N, and has no esterase activity on hippuryl- l -argininic acid. Its activity was inhibited by o-phenanthroline, dl -2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, CoCl2, 2-mercaptoethanol, dithiothreitol and 4-chloromercuribenzoic acid. These characteristics differentiate Carboxypeptidase U from Carboxypeptidase N and other known Carboxypeptidase.
Marc Van Sande - One of the best experts on this subject based on the ideXlab platform.
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purification and characterization of a new arginine Carboxypeptidase in human serum
Biochimica et Biophysica Acta, 1990Co-Authors: Dirk Hendriks, Marie-paule Lommaert, Simon Scharpe, W. Wang, Marc Van SandeAbstract:Abstract A Carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates in present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its unstability at room temperature and at 37°C, we named it unstable Carboxypeptidase (Carboxypeptidase U). Carboxypeptidase U was partially purified from fresh human serum by chromatography on DEAE-cellulose and Mono-Q sepharose and was found to be a 435 kDa protein. We compared this enzyme with Carboxypeptidase N, purified from human serum by a two-step affinity chromatography on arginine-Sepharose 4B, followed by ion-exchange chromatography on Mono-Q sepharose. Carboxypeptidase U cleaves hippuryl- l -arginine and hippuryl- l -lysine, but at a different relative rate than Carboxypeptidase N, and has no esterase activity on hippuryl- l -argininic acid. Its activity was inhibited by o-phenanthroline, dl -2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, CoCl2, 2-mercaptoethanol, dithiothreitol and 4-chloromercuribenzoic acid. These characteristics differentiate Carboxypeptidase U from Carboxypeptidase N and other known Carboxypeptidase.