Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We functionally characterized human skin mast cell carboxypeptidase A (MC-CPA), and explored its evolutionary relationship to other carboxypeptidases to understand further the structural basis for the substrate preferences of this enzyme. Purified human skin MC-CPA displayed more activity than did bovine pancreatic carboxypeptidase A (CPA) against carboxyl-terminal leucine residues, about equal activity with phenylalanine and tyrosine residues, and no activity with tryptophan or alanine. To correlate kinetic data with structure, we isolated and sequenced a cDNA encoding MC-CPA from human skin, and directly sequenced 30% of the purified protein. These sequences agreed with that of human lung MC-CPA, and further support the evidence for a single MC-CPA gene in humans. Four amino acid replacements, resulting in a net positive change in non-hydrogen atoms in the S1' subsite of MC-CPA, were associated with less alteration in substrate specificity, relative to bovine CPA, than might be expected from studies using rat CPA1 and CPA2. We noted two consensus N-linked glycosylation sites in human MC-CPA that are not found in rat and mouse MC-CPA, or in bovine CPA; that at least one of these sites is glycosylated in vivo was verified by N-glycosidase F treatment, lentil lectin binding, and Concanavalin A-Sepharose chromatography. Evolutionary trees constructed from the known carboxypeptidase sequences suggested that MC-CPA most likely evolved from a carboxypeptidase B-like enzyme, independent of the pancreatic CPA. Thus, in the carboxypeptidase gene family, MC-CPA displays a unique genealogy and several amino acid replacements in its S1' binding pocket that result in substrate specificity quite similar to bovine CPA.
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PMID:Human skin mast cell carboxypeptidase: functional characterization, cDNA cloning, and genealogy. 162 26

No gene for a hematopoietic cell carboxypeptidase has previously been characterized. Mast cell carboxypeptidase A (MC-CPA) is a prominent secretory granule marker of mast cell differentiation and phenotype. The 32-kb human MC-CPA gene was isolated, localized to chromosome 3, and found to contain 11 exons. No significant homology was found between the 5' flanking region of the MC-CPA gene and those of three rat pancreatic carboxypeptidase genes (carboxypeptidase A1 and A2, and carboxypeptidase B [CPB]). In contrast, the intron/exon organization of the MC-CPA gene was conserved, most closely resembling the CPB gene. MC-CPA is unique among carboxypeptidases in having a CPA-like substrate-binding pocket and enzymatic activity despite overall protein and gene structures more similar to CPB. Evolutionary tree analysis of the carboxypeptidase gene family showed that, before the mammalian species radiation, a common MC-CPA/CPB ancestor diverged by gene duplication from the lineage leading to CPA, and then underwent another gene duplication to form separate but similar gene structures for MC-CPA and CPB. MC-CPA mRNA was prominent in dispersed lung cells enriched for mast cells but was undetectable in other nontransformed populations of several lineages, demonstrating that transcription of MC-CPA, a novel carboxypeptidase gene, provides a specific molecular marker for mast cells among normal hematopoietic cell populations.
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PMID:Cloning and characterization of the novel gene for mast cell carboxypeptidase A. 172 76

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.
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PMID:Structural evolution of an enzyme specificity. The structure of rat carboxypeptidase A2 at 1.9-A resolution. 176 58

Crystallographic studies suggest that Arg-127 is a key amino acid in the hydrolysis of peptides and esters by carboxypeptidase A. The guanidinium group of Arg-127 is hypothesized to stabilize the oxyanion of the tetrahedral intermediate formed by the attack of water on the scissile carbonyl bond. We have replaced this amino acid in rat carboxypeptidase A1 with lysine (R127K), methionine (R127M), and alanine (R127A), in order to define the role of Arg-127 in carboxypeptidase catalyzed hydrolysis. The wild-type and mutant enzymes were expressed in yeast and purified. Kinetic studies show that Arg-127 substitution decreases kcat for both ester and amide substrates, whereas Km is relatively unchanged; for R127M and R127A this corresponds to a 6 kcal/mol decrease in transition state stabilization of the rate-limiting step. The binding affinity for the phosphonate transition state analog, Cbz-Phe-Ala(P)-OAla, was decreased by 5.4 kcal/mol, whereas binding affinity for the ground state inhibitor, DL-benzylsuccinic acid, was decreased by only 1.7 kcal/mol for R127M. Electrostatic calculations employing a finite difference solution to the Poisson-Boltzmann equation predict that the positive charge of Arg-127 should stabilize the transition state by 6-8 kcal/mol. Therefore, the experimental and theoretical data suggest that the primary role of Arg-127 is stabilization of the transition state through electrostatic interaction with the oxyanion.
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PMID:Arginine 127 stabilizes the transition state in carboxypeptidase. 224 16

A method is reported for the preparative isolation of the two forms of pro-(carboxypeptidase A) from pig pancreas: the monomer and the binary complex with pro-(proteinase E). This method, which is mainly based on chromatography on DEAE-Sepharose at pH 5.7, allows these proenzymes to be prepared more quickly and in safer conditions than with other reported methods. Undegraded and homogeneous carboxypeptidase A1 and A2 species (peptidyl-L-amino acid hydrolase, EC 3.4.17.1), in monomeric forms with high specific activity, are also obtained in high yield by controlled trypsin activation of either of the pro-(carboxypeptidases A) followed by chromatography on DEAE-Sepharose at pH 5.8 under dissociating conditions (7 M-urea).
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PMID:Preparative isolation of the two forms of pig pancreatic pro-(carboxypeptidase A) and their monomeric carboxypeptidases A. 405 13

The presence of carboxypeptidase A (EC 3.4.17.1; CPA) gene transcripts and corresponding catalytic activity was investigated in brain and other extradigestive rat tissues in which presence of the pancreatic enzyme had not been reported so far. Transcripts of two known CPA genes, CPA1 and CPA2, were identified in extremely low abundance in brain and several other extrapancreatic tissues using Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Whereas the CPA1 gene transcripts in brain, heart, stomach, or colon had a size similar to that in pancreas (1.35 kilobases), the CPA2 gene transcripts in brain, testis, or lung were of a smaller size (1.1 kilobases). Northern blot analysis using various probes, RT-PCR, and 5'-rapid amplification of cDNA 5'-end (5' RACE analysis) all indicated that this smaller size of the brain transcript was attributable to production by alternative splicing of the pro-mRNA. This process corresponds to deletion of the first four exons, leading to a mRNA encoding a protein in which the signal peptide and activation peptide of prepro-CPA2 are absent but the active site remains. The prediction that the shorter CPA2 isoform, designated CPA2(S), should correspond to a cytoplasmic metallopeptidase that does not require tryptic activation was verified by characterization of the recombinant protein and comparing it with the native CPA-like activity in brain. Both recombinant CPA2(S) generated in Escherichia coli and a soluble protein from brain displayed similar sizes on Western blots (32 kDa to be compared to 34 kDa for pancreatic CPA2). Recombinant CPA2(S) and a soluble CPA-like activity from brain displayed similar sensitivity to a series of inhibitors, contrasting with that of the pancreatic enzyme. It is concluded that alternative splicing produces a truncated CPA2 with distinct subcellular localization and modified catalytic activity. In spite of the presence of the CPA1 mRNA, no corresponding CPA activity could be detected in brain extracts, even after tryptic activation. This apparent discrepancy seems attributable to the presence of an endogenous peptide inhibitor which remains to be identified.
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PMID:Carboxypeptidase A isoforms produced by distinct genes or alternative splicing in brain and other extrapancreatic tissues. 765 30

The recent demonstration of the occurrence in rat brain and other nonpancreatic tissues of carboxypeptidase A (CPA) gene transcripts without associated catalytic activity could be ascribed to the presence of a soluble endogenous protein inhibitor. This tissue carboxypeptidase inhibitor (TCI), detected by the inhibition of added bovine pancreatic CPA, was purified from rat brain. Peptides were obtained by partial proteolysis of purified TCI, a protein of approximately 30 kDa, and starting from their sequences, a full-length cDNA encoding a 223-amino acid protein containing three potential phosphorylation sites was cloned from a cDNA library. Its identity with TCI was shown by expression in Escherichia coli of a recombinant protein recognized by antibodies raised against native TCI and display characteristic CPA-inhibiting activity. TCI appears as a hardly reversible, non-competitive, and potent inhibitor of CPA1 and CPA2 (Ki approximately 3 nM) and mast-cell CPA (Ki = 16 nM) and inactive on various other proteases. This pattern of selectivity might be attributable to a limited homology of a 11-amino acid sequence with sequences within the activation segments of CPA and CPB known to interact with residues within their active sites. The widespread expression of TCI in a number of tissues (e.g., brain, lung, or digestive tract) and its apparently cytosolic localization point to a rather general functional role, e.g., in the control of cytosolic protein degradation.
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PMID:Purification, cDNA cloning, functional expression, and characterization of a 26-kDa endogenous mammalian carboxypeptidase inhibitor. 861 74

Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stable in vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine- and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human carboxypeptidases A. Two of these analogs are MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-alpha-phenylalanine). Thus, the kcat/Km value for the wild type hCPA1 with MTX-alpha-phenylalanine is 0.44 microM-1 s-1, and kcat/Km values for hCPA1-T268G with MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine are 1.8 and 0.16 microM-1 s-1, respectively. The cytotoxic efficiency of hCPA1-268G was tested in an in vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B cell antigen CDw52. These conjugates were then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath 1H antigen) followed by incubation of the cells with the in vivo stable prodrugs. The results showed that the targeted ING-1:hCPA1-T268G conjugate produced excellent activation of the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By contrast, the enzyme-Campath 1H conjugate was without effect. These data strongly support the feasibility of ADEPT using a mutated human enzyme with a single amino acid change.
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PMID:Toward antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate. 918 78

Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described human plasma zymogen that is related to pancreatic carboxypeptidase B. The active form of TAFI (TAFIa), which is formed by thrombin cleavage of the zymogen, likely inhibits fibrinolysis by removal from partially degraded fibrin of the carboxyl-terminal lysine residues which act to stimulate plasminogen activation. We have isolated and characterized genomic clones which encompass the entire human TAFI gene from lambda phage and bacterial artificial chromosome genomic libraries. The complete TAFI gene contains 11 exons and spans approximately 48 kb of genomic DNA. The positions of intron/exon boundaries are conserved between the TAFI gene and the rat pancreatic carboxypeptidase A1, A2, and B and the human mast cell carboxypeptidase A genes, indicating that these carboxypeptidases arose from a common ancestral gene. However, the intron lengths diverge significantly among all of these genes. The TAFI promoter lacks a consensus TATA sequence, and transcription is initiated from multiple sites. Transient transfection of reporter plasmids containing portions of the TAFI 5'-flanking region into mammalian cells allowed localization of the promoter and identified a approximately 70 bp region crucial for liver-specific transcription. Sequence analysis of cDNA clones obtained from human liver RNA indicated that the TAFI transcript is polyadenylated at three different sites. Our findings will facilitate the assessment of the regulation of TAFI expression by transcriptional and/or posttranscriptional mechanisms. Furthermore, knowledge of the genomic structure of the TAFI gene will aid in the identification of mutations that may be associated with the tendency to either bleed or thrombose.
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PMID:Characterization of the gene encoding human TAFI (thrombin-activable fibrinolysis inhibitor; plasma procarboxypeptidase B). 1035 Apr 73

Imprinted gene(s) on human chromosome 7q32-qter have been postulated to be involved in intrauterine growth restriction associated with Silver-Russell syndrome (SRS) as 7-10% of patients have mUPD(7). Three imprinted genes, MEST, MESTIT1, and COPG2IT1 on chromosome 7q32, are unlikely to cause SRS since epigenetic and sequence mutation analyses have not shown any changes. One hundred kilobases proximal to MEST lies a group of four carboxypeptidase A (CPA) genes. Since most imprinted genes are found in clusters, this study focuses on analysing these CPAs for imprinting effects based on their proximity to an established imprinted domain. Firstly, a replication timing study across 7q32 showed that an extensive genomic region including the CPAs, MEST, MESTIT1, and COPG2IT1 replicates asynchronously. Subsequently, SNP analysis by sequencing RT-PCR products of CPA1, CPA2, CPA4, and CPA5 indicated preferential expression of CPA4. Pyrosequencing was used as a quantitative approach, which confirmed predominantly preferential expression of the maternal allele and biallelic expression in brain. CPA5 expression levels were too low to allow reliable evaluation of allelic expression, while CPA1 and CPA2 both showed biallelic expression. CPA4 was the only gene from this family in which an imprinting effect was shown despite the location of this family of genes next to an imprinted cluster. As CPA4 has a potential role in cell proliferation and differentiation, two preferentially expressed copies in mUPD patients with SRS syndrome would result in excess expression and could alter the growth profiles of these subjects and give rise to intrauterine growth restriction.
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PMID:The imprinted region on human chromosome 7q32 extends to the carboxypeptidase A gene cluster: an imprinted candidate for Silver-Russell syndrome. 1267 94


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