Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.17.21 (prostate-specific membrane antigen)
 1,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutamate carboxypeptidase II may modulate excitatory neurotransmission through the catabolism of the neuropeptide N-acetylaspartylglutamate (NAAG) and possibly other endogenous peptide substrates. To investigate the molecular properties of cloned human GCP II (hGCP II), we analyzed the NAAG-hydrolytic activity conveyed by transfection of a full-length hGCP II cDNA into PC3 cells, which do not express GCP II endogenously. Membrane fractions from these cells demonstrated activity with an apparent Km of 73 nM and Vmax of 35 pmol/(mg protein*min). Activity was inhibited by EDTA and stimulated by the addition of CoCl2. Addition of GCP II inhibitors beta-NAAG, quisqualic acid and 2-(phosphonomethyl)pentanedioic acid (PMPA) inhibited hydrolysis of 2.5 nM NAAG with IC50s of 201 nM, 155 nM and 98 pM, respectively. In competition experiments designed to infer aspects of hGCP II substrate selectivity, NAAG was the most potent alpha peptide tested, with an IC50 of 26 nM. Folate derivatives and some other gamma-glutamyl peptides showed comparable affinity to that of NAAG, also displaying IC50s in the low nM range. Taken together with previous evidence demonstrating their presence in GCP II-expressing tissues, these data suggest that both NAAG and folates are good candidate substrates for GCP II in vivo.
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PMID:Hydrolysis of the neuropeptide N-acetylaspartylglutamate (NAAG) by cloned human glutamate carboxypeptidase II. 962 70

Glutamate carboxypeptidase II (GCP II) catalyzes the extracellular hydrolysis of the neuromodulator N-acetyl-aspartylglutamate to N-acetyl-aspartate and glutamate. GCP II also hydrolyzes gamma-glutamyl bonds in folylpolyglutamate. The predicted amino acid sequence of GCP II displays similarities to aminopeptidases from Streptomyces griseus and Vibrio proteolyticus, whose crystal structures have been determined. These aminopeptidases are cocatalytic zinc metallopeptidases belonging to the peptidase family M28. Specific zinc and substrate ligands have been proposed in GCP II based on the amino acid sequence alignment to these M28 family members. In the present study, site-directed mutagenesis has been used to test the assignment of these putative ligands in human GCP II. Substitutions to the five putative zinc ligands resulted in severely reduced enzyme activity, although mutant protein was expressed as demonstrated by immunoblot analysis. In addition, substitutions of amino acids near the putative zinc ligands have identified other specific residues important for enzyme structure and/or function. Substitutions to putative substrate ligands were less perturbing, and increases in Km were observed for substitutions that introduced a large charge perturbation (e.g., Lys to Glu). The results from substitutions at the proposed zinc and substrate ligands are consistent with the assignment of these residues and suggest that GCP II has a three-dimensional structure similar to other members of the peptidase family M28.
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PMID:Site-directed mutagenesis of predicted active site residues in glutamate carboxypeptidase II. 988 12

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44-750) in Drosophila Schneider's cells and its purification to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on fluorimetric detection of released alpha-amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV-like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate specificity of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac-Glu-Met, Ac-Asp-Met and, surprisingly, Ac-Ala-Glu and Ac-Ala-Met were identified as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity.
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PMID:Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II. 1190 94

Glutamate carboxypeptidase II (GCPII) hydrolyzes polyglutamyl folates before their absorption. Recently, a 1561 C>T polymorphism in the GCPII gene was reported to be associated with lower folate and higher homocysteine plasma concentrations in a small (n = 75) selected elderly population. In this study, we examined the effect of this polymorphism in 680 men and 644 women attending the fifth examination of the Framingham Offspring Study. At the time of sample collection, subjects were not taking any supplements and were not exposed to food folate fortification. GCPII genotypes were determined by allelic discrimination using Taqman probes. In the population as a whole, this mutation was not associated with lower plasma folate level or with elevated plasma homocysteine. In men, plasma folate concentrations were higher in carriers of the T allele compared with those homozygotes of the wild-type allele (P < 0.05), whereas in women folate concentrations did not differ between genotypes (P = 0.8). In its relationship to plasma folate, this mutation exhibited a weak interaction with age and gender only in older women (P = 0.05). Overall, our data show that the GCPII C1561T polymorphism is not a determinant of plasma folate or total homocysteine concentrations in this large cohort of participants from the Framingham Offspring Study.
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PMID:The glutamate carboxypeptidase gene II (C>T) polymorphism does not affect folate status in the Framingham Offspring cohort. 1204 30

Elevated levels of total homocysteine and low folate in blood are independent and graded risk factors for arterial occlusive disease. An impairment of folate distribution can be an important cause of hyperhomocysteinemia. Glutamate carboxypeptidase II (GCPII) regulates the absorption of dietary folates. In the present study, we examined the relationship of a 1561C-->T variant in the GCPII gene with fasting, post-methionine load plasma homocysteine, folate and vitamin B(12) levels and the risk of cardiovascular disease (CVD) in 190 vascular disease patients and in 601 apparently healthy controls. Fasting as well as post-load homocysteine concentrations associated with the 1561TT genotype tended to be lower, whereas the homocysteine concentrations of the 1561CT individuals were not different from their 1561CC peers. The 1561C-->T polymorphism significantly increased both red blood cell folate and plasma folate concentrations (ANOVA P=0.013; test for linear trend P=0.03, respectively), but had no effect on vitamin B(12) levels (ANOVA P=0.35). Since not only homocysteine itself is considered to be positively associated with the risk of CVD, but also a decreased folate status, the results of this study indicate that the 1561C-->T polymorphism may affect the predisposition to CVD.
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PMID:Influence of a glutamate carboxypeptidase II (GCPII) polymorphism (1561C-->T) on plasma homocysteine, folate and vitamin B(12) levels and its relationship to cardiovascular disease risk. 1220 97

Glutamate carboxypeptidase II (GCPII, NAALADase, or NAAG peptidase) is a catalytic zinc metallopeptidase. Its extracellular domain hydrolyzes the abundant neuropeptide, N-acetyl-L-aspartyl-L-glutamate (NAAG), to produce N-acetylaspartate and glutamate following the synaptic release of this transmitter. Thus, GCPII influences the extracellular concentrations of both glutamate and NAAG. NAAG activates group II metabotropic glutamate receptors, and activation of this receptor has been found to protect against anoxia-induced excitotoxic nerve cell death. In contrast, high levels of glutamate can be neurotoxic. Thus, GCPII is a potential therapeutic target for the reduction of excitotoxic levels of glutamate and enhancement of extracellular NAAG. To explore the structural basis of the interaction between GCPII and its inhibitors, we modeled the three-dimensional structure of the GCPII extracellular domain using a homology modeling approach. On the basis of the GCPII model, the structures of GCPII in complex with its potent inhibitors 2-(phosphonomethyl)pentanedioic acid (PMPA) and 4,4'-phosphinicobis(butane-1,3-dicarboxylic acid) (PBDA) were built by a computational docking method. The model of GCPII mainly consists of two alpha/beta/alpha sandwiches, between which two zinc ions are quadrivalently coordinated by the His379-Asp389-Asp455-H(2)O and the Asp389-Glu427-His555-H(2)O clusters, respectively. The ligand binding pocket is situated between these two sandwiches and is comprised of two subpockets: one is a surface-exposed highly positively charged subpocket; the other is a buried hydrophobic subpocket. The positively charged subpocket can accommodate the pharmacophore groups of inhibitor molecules (PMPA and PBDA) through the coordination of Zn(2+) with their phosphorus functionality and hydrogen-bonding interactions with Arg536, Arg538, and Ser456 (or Asn521), while the hydrophobic subpocket is engaged in hydrophobic and hydrogen-bonding interactions with the nonpharmacophore groups of PBDA. The predicted binding mode is consistent with the experimental data obtained from site-directed mutagenesis. On the basis of the predicted interaction mode, our structure-based design has led to a series of highly potent GCPII inhibitors.
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PMID:Molecular modeling of the interactions of glutamate carboxypeptidase II with its potent NAAG-based inhibitors. 1221 57

Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face ofglia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT-PCR analysis of RNA from the brains of -/- mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII nullmutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nm and 2494 nm, respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4'-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) inhibited the residual activity. The IC50 value for 2-PMPA was about 1 nm for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned ratand human GCPII, and 88 nm for the activity in brain membranes of the null mutants.
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PMID:Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamate. 1235 25

Glutamate carboxypeptidase II (GCPII or prostate-specific membrane antigen or NAALADase) is an enzyme that catalyzes the hydrolysis of the neuropeptide N-acetylaspartylglutamate (NAAG) to N-acetylaspartate (NAA) and glutamate (G). Inhibitors of GCPII provide neuroprotection in a variety of animal models of central nervous system disorders. Neuroprotection is probably the result of increased NAAG concentrations and decreased levels of excess toxic glutamate. Consequently, GCPII inhibitors could be useful therapeutic agents where increased glutamate levels are the result of increased GCPII activity. Current GCPII in vitro activity assays are cumbersome or have limited sensitivity. In this report we describe a microplate assay to study GCPII inhibition that is most sensitive, efficient, and generates little waste. GCPII turnover number (k(cat)) was 4s(-1) and the binding constant (K(m)) for NAAG and GCPII was 130nM. The apparent association rate constant for GCPII and NAAG (k(cat)/K(m)) was 3 x 10(7)M(-1)s(-1). Inhibition studies with the GCPII inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) demonstrated competitive inhibition with a K(i)=0.2nM.
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PMID:Kinetics and inhibition of glutamate carboxypeptidase II using a microplate assay. 1241 72

Glutamate carboxypeptidase II (EC 3.4.17.21) catalyzes the hydrolysis (Km = 0.2 microM) of the neuropeptide N-acetylaspartylglutamate to yield N-acetylaspartate and glutamate and also serves as a high-affinity folate hydrolase in the gut, cleaving the polyglutamate chain to permit the absorption of folate. N-acetylaspartylglutamate is an agonist at the mGluR3 metabotropic receptor and a source of extracellular glutamate through hydrolysis by glutamate carboxypeptidase II. Given the important role of glutamate in brain development and function, we were interested in the effects of a null mutation of glutamate carboxypeptidase II that would potentiate the effects of N-acetylaspartylglutamate. The PGK-Neomycin cassette was inserted to delete exons 9 and 10, which we previously demonstrated encode for the zinc ligand domain essential for enzyme activity. Successful germline transmission was obtained from chimeras derived from embryonic stem cells with the targeted mutation of glutamate carboxypeptidase II. Homozygous null mutants did not survive beyond embryonic day 8. Folate supplementation of the heterozygous mothers did not rescue the homozygous embryos. Mice heterozygous for the null mutation appeared grossly normal and expressed both mutated and wild-type mRNA but the activity of glutamate carboxypeptidase II is comparable to the wild-type mice. The results indicate that the expression of glutamate carboxypeptidase II is upregulated when one allele is inactivated and that its activity is essential for early embryogenesis.
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PMID:Early embryonic death of glutamate carboxypeptidase II (NAALADase) homozygous mutants. 1455 33

Glutamate carboxypeptidase II (GCP II) inhibition has previously been shown to be protective against long-term neuropathy in diabetic animals. In the current study, we have determined that the GCP II inhibitor 2-(phosphonomethyl) pentanedioic acid (2-PMPA) is protective against glucose-induced programmed cell death (PCD) and neurite degeneration in dorsal root ganglion (DRG) neurons in a cell culture model of diabetic neuropathy. In this model, inhibition of caspase activation is mediated through the group II metabotropic glutamate receptor, mGluR3. 2-PMPA neuroprotection is completely reversed by the mGluR3 antagonist (S)-alpha-ethylglutamic acid (EGLU). In contrast, group I and III mGluR inhibitors have no effect on 2-PMPA neuroprotection. Furthermore, we show that two mGluR3 agonists, the direct agonist (2R,4R)-4-aminopyrrolidine-2, 4-dicarboxylate (APDC) and N-acetyl-aspartyl-glutamate (NAAG) provide protection to neurons exposed to high glucose conditions, consistent with the concept that 2-PMPA neuroprotection is mediated by increased NAAG activity. Inhibition of GCP II or mGluR3 may represent a novel mechanism to treat neuronal degeneration under high-glucose conditions.
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PMID:Protection against glucose-induced neuronal death by NAAG and GCP II inhibition is regulated by mGluR3. 1503 Mar 92


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