GCPII Variants, Paralogs and Orthologs

Hlouchová, Klára; Tykvart, Jan; Šácha, Pavel; Konvalinka, Jan

doi:10.2174/092986712799462676

Cited by 14 publications

(17 citation statements)

References 40 publications

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“…Glutamate carboxypeptidase III (GCPIII) is a binuclear zinc metallopeptidase that belongs to the MEROPS M28B peptidase subfamily (ID M28.012) . GCPIII shares 67% sequence identity and 81% sequence similarity with glutamate carboxypeptidase II (GCPII, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/17/21.html), one of the most well‐characterized members of the M28B subfamily .…”

Section: Introductionmentioning

confidence: 99%

Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificities

et al. 2016

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Glutamate carboxypeptidase III (GCPIII) is best known as a homologue of glutamate carboxypeptidase II [GCPII; also known as prostate-specific membrane antigen (PSMA)], a protease involved in neurological disorders and overexpressed in a number of solid cancers. However, mouse GCPIII was recently shown to cleave b-citrylglutamate (BCG), suggesting that these two closely related enzymes have distinct functions. To develop a tool to dissect, evaluate and quantify the activities of human GCPII and GCPIII, we analysed the catalytic efficiencies of these enzymes towards three physiological substrates. We observed a high efficiency of BCG cleavage by GCPIII but not GCPII. We also identified a strong modulation of GCPIII enzymatic activity by divalent cations, while we did not observe this effect for GCPII. Additionally, we used X-ray crystallography and computational modelling (quantum and molecular mechanical calculations) to describe the mechanism of BCG binding to the active sites of GCPII and GCPIII, respectively. Finally, we took advantage of the substantial differences in the enzymatic efficiencies of GCPII and GCPIII towards their substrates, using enzymatic assays for specific detection of these proteins in human tissues. Our findings suggest that GCPIII may not act merely as a complementary enzyme to GCPII, and it more likely possesses a specific physiological function related to BCG metabolism in the human body. DatabaseThe X-ray structure of GCPII Glu424Ala in complex with BCG has been deposited in the RCSB Protein Data Bank under accession code 5F09. Abbreviations 3D, three-dimensional; AAS, atomic absorption spectroscopy; ABS, arene-binding site; ACN, acetonitrile; BCG, b-citryl-L-glutamic acid (bcitrylglutamate, beta-citrylglutamate, beta-citryl-L-glutamic acid, beta-citryl-L-glutamate); BSA, bovine serum albumin; C12E8, octaethylene glycol monododecyl ether; DBU, 1,8-Diazabicycloundec-7-ene; DIEA, N,N-Diisopropylethylamine; EtOAc, ethylacetate; FolGlu n , folyl-n-c-Lglutamic acid; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; GCPII, glutamate carboxypeptidase II; GCPIII, glutamate carboxypeptidase III; HPLC, high-performance liquid chromatography; HRMS, high-resolution mass spectrometry; MeOH, methanol; MD/ MM, molecular dynamical/molecular mechanical calculations; NAAG, N-acetyl-L-aspartyl-L-glutamic acid; NMR, nuclear magnetic resonance; OPA, orthophtalaldehyde; ORF, open reading frame; Pd(C), palladium on activated charcoal; PSMA, prostate-specific membrane antigen; QM/MM, quantum mechanical and molecular mechanical calculations; QM/MM/MD, quantum mechanical, molecular mechanical and molecular dynamical calculations; qPCR, quantitative polymerase chain reaction; rhGCPII, recombinant human glutamate carboxypeptidase II; SDS/PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; TLC, thin-layer chromatography.

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Section: Introductionmentioning

confidence: 99%

Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificities

et al. 2016

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“…The same enzyme also exerts folyl-poly-g-Glu carboxypeptidase activity, which is thought to support cellular folate uptake (Pinto et al, 1996). Moreover, GCPII is used as a tumor marker because it is strongly up-regulated in most solid tumors, yet its role in cancer development is unclear (Hlouchová et al, 2012). To which extent these GCPII-specific catalytic activities are conserved in AMP1 has not been analyzed in detail; however, the limited level of structural conservation in the putative substrate-binding domains rather support different substrate specificities (Davis et al, 2005).…”

mentioning

confidence: 99%

ALTERED MERISTEM PROGRAM1Suppresses Ectopic Stem Cell Niche Formation in the Shoot Apical Meristem in a Largely Cytokinin-Independent Manner

et al. 2015

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Plants are able to reiteratively form new organs in an environmentally adaptive manner during postembryonic development. Organ formation in plants is dependent on stem cell niches (SCNs), which are located in the so-called meristems. Meristems show a functional zonation along the apical-basal axis and the radial axis. Shoot apical meristems of higher plants are dome-like structures, which contain a central SCN that consists of an apical stem cell pool and an underlying organizing center. Organ primordia are formed in the circular peripheral zone (PZ) from stem cell descendants in which differentiation programs are activated. One mechanism to keep this radial symmetry integrated is that the existing SCN actively suppresses stem cell identity in the PZ. However, how this lateral inhibition system works at the molecular level is far from understood. Here, we show that a defect in the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) causes the formation of extra SCNs in the presence of an intact primary shoot apical meristem, which at least partially contributes to the enhanced shoot meristem size and leaf initiation rate found in the mutant. This defect appears to be neither a specific consequence of the altered cytokinin levels in amp1 nor directly mediated by the WUSCHEL/CLAVATA feedback loop. De novo formation of supernumerary stem cell pools was further enhanced in plants mutated in both AMP1 and its paralog LIKE AMP1, indicating that they exhibit partially overlapping roles to suppress SCN respecification in the PZ.

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“…⌲im et al (11) attributed the A␤ 1-42 -hydrolyzing activity of GCPII to an unknown, alternative catalytic mechanism of the enzyme, noting that the observed activity was partially inhibited by 2-PMPA, a very potent GCPII inhibitor. This suggestion is, however, incompat-ible with the currently available, very detailed structural information about the mechanism of action of GCPII (12,30,31). GCPII possesses a single active site, and a major structural change leading to an alternative mechanism of action is difficult to imagine.…”

Section: Discussionmentioning

confidence: 98%

Glutamate carboxypeptidase II does not process amyloid‐β peptide

Sedlák¹,

Šácha

Blechová

et al. 2013

FASEB j.

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The accumulation of amyloid-β (Aβ) peptide is thought to be a major causative mechanism of Alzheimer's disease. Aβ accumulation could be caused by dysregulated processing of amyloid precursor protein, yielding excessive amounts of Aβ, and/or by inefficient proteolytic degradation of the peptide itself. Several proteases have been described as Aβ degradation enzymes, most notably metalloendopeptidases, aspartic endopeptidases, and some exopeptidases. Recently a report suggested that another metallopeptidase, glutamate carboxypeptidase II (GCPII), can also cleave Aβ. GCPII is a zinc exopeptidase that cleaves glutamate from N-acetyl-L-aspartyl-L-glutamate in the central nervous system and from pteroylpoly-γ-glutamate in the jejunum. GCPII has been proposed as a promising therapeutic target for disorders caused by glutamate neurotoxicity. However, an Aβ-degrading activity of GCPII would compromise potential pharmaceutical use of GCPII inhibitors, because the enzyme inhibition might lead to increased Aβ levels and consequently to Alzheimer's disease. Therefore, we analyzed the reported Aβ-degrading activity of GCPII using highly purified recombinant enzyme and synthetic Aβ. We did not detect any Aβ degradation activity of GCPII or its homologue even under prolonged incubation at a high enzyme to substrate ratio. These results are in good agreement with the current detailed structural understanding of the substrate specificity and enzyme-ligand interactions of GCPII.

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GCPII Variants, Paralogs and Orthologs

Cited by 14 publications

References 40 publications

Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificities

Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificities

ALTERED MERISTEM PROGRAM1Suppresses Ectopic Stem Cell Niche Formation in the Shoot Apical Meristem in a Largely Cytokinin-Independent Manner

Glutamate carboxypeptidase II does not process amyloid‐β peptide

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