Structural and biochemical characterization of the folyl‐poly‐γ‐<scp>l</scp>‐glutamate hydrolyzing activity of human glutamate carboxypeptidase <scp>II</scp>

Navratil, M.; Ptacek, Jakub; Šácha, Pavel; Starková, Jana; Łubkowski, J.; Bařinka, Cyril; Konvalinka, Jan

doi:10.1111/febs.12857

Cited by 27 publications

(31 citation statements)

References 35 publications

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“…B,C). These results are in good agreement with previously reported structural data showing that GCPII is unable to fully utilize the ABS for binding of the pteroate moiety of FolGlu 1 , while the FolGlu 2–3 substrates are able to bind into this exosite .…”

Section: Resultssupporting

confidence: 93%

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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: Resultssupporting

confidence: 93%

“…A. The glutamate moiety of BCG binds into the S1′ pocket of GCPII in an identical manner as described for FolGlu n or NAAG substrates . It forms hydrogen bonds with the side chains of Arg210, Asn257 and Tyr552, and also with Lys699 and Tyr700 forming the so‐called ‘glutarate sensor’ .…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2016

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“…Of interest, our laboratory recently found that GCPII is highly upregulated in microglial cells (e.g., the immune cells of the brain) following inflammation and its inhibition prevents neurological damage (27). With respect to enzymatic mechanism, to date there are 3 known FOLH1 /GCPII substrates that could be implicated as possible mediators of the therapeutic effect, including N-acetylaspartylglutamate (NAAG) (28), folate polyglutamate (29–31), and laminin-derived peptides (32, 33). NAAG is one of the most abundant peptides in nervous tissues where it is thought to participate in glutamatergic transmission, acting through NMDA and mGlu3 receptors (20, 34, 35).…”

Section: Discussionmentioning

confidence: 99%

FOLH1/GCPII is elevated in IBD patients, and its inhibition ameliorates murine IBD abnormalities

Rais¹,

Jiang²,

Zhai³

et al. 2016

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Recent gene-profiling analyses showed significant upregulation of the folate hydrolase (FOLH1) gene in the affected intestinal mucosa of patients with inflammatory bowel disease (IBD). The FOLH1 gene encodes a type II transmembrane glycoprotein termed glutamate carboxypeptidase II (GCPII). To establish that the previously reported increased gene expression was functional, we quantified the glutamate carboxypeptidase enzymatic activity in 31 surgical specimens and report a robust 2.8- to 41-fold increase in enzymatic activity in the affected intestinal mucosa of IBD patients compared with an uninvolved area in the same patients or intestinal mucosa from healthy controls. Using a human-to-mouse approach, we next showed a similar enzymatic increase in two well-validated IBD murine models and evaluated the therapeutic effect of the potent FOLH1/ GCPII inhibitor 2-phosphonomethyl pentanedioic acid (2-PMPA) (IC50 = 300 pM). In the dextran sodium sulfate (DSS) colitis model, 2-PMPA inhibited the GCPII activity in the colonic mucosa by over 90% and substantially reduced the disease activity. The significance of the target was confirmed in FOLH1−/− mice who exhibited resistance to DSS treatment. In the murine IL-10−/− model of spontaneous colitis, daily 2-PMPA treatment also significantly reduced both macroscopic and microscopic disease severity. These results provide the first evidence of FOLH1/GCPII enzymatic inhibition as a therapeutic option for IBD.

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“…NAAG is the most abundant neuropeptide in the brain and is thought to act as a reservoir for GCPII-dependent release of the neurotransmitter L-Glu. In the brush border of the small intestine the same enzyme also exerts folyl-poly-g-Glu carboxypeptidase activity, which is thought to mediate dietary folate uptake (Pinto et al, 1996;Navrátil et al, 2014). The in vivo requirement of these biochemical activities could not be clearly demonstrated since knock-out mice of GCPII show normal development and behavior, most likely because of functional redundancy with the close paralog GCPIII (Bacich et al, 2002;Gao et al, 2015).…”

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The small molecule hyperphyllin enhances leaf formation rate and mimics shoot meristem integrity defects associated with AMP1 deficiency

et al. 2016

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) is a member of the M28 family of carboxypeptidases with a pivotal role in plant development and stress adaptation. Its most prominent mutant defect is a unique hypertrophic shoot phenotype combining a strongly increased organ formation rate with enhanced meristem size and the formation of ectopic meristem poles. However, so far the role of AMP1 in shoot development could not be assigned to a specific molecular pathway nor is its biochemical function resolved. In this work we evaluated the level of functional conservation between AMP1 and its human homolog HsGCPII, a tumor marker of medical interest. We show that HsGCPII cannot substitute AMP1 in planta and that an HsGCPII-specific inhibitor does not evoke amp1-specific phenotypes. We used a chemical genetic approach to identify the drug hyperphyllin (HP), which specifically mimics the shoot defects of amp1, including plastochron reduction and enlargement and multiplication of the shoot meristem. We assessed the structural requirements of HP activity and excluded that it is a cytokinin analog. HP-treated wild-type plants showed amp1-related tissue-specific changes of various marker genes and a significant transcriptomic overlap with the mutant. HP was ineffective in amp1 and elevated the protein levels of PHAVOLUTA, consistent with the postulated role of AMP1 in miRNA-controlled translation, further supporting an AMP1-related mode of action. Our work suggests that plant and animal members of the M28 family of proteases adopted unrelated functions. With HP we provide a tool to characterize the plant-specific functions of this important class of proteins.Arabidopsis ALTERED MERISTEM PROGRAM1 (AMP1, At3G54720, MEROPS ID: M28.007) belongs to the Zn 2+

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Structural and biochemical characterization of the folyl‐poly‐γ‐l‐glutamate hydrolyzing activity of human glutamate carboxypeptidase II

Cited by 27 publications

References 35 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

FOLH1/GCPII is elevated in IBD patients, and its inhibition ameliorates murine IBD abnormalities

The small molecule hyperphyllin enhances leaf formation rate and mimics shoot meristem integrity defects associated with AMP1 deficiency

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