Glutamate carboxypeptidases II and III (GCPII and GCPIII) are highly homologous di-zinc metallopeptidases belonging to the M28 family. These enzymes are expressed in a variety of tissues, including the brain, prostate, kidney, testis and jejunum. GCPII has been recognized as a neuropeptidase in the central nervous system, as a folate hydrolase participating in absorption of folates in the jejunum and, most importantly, as a prostate-specific membrane antigen that is highly expressed in prostate adenocarcinoma. Furthermore, it has been identified in the neovasculature of most human solid tumors. In contrast, GCPIII has not been associated with any specific physiological function or pathology, and its expression, activity and inhibition have not been as well-studied. In this review, we provide an overview of the current understanding of the structure, enzymatic activity, substrate specificity, and tissue distribution of these two homologous enzymes. We discuss their potential physiological functions and describe the available animal models, including genetically modified mice. We also review the potential use of specific monoclonal antibodies and small-molecule inhibitors recognizing GCPII/III for diagnosis, imaging and experimental therapy of human cancers and other pathologies.

• MeSH
• adenokarcinom metabolismus   MeSH
• antigeny povrchové metabolismus   MeSH
• fenotyp MeSH
• glutamátkarboxypeptidasa II metabolismus   MeSH
• glutamáty chemie   MeSH
• hydrolýza MeSH
• idiopatické střevní záněty metabolismus   MeSH
• jejunum metabolismus   MeSH
• karboxypeptidasy metabolismus   MeSH
• krysa rodu rattus MeSH
• kyselina aspartová analogy a deriváty   chemie   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• monoklonální protilátky chemie   MeSH
• mozek metabolismus   MeSH
• mutantní kmeny myší MeSH
• myši transgenní MeSH
• myši MeSH
• nádorové biomarkery metabolismus   MeSH
• nádory prostaty metabolismus   MeSH
• neuropeptidy chemie   MeSH
• proteasy metabolismus   MeSH
• tenké střevo metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Glutamate carboxypeptidase III (GCPIII) is a metalloenzyme that belongs to the transferrin receptor/glutamate carboxypeptidase II (GCPII; EC 3.4.17.21) superfamily. GCPIII has been studied mainly because of its evolutionary relationship to GCPII, an enzyme involved in a variety of neuropathologies and malignancies, such as glutamatergic neurotoxicity and prostate cancer. Given the potential functional and pharmacological overlap between GCPIII and GCPII, studies addressing the structural and physiological properties of GCPIII are crucial for obtaining a deeper understanding of the GCPII/GCPIII system. In the present study, we report high-resolution crystal structures of the human GCPIII ectodomain in a 'pseudo-unliganded' state and in a complex with: (a) L-glutamate (a product of hydrolysis); (b) a phosphapeptide transition state mimetic, namely (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphinoyl]methyl}-pentanedioic acid; and (c) quisqualic acid, a glutamate biostere. Our data reveal the overall fold and quaternary arrangement of the GCPIII molecule, define the architecture of the GCPIII substrate-binding cavity, and offer an experimental evidence for the presence of Zn(2+) ions in the bimetallic active site. Furthermore, the structures allow us to detail interactions between the enzyme and its ligands and to characterize the functional flexibility of GCPIII, which is essential for substrate recognition. A comparison of these GCPIII structures with the equivalent GCPII complexes reveals differences in the organization of specificity pockets, in surface charge distribution, and in the occupancy of the co-catalytic zinc sites. The data presented here provide information that should prove to be essential for the structurally-aided design of GCPIII-specific inhibitors and might comprise guidelines for future comparative GCPII/GCPIII studies.

• MeSH
• antigeny povrchové chemie   MeSH
• fosfopeptidy chemie   MeSH
• glutamátkarboxypeptidasa II chemie   MeSH
• karboxypeptidasy chemie   MeSH
• katalytická doména MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• kvarterní struktura proteinů MeSH
• kyselina chischalová chemie   MeSH
• kyselina glutamová chemie   MeSH
• lidé MeSH
• ligandy MeSH
• molekulární mimikry MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

UNLABELLED: 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 β-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. DATABASE: The X-ray structure of GCPII Glu424Ala in complex with BCG has been deposited in the RCSB Protein Data Bank under accession code 5F09.

• MeSH
• antigeny povrchové chemie   metabolismus   MeSH
• glutamátkarboxypeptidasa II chemie   metabolismus   MeSH
• glutamáty chemie   metabolismus   MeSH
• karboxypeptidasy chemie   metabolismus   MeSH
• katalytická doména MeSH
• lidé MeSH
• molekulární struktura MeSH
• substrátová specifita MeSH
• termodynamika MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Human glutamate carboxypeptidase II (GCPII) is a transmembrane metallopeptidase found mainly in the brain, small intestine, and prostate. In the brain, it cleaves N-acetyl-L-aspartyl-glutamate, liberating free glutamate. Inhibition of GCPII has been shown to be neuroprotective in models of stroke and other neurodegenerations. In prostate, it is known as prostate-specific membrane antigen, a cancer marker. Recently, human glutamate carboxypeptidase III (GCPIII), a GCPII homolog with 67% amino acid identity, was cloned. While GCPII is recognized as an important pharmaceutical target, no biochemical study of human GCPIII is available at present. Here, we report the cloning, expression, and characterization of recombinant human GCPIII. We show that GCPIII lacks dipeptidylpeptidase IV-like activity, its activity is dependent on N-glycosylation, and it is effectively inhibited by several known inhibitors of GCPII. In comparison to GCPII, GCPIII has lower N-acetyl-L-aspartyl-glutamate-hydrolyzing activity, different pH and salt concentration dependence, and distinct substrate specificity, indicating that these homologs might play different biological roles. Based on a molecular model, we provide interpretation of the distinct substrate specificity of both enzymes, and examine the amino acid residues responsible for the differences by site-directed mutagenesis. These results may help to design potent and selective inhibitors of both enzymes.

• MeSH
• antigeny povrchové analýza   genetika   metabolismus   MeSH
• financování organizované MeSH
• glutamátkarboxypeptidasa II analýza   genetika   chemická syntéza   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• karboxypeptidasy analýza   genetika   chemická syntéza   metabolismus   MeSH
• klonování DNA metody   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH

Recent years witnessed rapid expansion of our knowledge about structural features of human glutamate carboxypeptidase II (GCPII). There are over thirty X-ray structures of human GCPII (and of its close ortholog GCPIII) publicly available at present. They include structures of ligand-free wild-type enzymes, complexes of wild-type GCPII/GCPIII with structurally diversified inhibitors as well as complexes of the GCPII(E424A) inactive mutant with several substrates. Combined structural data were instrumental for elucidating the catalytic mechanism of the enzyme. Furthermore the detailed knowledge of the GCPII architecture and protein-inhibitor interactions offers mechanistic insight into structure-activity relationship studies and can be exploited for the rational design of novel GCPII-specific compounds. This review presents a summary of structural information that has been gleaned since 2005, when the first GCPII structures were solved.

• MeSH
• glutamátkarboxypeptidasa II antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• inhibitory enzymů chemie   farmakologie   MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• polymorfismus genetický MeSH
• racionální návrh léčiv MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

We present here a structure-aided design of inhibitors targeting the active site as well as exosites of glutamate carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active toward GCPII than its closest human homologue, glutamate carboxypeptidase III (GCPIII). Additionally, we demonstrate that the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells.

• MeSH
• glutamátkarboxypeptidasa II antagonisté a inhibitory   chemie   metabolismus   MeSH
• HEK293 buňky účinky léků   MeSH
• inhibitory enzymů chemie   metabolismus   farmakologie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• močovina chemie   MeSH
• molekulární struktura MeSH
• preklinické hodnocení léčiv metody   MeSH
• racionální návrh léčiv MeSH
• techniky syntetické chemie MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein expressed in various tissues. When expressed in the brain it cleaves the neurotransmitter N-acetylaspartylglutamate (NAAG), yielding free glutamate. In jejunum it hydrolyzes folylpoly-gamma-glutamate, thus facilitating folate absorption. The prostate form of GCPII, known as prostate specific membrane antigen (PSMA), is an established cancer marker. The NAAG-hydrolyzing activity of GCPII has been implicated in a number of pathological conditions in which glutamate is neurotoxic (e.g. amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, epilepsy, schizophrenia, and stroke). Inhibition of GCPII was shown to be neuroprotective in tissue culture and in animal models. GCPII is therefore an interesting putative therapeutic target. However, only very limited and controversial data on the expression and localization of GCPII in human brain are available. Therefore, we set out to analyze the activity and expression of GCPII in various compartments of the human brain using a radiolabeled substrate of the enzyme and the novel monoclonal antibody GCP-04, which recognizes an epitope on the extracellular portion of the enzyme and is more sensitive to GCPII than to the homologous GCPIII. We show that this antibody is more sensitive in immunoblots than the widely used antibody 7E11. By Western blot, we show that there are approximately 50-300 ng of GCPII/mg of total protein in human brain, depending on the specific area. Immunohistochemical analysis revealed that astrocytes specifically express GCPII in all parts of the brain. GCPII is enzymatically active and the level of activity follows the expression pattern. Using pure recombinant GCPII and homologous GCPIII, we conclude that GCPII is responsible for the majority of overall NAAG-hydrolyzing activity in the human brain.

• MeSH
• aktivace enzymů genetika   MeSH
• antigeny povrchové analýza   imunologie   metabolismus   MeSH
• astrocyty enzymologie   MeSH
• dipeptidy metabolismus   MeSH
• financování organizované MeSH
• glutamátkarboxypeptidasa II analýza   imunologie   metabolismus   MeSH
• imunohistochemie metody   MeSH
• kyselina glutamová biosyntéza   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mapování epitopu metody   MeSH
• molekulární modely MeSH
• mozek anatomie a histologie   enzymologie   MeSH
• protilátky imunologie   MeSH
• radioligandová zkouška metody   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• terciární struktura proteinů genetika   MeSH
• western blotting MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH

Glutamate carboxypeptidase II (GCPII) and its splice variants, paralogs and human homologs represent a family of proteins with diverse tissue distribution, cellular localization and largely unknown function which have been explored only recently. While GCPII itself has been thoroughly studied from different perspectives, as clearly documented in this series of reviews, very little is known about other members of its family, even though they might be biologically relevant. Differential expression of individual GCPII splice variants is associated with tumor progression and prognosis of prostate cancer. The best studied GCPII homolog, GCPIII or NAALADase II, may be a valid pharmaceutical target for itself since it may compensate for a lack of normal GCPII enzymatic activity. Detailed molecular characterization of this family of proteins is thus very important not only with respect to the potential therapeutic use of GCPII inhibitors, but also for better understanding of the biological role of GCPII within as well as outside the nervous system.

• MeSH
• glutamátkarboxypeptidasa II analýza   antagonisté a inhibitory   genetika   metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• protein - isoformy analýza   antagonisté a inhibitory   genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

BACKGROUND: Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is generally recognized as a diagnostic and therapeutic cancer antigen and a molecular address for targeted imaging and drug delivery studies. Due to its significance in cancer research, numerous monoclonal antibodies (mAbs) against GCPII have been described and marketed in the past decades. Unfortunately, some of these mAbs are poorly characterized, which might lead to their inappropriate use and misinterpretation of the acquired results. METHODS: We collected the 13 most frequently used mAbs against GCPII and quantitatively characterized their binding to GCPII by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Using a peptide library, we mapped epitopes recognized by a given mAb. Finally, we assessed the applicability of these mAbs to routine experimental setups, including Western blotting, immunohistochemistry, and flow cytometry. RESULTS: ELISA and SPR analyses revealed that mAbs J591, J415, D2B, 107-1A4, GCP-05, and 2G7 bind preferentially to GCPII in native form, while mAbs YPSMA-1, YPSMA-2, GCP-02, GCP-04, and 3E6 bind solely to denatured GCPII. mAbs 24.4E6 and 7E11-C5.3 recognize both forms of GCPII. Additionally, we determined that GCP-02 and 3E6 cross-react with mouse GCPII, while GCP-04 recognizes GCPII and GCPIII proteins from both human and mouse. CONCLUSION: This comparative analysis provides the first detailed quantitative characterization of the most commonly used mAbs against GCPII and can serve as a guideline for the scientific community to use them in a proper and efficient way.

• MeSH
• adenokarcinom diagnóza   imunologie   metabolismus   MeSH
• ELISA MeSH
• imunohistochemie MeSH
• lidé MeSH
• mapování epitopu MeSH
• monoklonální protilátky analýza   diagnostické užití   imunologie   MeSH
• nádorové buněčné linie MeSH
• nádory prostaty diagnóza   imunologie   metabolismus   MeSH
• prostatický specifický antigen imunologie   metabolismus   MeSH
• průtoková cytometrie MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH