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Preparation, expression and characterization of mouse GCPIII
Bäumlová, Adriana ; Konvalinka, Jan (advisor) ; Šebo, Peter (referee)
English abstract Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a type II transmembrane glycoprotein which has been discovered in nervous system as an enzyme responsible for the hydrolysis of neuropeptide N-acetyl-L-aspartyl-L-glutamate to N-acetyl-L-aspartate and L-glutamate and that has been hypothesized to influence glutamatergic signaling processes. Except for brain, GCPII was mainly found in prostate, kidney, and small intestine. In small intestine, GCPII cleaves terminal glutamates from polyglutamylated folates facilitating thus absorption of dietary folates. In prostate, this enzyme is known as prostate-specific membrane antigen and is used as a cancer marker. Mus musculus is an important model for studing GCPII and its homologs as a therapeutic target. While human GCPII and its paralog GCPIII are relatively well characterized, no biochemical study of their mouse orthologs is available. That is why mouse glutamate carboxypeptidase III (mGCPIII) was cloned, prepared by recombinant expression in insect cells and characterized. We show that pure mouse GCPIII possesses α-glutamate carboxypeptidase activity which is effectively inhibited by specific inhibitor GCPII, 2-PMPA. We also analyzed sensitivity and specifity of monoclonal antibodies against mouse GCPIII. Immunoblots demonstrate that...
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Preparation of glutamate carboxypeptidase III using mammalian expression system
Šimonová, Lenka ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
The role of glutamate carboxypeptidase II in mammalian organism is already known quite well but only little is known about its homologue glutamate carboxypeptidase III. For structural and functional characterization of any protein, a large amount of protein is required. Protein could be obtained by expression in tissue culture. Properties of the protein may be affected by post translational modifications, where different organisms create different modifications. Therefore, we set to develop a system for recombinant expression of GCPIII in mammalian cells. First the mammalian expression system HEK 293-6E was introduced as a substitute for the current insect expression system. The advantage of this mammalian expression system is its option of transient transfection and that it is easy to cultivate cells under suspension conditions. Further, transfection conditions for this system were optimized by green fluorescent protein expression, for easy detection by flow cytometry. DNA encoding the extracellular part of mouse GCPIII (mEXSTII) was cloned into five expression plasmids with His or Fc tags attached to N- or C-termini. Cells were transfected with prepared plasmids. The presence of mEXSTII in media was tested using Western blot and subsequently the activity of GCPIII was tested by cleaving its...
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Development of analytical tools for quantification and screening for inhibitors of glutamate carboxypeptidases II and III
Navrátil, Václav ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee) ; Šedo, Aleksi (referee)
Glutamate carboxypeptidase II (GCPII) usually called prostate specific membrane antigen (PSMA) is membrane bound metallopeptidase expressed mainly in prostate carcinoma (PCa). Agents targeting GCPII suitable for both imaging and treatment of PCa are in development and they show promising results in advanced clinical trials. Some studies showed that GCPII may serve also as PCa blood serum marker, but this has not been validated due to the lack of methods suitable for accurate detection of GCPII in human blood. Moreover, GCPII is also expressed in brain, where it cleaves inhibitory N-acetyl-α-L- aspartyl-L-glutamate (NAAG) to release excitatory L-glutamate and GCPII inhibition has been shown to be neuroprotective in animal models of several neuropathies. Tight binding inhibitors of GCPII have been identified by rational design, but all have poor bioavailability and thus cannot be used in clinics. Identifying new scaffolds by 'brute force' screening methods is thus essential; however, no such method for GCPII has been developed so far. Glutamate carboxypeptidase III (GCPIII) is also expressed in brain and cleaves NAAG. It is thus an important protein for understanding of GCPII function as well as GCPII targeting in medicine. Here, we focused on development of novel methods for quantification of both...
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Preparation of glutamate carboxypeptidase III using mammalian expression system
Šimonová, Lenka ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
The role of glutamate carboxypeptidase II in mammalian organism is already known quite well but only little is known about its homologue glutamate carboxypeptidase III. For structural and functional characterization of any protein, a large amount of protein is required. Protein could be obtained by expression in tissue culture. Properties of the protein may be affected by post translational modifications, where different organisms create different modifications. Therefore, we set to develop a system for recombinant expression of GCPIII in mammalian cells. First the mammalian expression system HEK 293-6E was introduced as a substitute for the current insect expression system. The advantage of this mammalian expression system is its option of transient transfection and that it is easy to cultivate cells under suspension conditions. Further, transfection conditions for this system were optimized by green fluorescent protein expression, for easy detection by flow cytometry. DNA encoding the extracellular part of mouse GCPIII (mEXSTII) was cloned into five expression plasmids with His or Fc tags attached to N- or C-termini. Cells were transfected with prepared plasmids. The presence of mEXSTII in media was tested using Western blot and subsequently the activity of GCPIII was tested by cleaving its...
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Human glutamate carboxypeptidases II and III
Navrátil, Michal ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee) ; Pavlíček, Jiří (referee)
The herein presented Ph.D. dissertation describes kinetic and structural characterization of human glutamate carboxypeptidases II and III (GCPII and GCPIII) using a complete panel of their natural substrates. These enzymes hydrolyze C-terminal glutamate from their substrates. They share 67 % sequence identity and also similar enzymatic activities. This thesis quantitatively compares human GCPII and GCPIII in terms of their ability to hydrolyze the substrates N-acetyl-L-aspartyl-L-glutamate (NAAG), folyl-poly-γ-L-glutamic acids (FolGlun) and β-citryl-L-glutamate (BCG). We demonstrated that GCPIII hydrolyzes its substrates in a metal- dependent manner, that BCG is a specific substrate of GCPIII, and that NAAG and FolGlun are specific substrates of GCPII. We also provide indirect biochemical evidence that GCPIII might feature a heterometallic active-site cluster. Additionally, we characterized the relevance of a surface exosite of GCPII, the arene-binding site (ABS), for the hydrolysis of FolGlun substrates using mutagenesis and enzyme kinetics and showed that polymorphic His475Tyr variant of GCPII hydrolyzes FolGlun substrates with the same kinetic parameters as the wild-type enzyme. Furthermore, this thesis focuses on structural aspects of the substrate specificities of GCPII and GCPIII: we present...
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Preparation, expression and characterization of mouse GCPIII
Bäumlová, Adriana ; Šebo, Peter (referee) ; Konvalinka, Jan (advisor)
English abstract Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a type II transmembrane glycoprotein which has been discovered in nervous system as an enzyme responsible for the hydrolysis of neuropeptide N-acetyl-L-aspartyl-L-glutamate to N-acetyl-L-aspartate and L-glutamate and that has been hypothesized to influence glutamatergic signaling processes. Except for brain, GCPII was mainly found in prostate, kidney, and small intestine. In small intestine, GCPII cleaves terminal glutamates from polyglutamylated folates facilitating thus absorption of dietary folates. In prostate, this enzyme is known as prostate-specific membrane antigen and is used as a cancer marker. Mus musculus is an important model for studing GCPII and its homologs as a therapeutic target. While human GCPII and its paralog GCPIII are relatively well characterized, no biochemical study of their mouse orthologs is available. That is why mouse glutamate carboxypeptidase III (mGCPIII) was cloned, prepared by recombinant expression in insect cells and characterized. We show that pure mouse GCPIII possesses α-glutamate carboxypeptidase activity which is effectively inhibited by specific inhibitor GCPII, 2-PMPA. We also analyzed sensitivity and specifity of monoclonal antibodies against mouse GCPIII. Immunoblots demonstrate that...
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