Pavel Šácha

Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II

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 Schneiders 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.

Expression of glutamate carboxypeptidase II in human brain.

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, Huntingtons disease, Alzheimers 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.

Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity.

Glutamate carboxypeptidase II (GCPII) is a membrane peptidase expressed in the prostate, central and peripheral nervous system, kidney, small intestine, and tumor‐associated neovasculature. The GCPII form expressed in the central nervous system, termed NAALADase, is responsible for the cleavage of N‐acetylL‐aspartyl‐L‐glutamate (NAAG) yielding free glutamate in the synaptic cleft, and is implicated in various pathologic conditions associated with glutamate excitotoxicity. The prostate form of GCPII, termed prostate‐specific membrane antigen (PSMA), is up‐regulated in cancer and used as an effective prostate cancer marker. Little is known about the structure of this important pharmaceutical target. As a type II membrane protein, GCPII is heavily glycosylated. In this paper we show that N‐glycosylation is vital for proper folding and subsequent secretion of human GCPII. Analysis of the predicted N‐glycosylation sites also provides evidence that these sites are critical for GCPII carboxypeptidase activity. We confirm that all predicted N‐glycosylation sites are occupied by an oligosaccharide moiety and show that glycosylation at sites distant from the putative catalytic domain is critical for the NAAG‐hydrolyzing activity of GCPII calling the validity of previously described structural models of GCPII into question.

Biochemical characterization of human glutamate carboxypeptidase III

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.

Comparative analysis of monoclonal antibodies against prostate-specific membrane antigen (PSMA).

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.

Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human Glutamate Carboxypeptidase II

Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo. We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.

Rational design of urea-based glutamate carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery

Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is an established prostate cancer marker and is considered a promising target for specific anticancer drug delivery. Low-molecular-weight inhibitors of GCPII are advantageous specific ligands for this purpose. However, they must be modified with a linker to enable connection of the ligand with an imaging molecule, anticancer drug, and/or nanocarrier. Here, we describe a structure-activity relationship (SAR) study of GCPII inhibitors with linkers suitable for imaging and drug delivery. Structure-assisted inhibitor design and targeting of a specific GCPII exosite resulted in a 7-fold improvement in Ki value compared to the parent structure. X-ray structural analysis of the inhibitor series led to the identification of several inhibitor binding modes. We also optimized the length of the inhibitor linker for effective attachment to a biotin-binding molecule and showed that the optimized inhibitor could be used to target nanoparticles to cells expressing GCPII.

Prostate‐specific membrane antigen and its truncated form PSM′

Prostate specific membrane antigen (PSMA) is a type II transmembrane protein overexpressed in prostate cancer as well as in the neovasculature of several non‐prostatic solid tumors. In addition to full‐length PSMA, several splice variants exist in prostatic tissue. Notably, the N‐terminally truncated PSMA variant, termed PSM′, is prevalent in healthy prostate, and the ratio of PSMA/PSM′ mRNA has been shown to correlate with cancer progression. The widely accepted hypothesis is that the PSM′ protein is a translation product arising from the alternatively spliced PSM′ mRNA.

iBodies: Modular Synthetic Antibody Mimetics Based on Hydrophilic Polymers Decorated with Functional Moieties

Abstract Antibodies are indispensable tools for biomedicine and anticancer therapy. Nevertheless, their use is compromised by high production costs, limited stability, and difficulty of chemical modification. The design and preparation of synthetic polymer conjugates capable of replacing antibodies in biomedical applications such as ELISA, flow cytometry, immunocytochemistry, and immunoprecipitation is reported. The conjugates, named “iBodies”, consist of an HPMA copolymer decorated with low‐molecular‐weight compounds that function as targeting ligands, affinity anchors, and imaging probes. We prepared specific conjugates targeting several proteins with known ligands and used these iBodies for enzyme inhibition, protein isolation, immobilization, quantification, and live‐cell imaging. Our data indicate that this highly modular and versatile polymer system can be used to produce inexpensive and stable antibody substitutes directed toward virtually any protein of interest with a known ligand.

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

In addition to its well‐characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII; Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (folyl‐n‐γ‐l‐glutamic acid), which are the provitamin form of folic acid (also known as vitamin B9). Despite the role of GCPII as a folate hydrolase, nothing is known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism. In the present study, we report: (a) a series X‐ray structures of complexes between a catalytically inactive GCPII mutant (Glu424Ala) and a panel of naturally occurring polyglutamylated folates; (b) the X‐ray structure of the His475Tyr variant at a resolution of 1.83 Å; (c) the study of the recently identified arene‐binding site of GCPII through mutagenesis (Arg463Leu, Arg511Leu and Trp541Ala), inhibitor binding and enzyme kinetics with polyglutamylated folates as substrates; and (d) a comparison of the thermal stabilities and folate‐hydrolyzing activities of GCPII wild‐type and His475Tyr variants. As a result, the crystallographic data reveal considerable details about the binding mode of polyglutamylated folates to GCPII, especially the engagement of the arene binding site in recognizing the folic acid moiety. Additionally, the combined structural and kinetic data suggest that GCPII wild‐type and His475Tyr variant are functionally identical.