Klára Hlouchová

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.

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.

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol

Significance Microbes in contaminated environments often evolve new metabolic pathways for detoxification or degradation of pollutants. In some cases, intermediates in newly evolving pathways are more toxic than the initial compound. The initial step in the degradation of pentachlorophenol by Sphingobium chlorophenolicum generates a particularly toxic intermediate, tetrachlorobenzoquinone (TCBQ). This paper describes how the bacterium is protected from the toxic effects of TCBQ. In the presence of tetrachlorobenzoquinone reductase, TCBQ produced by pentachlorophenol hydroxylase is sequestered until it is reduced to the less toxic tetrachlorohydroquinone. Microbes in contaminated environments often evolve new metabolic pathways for detoxification or degradation of pollutants. In some cases, intermediates in newly evolved pathways are more toxic than the initial compound. The initial step in the degradation of pentachlorophenol by Sphingobium chlorophenolicum generates a particularly reactive intermediate; tetrachlorobenzoquinone (TCBQ) is a potent alkylating agent that reacts with cellular thiols at a diffusion-controlled rate. TCBQ reductase (PcpD), an FMN- and NADH-dependent reductase, catalyzes the reduction of TCBQ to tetrachlorohydroquinone. In the presence of PcpD, TCBQ formed by pentachlorophenol hydroxylase (PcpB) is sequestered until it is reduced to the less toxic tetrachlorohydroquinone, protecting the bacterium from the toxic effects of TCBQ and maintaining flux through the pathway. The toxicity of TCBQ may have exerted selective pressure to maintain slow turnover of PcpB (0.02 s−1) so that a transient interaction between PcpB and PcpD can occur before TCBQ is released from the active site of PcpB.

Pentachlorophenol hydroxylase, a poorly functioning enzyme required for degradation of pentachlorophenol by Sphingobium chlorophenolicum

Several strains of Sphingobium chlorophenolicum have been isolated from soil that was heavily contaminated with pentachlorophenol (PCP), a toxic pesticide introduced in the 1930s. S. chlorophenolicum appears to have assembled a poorly functioning pathway for degradation of PCP by patching enzymes recruited via two independent horizontal gene transfer events into an existing metabolic pathway. Flux through the pathway is limited by PCP hydroxylase. PCP hydroxylase is a dimeric protein that belongs to the family of flavin-dependent phenol hydroxylases. In the presence of NADPH, PCP hydroxylase converts PCP to tetrachlorobenzoquinone (TCBQ). The k(cat) for PCP (0.024 s(-1)) is very low, suggesting that the enzyme is not well evolved for turnover of this substrate. Structure-activity studies reveal that substrate binding and activity are enhanced by a low pK(a) for the phenolic proton, increased hydrophobicity, and the presence of a substituent ortho to the hydroxyl group of the phenol. PCP hydroxylase exhibits substantial uncoupling; the C4a-hydroxyflavin intermediate, instead of hydroxylating the substrate, can decompose to produce H(2)O(2) in a futile cycle that consumes NADPH. The extent of uncoupling varies from 0 to 100% with different substrates. The extent of uncoupling is increased by the presence of bulky substituents at position 3, 4, or 5 and decreased by the presence of a chlorine in the ortho position. The effectiveness of PCP hydroxylase is additionally hindered by its promiscuous activity with tetrachlorohydroquinone (TCHQ), a downstream metabolite in the degradation pathway. The conversion of TCHQ to TCBQ reverses flux through the pathway. Substantial uncoupling also occurs during the reaction with TCHQ.

Structural insight into the evolutionary and pharmacologic homology of glutamate carboxypeptidases II and III

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 Zn2+ 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.

GCPII variants, paralogs and orthologs.

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.

Random protein sequences can form defined secondary structures and are well-tolerated in vivo

The protein sequences found in nature represent a tiny fraction of the potential sequences that could be constructed from the 20-amino-acid alphabet. To help define the properties that shaped proteins to stand out from the space of possible alternatives, we conducted a systematic computational and experimental exploration of random (unevolved) sequences in comparison with biological proteins. In our study, combinations of secondary structure, disorder, and aggregation predictions are accompanied by experimental characterization of selected proteins. We found that the overall secondary structure and physicochemical properties of random and biological sequences are very similar. Moreover, random sequences can be well-tolerated by living cells. Contrary to early hypotheses about the toxicity of random and disordered proteins, we found that random sequences with high disorder have low aggregation propensity (unlike random sequences with high structural content) and were particularly well-tolerated. This direct structure content/aggregation propensity dependence differentiates random and biological proteins. Our study indicates that while random sequences can be both structured and disordered, the properties of the latter make them better suited as progenitors (in both in vivo and in vitro settings) for further evolution of complex, soluble, three-dimensional scaffolds that can perform specific biochemical tasks.

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

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.

Chapter 3:Glutamate Carboxypeptidase II as a Therapeutic Target

Glutamate carboxypeptidase II (GCPII; EC 3.4.17.21) is a Zn2+-dependent metalloprotease of the M28 peptidase family. It is also known as prostate-specific membrane antigen, N-acetylated-α-linked acidic dipeptidase, or folate hydrolase I. In the brain, the enzyme cleaves the peptide neurotransmitter N-acetyl-l-aspartyl-l-glutamate (NAAG) into N-acetyl-l-aspartate and free glutamate, a potent and potentially neurotoxic neurotransmitter. The folate hydrolase activity of GCPII facilitates the absorption of dietary folates in the small intestine by cleaving γ-linked glutamates from folyl-poly-γ-glutamates. While the physiological function of GCPII in the prostate is not known, its expression levels are elevated in metastatic prostate carcinoma. Furthermore, the enzyme is also expressed in the cancer-associated neovasculature of most solid tumors. In the nervous system, GCPII inhibition leads to an increase in NAAG concentration and has been shown to be neuroprotective in animal models of ischemic or traumatic brain injury, neuropathic pain, or amyotrophic lateral sclerosis. In tumors, GCPII represents a very promising target for specific diagnostics, imaging, and targeted delivery of anticancer drugs. The present chapter reviews the expression of GCPII in various tissues, its splice variants, orthologs, and paralogs, and summarizes available information on the physiological function and proteolytic activity of the enzyme. Furtermore, its 3-D structure, substrate specificity and binding mode of available specific inhibitors are described in more detail. Finally, current and potential applications of inhibitors and ligands of GCPII for the diagnostics, imaging, and experimental therapy of neurodegenerative diseases and prostate cancer are discussed.