Petra Mlčochová

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.

Unusual Binding Mode of an HIV-1 Protease Inhibitor Explains its Potency against Multi-drug-resistant Virus Strains

Protease inhibitors (PIs) are an important class of drugs for the treatment of HIV infection. However, in the course of treatment, resistant viral variants with reduced sensitivity to PIs often emerge and become a major obstacle to successful control of viral load. On the basis of a compound equipotently inhibiting HIV-1 and 2 proteases (PR), we have designed a pseudopeptide inhibitor, QF34, that efficiently inhibits a wide variety of PR variants. In order to analyze the potency of the inhibitor, we constructed PR species harboring the typical (signature) mutations that confer resistance to commercially available PIs. Kinetic analyses showed that these mutated PRs were inhibited up to 1,000-fold less efficiently by the clinically approved PIs. In contrast, all PR species were effectively inhibited by QF34. In a clinical study, we have monitored 30 HIV-positive patients in the Czech Republic undergoing highly active antiretroviral therapy, and have identified highly PI resistant variants. Kinetic analyses revealed that QF34 retained its subnanomolar potency against multi-drug resistant PR variants. X-ray crystallographic analysis and molecular modeling experiments explained the wide specificity of QF34: this inhibitor binds to the PR in an unusual manner, thus avoiding contact sites that are mutated upon resistance development, and the unusual binding mode and consequently the binding energy is therefore preserved in the complex with a resistant variant. These results suggest a promising route for the design of second-generation PIs that are active against a variety of resistant PR variants.

Reaction Mechanism of Glutamate Carboxypeptidase II Revealed by Mutagenesis, X-ray Crystallography, and Computational Methods

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc-dependent exopeptidase and an important therapeutic target for neurodegeneration and prostate cancer. The hydrolysis of N-acetyl-l-aspartyl-l-glutamate (N-Ac-Asp-Glu), the natural dipeptidic substrate of the GCPII, is intimately involved in cellular signaling within the mammalian nervous system, but the exact mechanism of this reaction has not yet been determined. To investigate peptide hydrolysis by GCPII in detail, we constructed a mutant of human GCPII [GCPII(E424A)], in which Glu424, a putative proton shuttle residue, is substituted with alanine. Kinetic analysis of GCPII(E424A) using N-Ac-Asp-Glu as substrate revealed a complete loss of catalytic activity, suggesting the direct involvement of Glu424 in peptide hydrolysis. Additionally, we determined the crystal structure of GCPII(E424A) in complex with N-Ac-Asp-Glu at 1.70 A resolution. The presence of the intact substrate in the GCPII(E424A) binding cavity substantiates our kinetic data and allows a detailed analysis of GCPII/N-Ac-Asp-Glu interactions. The experimental data are complemented by the combined quantum mechanics/molecular mechanics calculations (QM/MM) which enabled us to characterize the transition states, including the associated reaction barriers, and provided detailed information concerning the GCPII reaction mechanism. The best estimate of the reaction barrier was calculated to be DeltaG(++) approximately 22(+/-5) kcal x mol(-1), which is in a good agreement with the experimentally observed reaction rate constant (k(cat) approximately 1 s(-1)). Combined together, our results provide a detailed and consistent picture of the reaction mechanism of this highly interesting enzyme at the atomic level.

Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis.

Human glutamate carboxypeptidase II [GCPII (EC 3.4.17.21)] is recognized as a promising pharmacological target for the treatment and imaging of various pathologies, including neurological disorders and prostate cancer. Recently reported crystal structures of GCPII provide structural insight into the organization of the substrate binding cavity and highlight residues implicated in substrate/inhibitor binding in the S1′ site of the enzyme. To complement and extend the structural studies, we constructed a model of GCPII in complex with its substrate, N‐acetyl‐l‐aspartyl‐l‐glutamate, which enabled us to predict additional amino acid residues interacting with the bound substrate, and used site‐directed mutagenesis to assess the contribution of individual residues for substrate/inhibitor binding and enzymatic activity of GCPII. We prepared and characterized 12 GCPII mutants targeting the amino acids in the vicinity of substrate/inhibitor binding pockets. The experimental results, together with the molecular modeling, suggest that the amino acid residues delineating the S1′ pocket of the enzyme (namely Arg210) contribute primarily to the high affinity binding of GCPII substrates/inhibitors, whereas the residues forming the S1 pocket might be more important for the ‘fine‐tuning’ of GCPII substrate specificity.

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.