RESUMO
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.
Assuntos
Adenocarcinoma/imunologia , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Antígeno Prostático Específico/imunologia , Neoplasias da Próstata/imunologia , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Western Blotting , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/metabolismoRESUMO
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.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glutamato Carboxipeptidase II/antagonistas & inibidores , Glutamato Carboxipeptidase II/química , Animais , Cristalografia por Raios X , Glutamato Carboxipeptidase II/genética , Glutamato Carboxipeptidase II/metabolismo , Humanos , Modelos Moleculares , Polimorfismo Genético , Conformação ProteicaRESUMO
Glutamate carboxypeptidase II (GCPII) is a membrane-bound binuclear zinc metallopeptidase with the highest expression levels found in the nervous and prostatic tissue. Throughout the nervous system, glia-bound GCPII is intimately involved in the neuron-neuron and neuron-glia signaling via the hydrolysis of N-acetylaspartylglutamate (NAAG), the most abundant mammalian peptidic neurotransmitter. The inhibition of the GCPII-controlled NAAG catabolism has been shown to attenuate neurotoxicity associated with enhanced glutamate transmission and GCPII-specific inhibitors demonstrate efficacy in multiple preclinical models including traumatic brain injury, stroke, neuropathic and inflammatory pain, amyotrophic lateral sclerosis, and schizophrenia. The second major area of pharmacological interventions targeting GCPII focuses on prostate carcinoma; GCPII expression levels are highly increased in androgen-independent and metastatic disease. Consequently, the enzyme serves as a potential target for imaging and therapy. This review offers a summary of GCPII structure, physiological functions in healthy tissues, and its association with various pathologies. The review also outlines the development of GCPII-specific small-molecule compounds and their use in preclinical and clinical settings.
Assuntos
Glutamato Carboxipeptidase II/metabolismo , Doenças do Sistema Nervoso/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Glutamato Carboxipeptidase II/antagonistas & inibidores , Humanos , Masculino , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/tratamento farmacológico , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/tratamento farmacológicoRESUMO
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.
Assuntos
Antígenos de Superfície/genética , Antígenos de Superfície/isolamento & purificação , Glutamato Carboxipeptidase II/genética , Glutamato Carboxipeptidase II/isolamento & purificação , Sequência de Aminoácidos , Animais , Antígenos de Superfície/química , Antígenos de Superfície/metabolismo , Biotina/química , Biotina/metabolismo , Biotinilação , Linhagem Celular , Cristalografia por Raios X , Drosophila/citologia , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Glutamato Carboxipeptidase II/química , Glutamato Carboxipeptidase II/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Antígenos de Superfície/metabolismo , Encéfalo/enzimologia , Dipeptídeos/metabolismo , Glutamato Carboxipeptidase II/metabolismo , Ácido Glutâmico/biossíntese , Idoso , Idoso de 80 Anos ou mais , Anticorpos/imunologia , Antígenos de Superfície/análise , Antígenos de Superfície/imunologia , Astrócitos/enzimologia , Western Blotting , Encéfalo/anatomia & histologia , Ativação Enzimática/fisiologia , Mapeamento de Epitopos/métodos , Feminino , Glutamato Carboxipeptidase II/análise , Glutamato Carboxipeptidase II/imunologia , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Estrutura Terciária de Proteína/fisiologia , Ensaio Radioligante/métodos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In order to identify inhibitors of various drug-resistant forms of the human immunodeficiency virus protease (HIV PR), we have designed and synthesized pseudopeptide libraries with a general structure Z-mimetic-Aa1-Aa2-NH2. Five different chemistries for peptide bond replacement have been employed and the resulting five individual sublibraries tested with the HIV PR and its drug-resistant mutants. Each mutant contains amino acid substitutions that have previously been shown to be associated with resistance to protease inhibitors, including Ritonavir, Indinavir, and Saquinavir. We have mapped the subsite preferences of resistant HIV PR species with the aim of selecting a pluripotent pharmaceutical lead. All of the enzyme species in this study manifest clear preference for an L-Glu residue in the P2' position. Slight, but significant, differences in P3' subsite specificity among individual resistant PR species have been documented. We have identified three compounds, combining the most favorable features of the inhibitor array, that exhibit low-nanomolar or picomolar Ki values for all three mutant PR species tested.