RESUMEN
OBJECTIVE: To investigate the effect of selenopeptide on phagocytosis, NO and H2O2 secretion of mouse peritoneal macrophages. METHODS: Mouse peritoneal macrophages induced by lipopolysaccharide (LPS) were cultured for 24 hours by various concentrations of Se-ZnCu-65P, which is a selenopeptide with double antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Then, the relative cell activity was determined by methyl thiazolyl tetrazolium (MTT) assays, the phagocytic ability of macrophages was evaluated by neutral red uptake assay, nitric oxide (NO) content was examined by nitrate reductase method, and hydrogen peroxide (H2O2) content was detected by molybdate colorimetric method. RESULTS: When Se-ZnCu-65P acted on macrophages alone, the relative cell viability was enhanced, the phagocytic ability was not affected, H2O2 content was reduced, and NO level was almost unchanged. However, the phagocytic ability of macrophages induced by LPS was raised, meanwhile, secretion of NO and H2O2 was promoted. When Se-ZnCu-65P acted on macrophages induced by LPS, the phagocytic ability were further enhanced, and secretion of NO and H2O2 was inhibited significantly. CONCLUSION: Se-ZnCu-65P can effectively improve the relative cell activity of mouse peritoneal macrophages induced by LPS, enhance the phagocytic ability of macrophages, and reduce the secretion levels of NO and H2O2.
Asunto(s)
Glutatión Peroxidasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Macrófagos Peritoneales/enzimología , Óxido Nítrico/metabolismo , Fagocitosis , Selenio/metabolismo , Superóxido Dismutasa/metabolismo , Animales , Femenino , Glutatión Peroxidasa/genética , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos BALB C , Superóxido Dismutasa/genéticaRESUMEN
In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH-S-2,4-dinitrophenyl t-butyl ester (GSH-S-DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH + VL single-chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv-encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv-B3) in Escherichia coli Rosetta. The scFv-B3 was purified by Ni(2+)-immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0-3.0 mg of proteins from 1 L culture. After the active site serines of scFv-B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se-scFv-B3) with GPX activity of 1288 U/micromol.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Glutatión Peroxidasa/metabolismo , Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/aislamiento & purificación , Catálisis , Evaluación Preclínica de Medicamentos , Glutatión/análogos & derivados , Glutatión/inmunología , Humanos , Fragmentos de Inmunoglobulinas/química , Biblioteca de Péptidos , Selenocisteína/químicaRESUMEN
Glutathione peroxidase (GPX) is a critical antioxidant selenoenzyme in organisms that protects cells against oxidative damage by catalyzing the reduction of hydroperoxides by glutathione (GSH). Thus, some GPX mimics have been generated because of their potential therapeutic value. The generation of a semisynthetic selenoenzyme with peroxidase activity, which matches the catalytic efficiencies of naturally evolved GPX, has been a great challenge. Previously, we semisynthesized a GPX mimetic with high catalytic efficiency using a rat theta class glutathione transferase (rGST T2-2) as a scaffold, in which the highly specific GSH-binding site is adjacent to an active site serine residue that can be chemically modified to selenocysteine (Sec). In this study, we have taken advantage of a new scaffold, hGSTZ1-1, in which there are two serine residues in the active site, to achieve both high thiol selectivity and highly catalytic efficiency. The GPX activity of Se-hGSTZ1-1 is about 1.5 times that of rabbit liver GPX, indicating that the selenium content at the active site plays an important role in enhancement of catalytic performance. Kinetic studies revealed that the catalytic mechanism of Se-hGSTZ1-1 belong in a ping-pong mechanism similar to that of the natural GPX.
Asunto(s)
Glutatión Peroxidasa/metabolismo , Glutatión Transferasa/metabolismo , Selenio/metabolismo , Catálisis/efectos de los fármacos , Humanos , Cinética , Serina/metabolismo , Análisis Espectral , Compuestos de Tosilo/farmacologíaRESUMEN
Glutathione peroxidase (GPX) is one of the most crucial antioxidant enzymes in a variety of organisms. Here we described a new strategy for generating a novel GPX mimic by combination of a phage-displayed random 15-mer peptide library followed by computer-aided rational design and chemical mutation. The novel GPX mimic is a homodimer consisting of a 15-mer selenopeptide with an appropriate catalytic center, a specific binding site for substrates, and high catalytic efficiency. Its steady state kinetics was also studied, and the values of k(cat)/K(m)(GSH) and k(cat)/ K(mH(2)O(2)) were found to be similar to that of native GPX and the highest among the existing GPX mimics. Moreover, the novel GPX mimic was confirmed to have a strong antioxidant ability to inhibit lipid peroxidation by measuring the content of malondialdehyde, cell viability, and lactate dehydrogenase activity. Importantly, the novel GPX mimic can penetrate into the cell membrane because of its small molecular size. These characteristics endue the novel mimic with potential perspective for pharmaceutical applications.
Asunto(s)
Glutatión Peroxidasa/metabolismo , Péptidos/metabolismo , Selenio/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Glutatión Peroxidasa/química , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Conformación ProteicaRESUMEN
The antioxidant effect of selenium-containing single-chain Fv catalytic antibody (Se-scFv2F3), a new mimic of glutathione peroxidase, was confirmed using a model system in which cultured rat skin epidermal cells were injured by ultraviolet B (UVB). The cell damage was characterized in terms of lipid peroxidation of the cells, cell viability, and cell membrane integrity. The injury effects of UVB and protection effects of Se-scFv2F3 on the cells were studied using the model system. UVB can damage the cells severely. Upon precultivation of the cells with 0.4U/ml Se-scFv2F3, however, the damage was significantly reduced as shown by the increase in cell viability, the decrease in the malondialdehyde and hydrogen peroxide levels, and the normalization of lactate dehydrogenase activity. In addition, a novel finding that Se-scFv2F3 can stimulate cultured epidermal cells to proliferate under certain conditions was observed.
Asunto(s)
Anticuerpos/química , Antioxidantes/farmacología , Células Epidérmicas , Epidermis/efectos de la radiación , Selenio/farmacología , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Catálisis , División Celular , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Radicales Libres , Glutatión Peroxidasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Región Variable de Inmunoglobulina/química , L-Lactato Deshidrogenasa/metabolismo , Metabolismo de los Lípidos , Malondialdehído/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno , Rayos UltravioletaRESUMEN
The expression vectors of the gene encoding ScFv-2F3 were transformed into E. coli BL21(DE3). Clones of higher expression were first selected, then were grown in the presence of IPTG at 37 degrees C to induce its expression. The culture conditions were carefully optimized. It was found that optimal conditions were as follows: the induction was started as OD590 reached to 1.0-1.8; the concentration of IPTG was 0.3-0.5 mmol/L and induction time is 7 h. The yield of ScFv-2F3 expressed in the selected clones is about 20% of the total proteins. The optimal culture conditions were successfully applied to fermenter of 50 L. The conditions of washing the inclusion bodies were also optimized. A two-step method was used to renature the inclusion body. The expression product of interest and its biological activities were characterized with Western blotting and ELISA. A novel selenium-containing single-chain abzyme with GPX activity was prepared.