RESUMEN
Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se-scFv-B3 (selenium-containing single-chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv-B3 was carried out. A three-dimensional (3D) structure of scFv-B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer-aided docking and energy minimization (EM) calculations of the antibody-GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni(2+)-immobilized metal affinity chromatography (IMAC), then transformed to selenium-containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se-scFv-B3-A180S was significantly increased compared to the original Se-scFv-B3.
Asunto(s)
Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/metabolismo , Glutatión Peroxidasa/metabolismo , Región Variable de Inmunoglobulina/metabolismo , Mutagénesis Sitio-Dirigida , Mutación/genética , Selenio/metabolismo , Secuencia de Aminoácidos , Anticuerpos Catalíticos/genética , Anticuerpos Catalíticos/aislamiento & purificación , Sitios de Unión , Western Blotting , Células Clonales , Biología Computacional , Electroforesis en Gel de Poliacrilamida , Glutatión/química , Glutatión Peroxidasa/química , Glutatión Peroxidasa/genética , Humanos , Enlace de Hidrógeno , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Selenocisteína/metabolismo , Alineación de Secuencia , TermodinámicaRESUMEN
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
GPX is a mammalian antioxidant selenoenzyme which protects biomembranes and other cellular components from oxidative damage by catalyzing the reduction of a variety of hydroperoxides (ROOH), using Glutathione (GSH) as the reducing substrate. The single-chain Fv fragment of the monoclonal antibody 2F3 (scFv2F3) can be converted into the selenium-containing Se-scFv2F3 by chemical modification of the serine. The new selenium-containing catalytic antibody Se-scFv2F3 acts as a glutathione peroxidase (GPX) mimic with high catalytic efficiency. In order to investigate which residue of scFv2F3 is converted into selenocysteine and to describe the proper reaction site of GSH to Se-scFv2F3, a three-dimensional structure of scFv2F3 is built by means of homology modeling. The 3D model is assessed by molecular dynamics (MD) simulation to determine its stability and by comparison with those of known protein structures. After the serine in the scFv2F3 is modified to selenocysteine, a catalytic antibody (abzyme) is obtained. From geometrical considerations, the solvent-accessible surface of the protein is examined. The computer-aided docking and energy minimization (EM) calculations of the abzyme-GSH complex are then carried out to explore the possible active site of the glutathione peroxidase mimic Se-scFv2F3. The structural information from the theoretically modeled complex can help us to further understand the catalytic mechanism of GPX.