title: Defining the immunoreactive epitope for the monoclonal anti-human glutathione peroxidase-4 antibody anti-hGPx4 Mab63-1.
abstract: Glutathione peroxidases (GPx) form a heterogeneous enzyme family and GPx4-isoforms have been implicated in anti-oxidative defense, brain development, neuroinjury and sperm maturation. In humans seven GPx isoforms (GPx1-GPx7) can be separated. To selectively quantify the expression of GPx4-isoforms we have raised a monoclonal antibody (anti-hGPx4 Mab63-1) against the pure recombinant Sec46Cys mutant of human cytosolic GPx4 and used it for immunoblotting, immunoprecipitation and immunohistochemistry. The antibody recognizes human GPx4, its mouse ortholog but neither reacted with rat GPx4 nor other human GPx-isoforms. Sequence alignment of human and rat GPx4 proteins indicated three different amino acids (S18, F35, K99 in humans, A18, C35, R99 in rats) and a S18A exchange in the human enzyme completely abolished immunoreactivity. To further characterize the immunological epitope we synthesized a set of 12-mer peptides flanking S18* of human GPx4 and found that the sequence SMHEFS*AKDIDG exhibited strongest immunoreactivity. Substitution analysis and peptide length variation narrowed down the essential epitope to FS*AKDI and indicated that most mutations in this region strongly impaired immunoreactivity. In silico blast searches of public protein databases failed to identify proteins with potential immunoreactivity suggesting that the antibody exhibits a high specificity for human and mouse GPx4 and may not cross-react with unrelated proteins.
authors.affiliation: Institute of Biochemistry, University Medicine Berlin - Charité, Monbijoustr. 2, D-10117 Berlin, Federal Republic of Germany.
authors.forename: Astrid
authors.initials: A
authors.lastname: Borchert
authors.forename: Gabi
authors.initials: G
authors.lastname: Küttner
authors.forename: Elke
authors.initials: E
authors.lastname: Giessmann
authors.forename: Chi Chiu
authors.initials: CC
authors.lastname: Wang
authors.forename: Helga
authors.initials: H
authors.lastname: Wessner
authors.forename: Rudolf
authors.initials: R
authors.lastname: Volkmer
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Höhne
authors.forename: Hartmut
authors.initials: H
authors.lastname: Kuhn
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D020650:Combinatorial Chemistry Techniques; D003429:Cross Reactions; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D050505:Mutant Proteins / Q000235:genetics; D018384:Oxidative Stress; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D051381:Rats; D019076:Transgenes / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D050505:Mutant Proteins; D010446:Peptide Fragments; D020033:Protein Isoforms; D005979:Glutathione Peroxidase