title: Characterization of a novel isoform of alpha-nascent polypeptide-associated complex as IgE-defined autoantigen.
abstract: The nascent polypeptide-associated complex is required for intracellular translocation of newly synthesized polypeptides in eukaryotic cells. It may also act as a transcriptional coactivator in humans and various eukaryotic organisms and binds to nucleic acids. Recently, we provided evidence that a component of nascent polypeptide-associated complex, alpha-nascent polypeptide-associated complex, represents an IgE-reactive autoantigen for atopic dermatitis patients. By oligonucleotide screening we isolated a complete cDNA coding for a so far unknown alpha-nascent polypeptide-associated complex isoform from a human epithelial cDNA library. Southern blot hybridization experiments provided further evidence that alpha-nascent polypeptide-associated complex is encoded by a gene family. Recombinant alpha-nascent polypeptide-associated complex was expressed in Escherichia coli as a soluble, His-tagged protein, and purified via nickel affinity chromatography. By circular dichroism analysis it is demonstrated that purified recombinant alpha-nascent polypeptide-associated complex represents a folded protein of mixed alpha-helical and beta-sheet conformation with unusual high thermal stability and remarkable refolding capacity. Complete recombinant alpha-nascent polypeptide-associated complex (215 amino acids) and its 86 amino acid C-terminal fragment specifically bound IgE autoantibodies. Recombinant alpha-nascent polypeptide-associated complex also inhibited IgE binding to natural alpha-nascent polypeptide-associated complex, demonstrating the presence of common IgE epitopes between the recombinant and natural protein. Furthermore, recombinant alpha-nascent polypeptide-associated complex induced specific lymphoproliferative responses in peripheral blood mononuclear cells of a sensitized atopic dermatitis patient. As has been proposed for environmental allergens it is possible that T cell responses to IgE-defined autoantigens may contribute to the chronic skin manifestations in atopic dermatitis.
authors.affiliation: Department of Pathophysiology, Clinical Institute for Medical and Chemical Laboratory Diagnostics, University of Vienna, Austria.
authors.forename: Roschanak
authors.initials: R
authors.lastname: Mossabeb
authors.forename: Susanne
authors.initials: S
authors.lastname: Seiberler
authors.forename: Irene
authors.initials: I
authors.lastname: Mittermann
authors.forename: Renate
authors.initials: R
authors.lastname: Reininger
authors.forename: Susanne
authors.initials: S
authors.lastname: Spitzauer
authors.forename: Susanne
authors.initials: S
authors.lastname: Natter
authors.forename: Petra
authors.initials: P
authors.lastname: Verdino
authors.forename: Walter
authors.initials: W
authors.lastname: Keller
authors.forename: Dietrich
authors.initials: D
authors.lastname: Kraft
authors.forename: Rudolf
authors.initials: R
authors.lastname: Valenta
mesh_terms: D000595:Amino Acid Sequence; D001324:Autoantigens / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D003876:Dermatitis, Atopic / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D008213:Lymphocyte Activation; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001324:Autoantigens; D018832:Molecular Chaperones; D020033:Protein Isoforms; D011994:Recombinant Proteins; D015534:Trans-Activators; C096099:nascent-polypeptide-associated complex; D007073:Immunoglobulin E