title: The molecular basis for development of proinflammatory autoantibodies to progranulin.
abstract: Recently we identified in a wide spectrum of autoimmune diseases frequently occurring proinflammatory autoantibodies directed against progranulin, a direct inhibitor of TNFR1 & 2 and of DR3. In the present study we investigated the mechanisms for the breakdown of self-tolerance against progranulin. Isoelectric focusing identified a second, differentially electrically charged progranulin isoform exclusively present in progranulin-antibody-positive patients. Alkaline phosphatase treatment revealed this additional progranulin isoform to be hyperphosphorylated. Subsequently Ser81, which is located within the epitope region of progranulin-antibodies, was identified as hyperphosphorylated serine residue by site directed mutagenesis of candidate phosphorylation sites. Hyperphosphorylated progranulin was detected exclusively in progranulin-antibody-positive patients during the courses of their diseases. The occurrence of hyperphosphorylated progranulin preceded seroconversions of progranulin-antibodies, indicating adaptive immune response. Utilizing panels of kinase and phosphatase inhibitors, PKCβ1 was identified as the relevant kinase and PP1 as the relevant phosphatase for phosphorylation and dephosphorylation of Ser81. In contrast to normal progranulin, hyperphosphorylated progranulin interacted exclusively with inactivated (pThr320) PP1, suggesting inactivated PP1 to cause the detectable occurrence of phosphorylated Ser81 PGRN. Investigation of possible functional alterations of PGRN due to Ser81 phosphorylation revealed, that hyperphosphorylation prevents the interaction and thus direct inhibition of TNFR1, TNFR2 and DR3, representing an additional direct proinflammatory effect. Finally phosphorylation of Ser81 PGRN alters the conversion pattern of PGRN. In conclusion, inactivated PP1 induces hyperphosphorylation of progranulin in a wide spectrum of autoimmune diseases. This hyperphosphorylation prevents direct inhibition of TNFR1, TNFR2 and DR3 by PGRN, alters the conversion of PGRN, and is strongly associated with the occurrence of neutralizing, proinflammatory PGRN-antibodies, indicating immunogenicity of this alternative secondary modification.
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany. Electronic address: lorenz.thurner@uks.eu.
authors.forename: Lorenz
authors.initials: L
authors.lastname: Thurner
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany.
authors.forename: Natalie
authors.initials: N
authors.lastname: Fadle
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany.
authors.forename: Evi
authors.initials: E
authors.lastname: Regitz
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Kemele
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Klemm
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany.
authors.forename: Marina
authors.initials: M
authors.lastname: Zaks
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Stöger
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Bette
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany.
authors.forename: Gabi
authors.initials: G
authors.lastname: Carbon
authors.affiliation: Department of Internal Medicine II, Saarland University Medical Center, Homburg, Saar, Germany.
authors.forename: Vincent
authors.initials: V
authors.lastname: Zimmer
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany.
authors.forename: Gunter
authors.initials: G
authors.lastname: Assmann
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany.
authors.forename: Niels
authors.initials: N
authors.lastname: Murawski
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany.
authors.forename: Boris
authors.initials: B
authors.lastname: Kubuschok
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Held
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany.
authors.forename: Klaus-Dieter
authors.initials: KD
authors.lastname: Preuss
authors.affiliation: Saarland University Medical School, José Carreras Center for Immuno- and Gene Therapy, Internal Medicine I, Homburg, Saar, Germany. Electronic address: michael.pfreundschuh@uks.eu.
authors.forename: Michael
authors.initials: M
authors.lastname: Pfreundschuh
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D000077153:Progranulins; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D064546:Protein Kinase C beta / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D053219:Receptors, Tumor Necrosis Factor, Member 25 / Q000276:immunology / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000276:immunology / Q000378:metabolism; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000276:immunology / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D001323:Autoantibodies; C492314:GRN protein, human; D036341:Intercellular Signaling Peptides and Proteins; D000077153:Progranulins; D020033:Protein Isoforms; D011498:Protein Precursors; D053219:Receptors, Tumor Necrosis Factor, Member 25; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; C506033:TNFRSF25 protein, human; D012694:Serine; D064546:Protein Kinase C beta
keywords: Alternative conversion; DR3; Hyperphosphorylation of Ser81; Neoantigen; PKCβ1; PP1; Progranulin; Proinflammatory autoantibody; TNFR1&2