title: Proteomic identification of heterogeneous nuclear ribonucleoprotein K as a novel cold-associated autoantigen in patients with secondary Raynaud's phenomenon.
abstract: OBJECTIVE
  The aim of this study was to identify cold-associated autoantibodies in patients with RP secondary to CTDs.
  
  
  METHODS
  Indirect immunofluorescence staining was performed on non-permeabilized cold-stimulated normal human dermal microvascular endothelial cells (dHMVECs), using patients' sera. Cold-induced alterations in cell surface proteomes were analysed by isobaric tag for relative and absolute quantitation (iTRAQ) analysis. Serological proteome analysis (SERPA) was applied to screen cold-associated autoantigens. The prevalence of the candidate autoantibody was determined by ELISA in 290 patients with RP secondary to CTDs (SSc, SLE or MCTD), 10 patients with primary RP and 27 healthy controls.
  
  
  RESULTS
  Enhanced cell surface immunoreactivity was detected in cold-stimulated dHMVECs when incubated with sera from patients with secondary RP. By iTRAQ analysis, many proteins, including heterogeneous nuclear ribonucleoprotein K (hnRNP-K), were found to be increased on the cell surface of dHMVECs after cold stimulation. By the SERPA approach, hnRNP-K was identified as a candidate autoantigen in patients with secondary RP. Cold-induced translocation of hnRNP-K to the cell surface was confirmed by immunoblotting and flow cytometry. By ELISA analysis, patients with secondary RP show a significantly higher prevalence of anti-hnRNP-K autoantibody (30.0%, 61/203) than patients without RP (9.2%, 8/87, P = 0.0001), patients with primary RP (0%, 0/10, P = 0.0314) or healthy controls (0%, 0/27, P = 0.0001).
  
  
  CONCLUSIONS
  By comprehensive proteomics, we identified hnRNP-K as a novel cold-associated autoantigen in patients with secondary RP. Anti-hnRNP-K autoantibody may potentially serve as a biomarker for RP secondary to various CTDs.
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan. Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Lingli
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Fujimoto
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Murota
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Serada
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Fujimoto
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Honda
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan. Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Kohji
authors.initials: K
authors.lastname: Yamada
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Suzuki
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Nishikawa
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Hosono
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Yoneda
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Takehara
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Imura
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Tsuneyo
authors.initials: T
authors.lastname: Mimori
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Takeuchi
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Katayama
authors.affiliation: Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan. tnaka@nibio.go.jp.
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Naka
mesh_terms: D001323:Autoantibodies / Q000378:metabolism; D001324:Autoantigens / Q000378:metabolism*; D015551:Autoimmunity / Q000502:physiology; D016022:Case-Control Studies; D002478:Cells, Cultured; D003080:Cold Temperature; D042783:Endothelial Cells / Q000378:metabolism; D005260:Female; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D040901:Proteomics / Q000379:methods; D011928:Raynaud Disease / Q000276:immunology*
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D034641:Heterogeneous-Nuclear Ribonucleoprotein K
keywords: Raynaud’s phenomenon; autoantibody; heterogeneous nuclear RNP-K; proteomics