Proteomic identification of heterogeneous nuclear ribonucleoprotein K as a novel cold-associated autoantigen in patients with secondary Raynaud's phenomenon

Lingli Yang, Minoru Fujimoto, Hiroyuki Murota, Satoshi Serada, Manabu Fujimoto, Hiromi Honda, Kohji Yamada, Katsuya Suzuki, Ayumi Nishikawa, Yuji Hosono, Yoshihiro Yoneda, Kazuhiko Takehara, Yoshitaka Imura, Tsuneyo Mimori, Tsutomu Takeuchi, Ichiro Katayama, Tetsuji Naka

Research output: Contribution to journalArticle

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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).

CONCLUSION: 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.

Original languageEnglish
Pages (from-to)349-358
Number of pages10
JournalRheumatology (Oxford, England)
Volume54
Issue number2
DOIs
Publication statusPublished - 2015 Feb 1

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Heterogeneous-Nuclear Ribonucleoprotein K
Raynaud Disease
Autoantigens
Proteomics
Autoantibodies
Proteome
Enzyme-Linked Immunosorbent Assay
Mixed Connective Tissue Disease
Indirect Fluorescent Antibody Technique
Serum
Immunoblotting
Flow Cytometry

Keywords

  • autoantibody
  • heterogeneous nuclear RNP-K
  • proteomics
  • Raynaud’s phenomenon

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Proteomic identification of heterogeneous nuclear ribonucleoprotein K as a novel cold-associated autoantigen in patients with secondary Raynaud's phenomenon. / Yang, Lingli; Fujimoto, Minoru; Murota, Hiroyuki; Serada, Satoshi; Fujimoto, Manabu; Honda, Hiromi; Yamada, Kohji; Suzuki, Katsuya; Nishikawa, Ayumi; Hosono, Yuji; Yoneda, Yoshihiro; Takehara, Kazuhiko; Imura, Yoshitaka; Mimori, Tsuneyo; Takeuchi, Tsutomu; Katayama, Ichiro; Naka, Tetsuji.

In: Rheumatology (Oxford, England), Vol. 54, No. 2, 01.02.2015, p. 349-358.

Research output: Contribution to journalArticle

Yang, L, Fujimoto, M, Murota, H, Serada, S, Fujimoto, M, Honda, H, Yamada, K, Suzuki, K, Nishikawa, A, Hosono, Y, Yoneda, Y, Takehara, K, Imura, Y, Mimori, T, Takeuchi, T, Katayama, I & Naka, T 2015, 'Proteomic identification of heterogeneous nuclear ribonucleoprotein K as a novel cold-associated autoantigen in patients with secondary Raynaud's phenomenon', Rheumatology (Oxford, England), vol. 54, no. 2, pp. 349-358. https://doi.org/10.1093/rheumatology/keu325
Yang, Lingli ; Fujimoto, Minoru ; Murota, Hiroyuki ; Serada, Satoshi ; Fujimoto, Manabu ; Honda, Hiromi ; Yamada, Kohji ; Suzuki, Katsuya ; Nishikawa, Ayumi ; Hosono, Yuji ; Yoneda, Yoshihiro ; Takehara, Kazuhiko ; Imura, Yoshitaka ; Mimori, Tsuneyo ; Takeuchi, Tsutomu ; Katayama, Ichiro ; Naka, Tetsuji. / Proteomic identification of heterogeneous nuclear ribonucleoprotein K as a novel cold-associated autoantigen in patients with secondary Raynaud's phenomenon. In: Rheumatology (Oxford, England). 2015 ; Vol. 54, No. 2. pp. 349-358.
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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).CONCLUSION: 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.",
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T1 - Proteomic identification of heterogeneous nuclear ribonucleoprotein K as a novel cold-associated autoantigen in patients with secondary Raynaud's phenomenon

AU - Yang, Lingli

AU - Fujimoto, Minoru

AU - Murota, Hiroyuki

AU - Serada, Satoshi

AU - Fujimoto, Manabu

AU - Honda, Hiromi

AU - Yamada, Kohji

AU - Suzuki, Katsuya

AU - Nishikawa, Ayumi

AU - Hosono, Yuji

AU - Yoneda, Yoshihiro

AU - Takehara, Kazuhiko

AU - Imura, Yoshitaka

AU - Mimori, Tsuneyo

AU - Takeuchi, Tsutomu

AU - Katayama, Ichiro

AU - Naka, Tetsuji

PY - 2015/2/1

Y1 - 2015/2/1

N2 - 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).CONCLUSION: 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.

AB - 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).CONCLUSION: 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.

KW - autoantibody

KW - heterogeneous nuclear RNP-K

KW - proteomics

KW - Raynaud’s phenomenon

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