Proteomic identification of heterogeneous nuclear ribonucleoprotein K as a novel cold-associated autoantigen in patients with secondary Raynaud's phenomenon.
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.
['Yang|Lingli|L|', 'Fujimoto|Minoru|M|', 'Murota|Hiroyuki|H|', 'Serada|Satoshi|S|', 'Fujimoto|Manabu|M|', 'Honda|Hiromi|H|', 'Yamada|Kohji|K|', 'Suzuki|Katsuya|K|', 'Nishikawa|Ayumi|A|', 'Hosono|Yuji|Y|', 'Yoneda|Yoshihiro|Y|', 'Takehara|Kazuhiko|K|', 'Imura|Yoshitaka|Y|', 'Mimori|Tsuneyo|T|', 'Takeuchi|Tsutomu|T|', 'Katayama|Ichiro|I|', 'Naka|Tetsuji|T|']
D001323:Autoantibodies D001324:Autoantigens D015551:Autoimmunity D016022:Case-Control Studies D002478:Cells, Cultured D003080:Cold Temperature D042783:Endothelial Cells D005260:Female D034641:Heterogeneous-Nuclear Ribonucleoprotein K D006801:Humans D008297:Male D008875:Middle Aged D040901:Proteomics D011928:Raynaud Disease
proteomics autoantibody Raynaud’s phenomenon heterogeneous nuclear RNP-K