Mapping epitopes of U1-70K autoantibodies at single-amino acid resolution

Abstract

The mechanisms underlying development of ribonucleoprotein (RNP) autoantibodies are unclear. The U1-70K protein is the predominant target of RNP autoantibodies, and the RNA binding domain has been shown to be the immunodominant autoantigenic region of U1-70K, although the specific epitopes are not known. To precisely map U1-70K epitopes, we developed silicon-based peptide microarrays with >5700 features, corresponding to 843 unique peptides derived from the U1-70K protein. The microarrays feature overlapping peptides, with single-amino acid resolution in length and location, spanning amino acids 110–170 within the U1-70K RNA binding domain. We evaluated the serum IgG of a cohort of patients with systemic lupus erythematosus (SLE; n = 26) using the microarrays, and identified multiple reactive epitopes, including peptides 116–121 and 143–148. Indirect peptide ELISA analysis of the sera of patients with SLE (n = 88) revealed that ∼14% of patients had serum IgG reactivity to 116–121, while reactivity to 143–148 appeared to be limited to a single patient. SLE patients with serum reactivity to 116–121 had significantly lower SLE Disease Activity Index (SLEDAI) scores at the time of sampling, compared to non-reactive patients. Minimal reactivity to the peptides was observed in the sera of healthy controls (n = 92). Competitive ELISA showed antibodies to 116–121 bind a common epitope in U1-70K (68–72) and the matrix protein M1 of human influenza B viruses. Institutional Review Boards approved this study. Knowledge of the precise epitopes of U1-70K autoantibodies may provide insight into the mechanisms of development of anti-RNP, identify potential clinical biomarkers and inform ongoing clinical trails of peptide-based therapeutics.

• Silicon-based peptide microarray
• systemic lupus erythematosus
• SLE
• ribonucleoprotein
• RNP

Acknowledgements

The authors would like to acknowledge the patients and volunteers who participated in this study. Alex Kuo for critical discussions relating to the article. Rohit Gupta, Robin Castel Navarro and the members of the Utz lab for support.

Declaration of interest

M.V. and G.C. are employees of Intel Inc. All the other authors declare that they have no competing interests. P.J.U. is the recipient of a Donald E. and Delia B. Baxter Foundation Career Development Award and is supported by National Heart, Lung, and Blood Institute (NHLBI) Proteomics contract HHSN288201000034C, Proteomics of Inflammatory Immunity and Pulmonary Arterial Hypertension; grants from the NIH (5 U19-AI082719, 5 U19-AI050864, 5 U19-AI056363, 1 U19 AI090019 and 4 U19 AI090019); Canadian Institutes of Health Research (2 OR-92141); Alliance for Lupus Research (grant number 21858); a gift from the Ben May Trust and a gift from the Floren Family Trust. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 261382. C.L.L. is a recipient of an NIH National Research Service Award Fellowship (5 F32 AI-080086-02). D.J.H. was supported by the Canadian Institutes of Health Research (CIHR). The trial in healthy volunteers enrolled by the Stanford-LPCH Vaccine Program was supported by NIH/NIAID U19AI090019 (M. Davis, P.I.) and an NIH/NCRR CTSA award number UL1 RR025744.

Supplementary material available online.

Tables S1 and S2 and Figures S1-S3.