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.

Keywords: RNP; SLE; Silicon-based peptide microarray; ribonucleoprotein; systemic lupus erythematosus.

Conflict of interest statement

Declaration of interest

All the other authors declare that they have no competing interests.

Figures

Figure 1

Layout and content of silicon-based U1-70K peptide microarrays. (A) Peptides representing sequences from amino acids 110–170 of the RNA-binding domain of the human U1-70K protein were synthesized using maskless photolithography on the surface of a silicon wafer. (B) The microarray consists of overlapping 3- to 10-mer peptides in triplicate, tiled with a one amino acid offset across the region (upper), and duplicate 21 × 21 feature blocks representing every possible 1- to 21-mer within the sequence ranges shown below (lower). (C) An expanded view of a block featuring peptides derived from amino acid sequence 110–130 is shown. The peptide sequences of features from the bottom row and right-most column are shown for illustration.

Figure 2

U1-70K peptide microarrays identify the minimal epitopes of an anti-U1-70K polyclonal antibody. (A) An anti-U1-70K polyclonal antibody, 70R-4091, and a nonspecific polyclonal control, were used to probe U1-70K microarrays at 1 μg/ml. Reactivity was detected with a Cy3-conjugated anti-rabbit secondary antibody, and the resulting fluorescent microarray images are shown, including an inset featuring block 110–130 in greater detail. A heatmap (right) shows the Median Fluorescence Intensity (MFI) of each feature in the inset block, and minimal epitopes 112–115 and 121–124 are highlighted. (B) Selected reactive peptides (111–124, 118–124 and 120–124) and a nonreactive control (143–154) were generated as C-terminal lysine-biotinylated peptides using traditional solid-phase synthesis. Indirect peptide ELISA on streptavidin-coated plates was performed using 70R-4091 followed by europium-labeled goat anti-rabbit IgG, and the time-resolved fluorescence counts for each condition are shown (error bars = SD). Reactive peptides used for ELISA are highlighted in the microarray inset in (A).

Figure 3

U1-70K peptide microarrays identify the serum antibody epitopes of patients with SLE. (A) Serum samples from patients with SLE (n = 26) were used to probe U1-70K microarrays, and antibody binding was detected using a Cy5-conjugated anti-human IgG (Fcγ specific) secondary antibody. A hierarchically clustered (unsupervised, Euclidean distance) heatmap of 216 peptides, filtered for features with a maximum Z-score <3 is shown. Black bars on the right indicate reactive peptide clusters. Peptide clusters 116–121 and 143–148 are shown in more detail in (B) and (C), respectively. The sequences corresponding to the microarray features are shown on the right, and the peptides used for indirect ELISA are underlined. Sample names are shown at the bottom of each column. The MFIs of each microarray were median centered and log transformed prior to clustering.

Figure 4

U1-70K microarrays reveal diverse peptide reactivity patterns when probed with SLE patient serum. (A) Fluorescence images from selected microarrays are shown with patient identifiers on the left. Patient samples 173-04 and 068-04 have high reactivity to 116–121 and 143–148, respectively. A less-reactive patient sample, 030-04, is shown for comparison. Insets highlighting reactivity to specific peptide features within the 110–130 (B) and 140–160 (C) blocks are shown. Peptide features corresponding to peptides used for validation by ELISA (116–121 and 143–148) are indicated with smaller squares, and their sequences are shown to the right of the most reactive samples. Peptide 128–129 PI is indicated with a magenta arrow.

Figure 5

ELISA analysis confirms serum reactivity to U1-70K peptide epitopes, and reveals distinct longitudinal patterns of reactivity between patients. (A) Selected reactive peptide sequences (116–121 and 143–148) were generated as C-terminal lysine-biotinylated peptides using traditional solid-phase synthesis, and were used to perform indirect peptide ELISA. The peptides were bound to streptavidin-coated plates, and the plates were then probed with a dilution series of serum samples from known reactive patients (173 and 068). Binding of the europium-labeled mouse anti-human IgG (Fcγ specific) secondary was quantified by time-resolved fluorescence. (B) Longitudinal serum samples, collected approximately every 3 months and corresponding to 1 year of clinic visits, were assayed by ELISA (error bars = SD).

Figure 6

Peptide ELISA reveals a subset of SLE patients with serum IgG reactivity to U1-70K epitope 116–121, and reactivity to 116–121 is associated with decreased disease activity. (A) Biotinylated peptides 116–121 and 143–148 were used to coat 96-well streptavidin coated ELISA plates. Serum samples from patients with SLE (n = 88) and healthy volunteers (n = 92) were used to probe the plates in duplicate, and Europium-labeled anti-human IgG (Fcγ specific) was used as a secondary reagent. Boxplots of the time-resolved fluorescent counts are shown above (vertical bars show 75th percentile + 1.58 × IQR × n−1/2). (B) Patients with SLE were defined as positive or negative if their reactivity to peptide 116–121 was greater or less than the maximum reactivity of the healthy controls, respectively. A Mann–Whitney test was used to compare SLEDAI scores at the time of serum collection between groups.

Figure 7

U1-70K microarray analysis of a 116–121 reactive SLE patient serum identified by ELISA. (A) Selected fluorescence images from microarrays are shown with patient identifiers on the left. Patient sample 216-03 was predicted to have high levels of reactivity to 116–121 by peptide ELISA. Patient 002-07 was predicted to be negative to both epitopes by peptides ELISA, and was used as a negative control. Insets highlighting reactivity to specific peptide features with the 110–130 (B) and 130–150 (C) blocks are shown. Sequences of peptides 116–121 and 131–137 are indicated with smaller yellow and green squares, respectively.

Figure 8

Antibodies to 116–121 bind a common epitope in U1-70K and the matrix protein M1 of human influenza B viruses. Serum samples from two patients, selected based on high reactivity to 116–121, were analyzed using competitive ELISA. The samples were incubated with U1-70K peptide 68–72 (ERKRR), a scrambled control peptide (RRERK) or vehicle. Biotinylated peptide 116–121 was used to coat a 96-well streptavidin coated ELISA plate. Following incubation, the samples were used to probe the plate in duplicate, and Europium-labeled anti-human IgG (Fcγ specific) was used as a secondary reagent. A bar graph of the time-resolved fluorescent counts is shown. Error bars represent SD of duplicate wells.