Autoimmunity to isomerized histone H2B in systemic lupus erythematosus

Abstract

Histone H2B is a common target of autoantibodies in both spontaneous and drug-induced systemic lupus erythematosus (SLE). Recent studies demonstrate that Asp(25) of histone H2B (H2B) spontaneously converts to an isoaspartic acid (isoAsp) in vivo. Our laboratory has demonstrated that the posttranslational modification of an aspartic acid to an isoaspartic acid within self-peptides renders otherwise ignored peptides immunogenic. Analysis of serum from lupus-prone mice and histone antibody positive SLE patients revealed antibodies specific to the Asp and isoAsp H2B(21-35) peptide, and that the expression of these antibodies is dependent on TLR9. IsoAsp H2B(21-35) is immunogenic in non-autoimmune prone mice and mice lacking the ability to repair isoAsp have significantly reduced levels of antibodies to H2B. Asp H2B(21-35) incubated at physiological temperatures and pH acquires the isoAsp modification, demonstrating that H2B(21-35) is prone to spontaneous isoAsp formation in vivo. Autoimmunity to isoAsp H2B suggests that this form of the autoantigen may be critical in the induction of anti-histone autoantibodies in human SLE and in murine models of disease.

Figure 1

Structure and formation of isoaspartic acid. Initial nucleophilic attack of the peptide bond nitrogen leads to the formation of a succinimide intermediate, which after hydrolysis under physiological conditions yields isoaspartic acid.

Figure 2

MRL lpr sera contain high titers of antibodies that react against Asp and isoAsp H2B_21–35. Sera from MRL lpr mice were diluted 1:1000 and IgG against (A) Asp and (B) isoAsp H2B_21–35 measured by ELISA. Horizontal line represents the mean. Results represent 4–20 mice per time point.

Figure 3

3H9 Tg MRL lpr mice have IgM antibodies against isoAsp H2B_21–35 that are regulated by TRL9. Sera from 3H9 Tg MRL lpr TLR9 wild-type or knockout mice were diluted 1:1000 and IgM against (A) isoAsp H2B_{21 –35} or a control peptide and (B) dsDNA measured by ELISA. Mice were 17–20 weeks of age with results representing 4 mice per group. Statistics were calculated with a 2-tailed Students t-test.

Figure 4

Mice immunized with isoAsp H2B_{21 – 35} develop antibodies against histone H2B and whole histones. Sera from B10.A mice immunized with either Asp H2B_{21 – 35}, isoAsp H2B_21–35 or injected with PBS-CFA were diluted 1:100 and IgG against histone H2B and whole histones measured by ELISA. Sera from MRL lpr mice served as a positive control. Results represent four mice per group.

Figure 5

Mice unable to repair isoAsp have reduced levels of antibody against H2B_21–35. Sera from mice lacking the isoAsp repair enzyme PIMT (Tg PIMT knockout) or Tg PIMT wild-type mice were diluted 1:100 and used to measure IgG against both isoforms of H2B_{21 –35} by ELISA. Horizontal line represents the mean. Results represent 10–15 mice per group. Statistics were calculated using the Mann-Whitney U-test.

Figure 6

Sera from individuals who have antibodies against histones react against Asp and isoAsp H2B_21–35. Sera (1:100 dilution) from histone antibody positive individuals, histone negative individuals and normal healthy individuals (NHS) were analyzed for IgG responses to both Asp and isoAsp H2B_21–35 by ELISA. Results are representative of 7–10 individuals per group. Experiment was repeated 3 times. Statistics were calculated by the Mann-Whitney U-test.

Figure 7

Asp H2B_21–35 undergoes spontaneous isomerization at Asp²⁵. The isoAsp content of Asp H2B_21–35 was determined using the ISOQUANT Isoaspartate Determination kit after incubation in PBS pH 7.4 at 37°C for either 14 or 26 days. Asp H2B_21–35 stored at – 80° C was used to determine the baseline amount of isoAsp within the peptide.