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Systemic Lupus Erythematosus Patients Contain Significantly Less IgM against Mono-Methylated Lysine than Healthy Subjects

PLoS ONE (2013) 8(7)
DOI: 10.1371/journal.pone.0068520
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Abstract

Post-translational modifications on proteins are important in biological processes but may create neo-epitopes that induce autoimmune responses. In this study, we measured the serum IgG and IgM response to a set of non-modified or acetyl- and methyl-modified peptides corresponding to residues 1-19 of the histone 3 N-terminal tail in systemic lupus erythematosus (SLE) patients and healthy subjects. Our results indicated that the SLE patients and healthy subjects produced antibodies (Abs) to the peptides, but the two groups had different Ab isotype and epitope preferences. Abs to the non-modified form, H31-19, were of the IgG isotype and produced by SLE patients. They could not recognize the scrambled H31-19, which contained the same amino acid composition but a different sequence as H31-19. In comparison, healthy subjects in general did not produce IgG against H31-19. However, about 70% of the healthy subjects produced IgM Abs against mono-methylated K9 of H31-19 (H31-19K9me). Our further studies revealed that ε-amine mono-methylated lysine could completely inhibit the IgM binding to H31-19K9me, but lysine had no inhibitory effect. In addition, the IgM Abs could bind peptides containing a mono-methylated lysine residue but with totally different sequences. Thus, mono-methylated lysine was the sole epitope for the IgM. Interestingly, SLE patients had much lower levels of this type of IgM. There was no obvious correlation between the IgM levels and disease activity and the decreased IgM was unlikely caused by medical treatments.We also found that the IgM Abs were not polyreactive to dsDNA, ssDNA, lipopolysaccharide (LPS) or insulin and they did not exist in umbilical cord serum, implying that they were not natural Abs. The IgM Abs against mono-methylated lysine are present in healthy subjects but are significantly lower in SLE patients, suggesting a distinct origin of production and special physiological functions. © 2013 Guo et al.

Figures

  • Table 1. Distribution of IgG and IgM anti-H31–19 and H31–19K9me in SLE patients and healthy controls.
  • Figure 1. Reactivity of serum IgG and IgM against non-modified and modified H3 peptides. Microtiter plates were coated with peptides conjugated to BSA. Serum samples from healthy controls or SLE patients were diluted 1:100 and tested. KT47 anti-human IgG and KT16 anti-human IgM mAbs were used as primary Abs, and HRP-conjugated goat anti-mouse IgG was used as a secondary Ab (see Materials and Methods). The data are presented by box-blot diagram. (A) IgG anti-peptide Abs of pediatric samples. (B) IgG anti-peptide Abs of adult samples. (C) IgM anti-peptide Abs of pediatric samples. (D) IgM anti-peptide Abs of adult samples. Healthy controls are represented in blue and SLE patients are represented in red. P values between the healthy control and SLE groups were calculated using the Student’s t test. P values within the same groups were calculated using the paired t test. doi:10.1371/journal.pone.0068520.g001
  • Figure 2. Comparison of IgM anti-H31–19K9me levels in patients and healthy subjects. Anti-H31–19K9me IgM levels in pSLE patients (n = 62), JIA patients (n = 36), other RD patients (n = 26, including juvenile ankylosing spondylitis, Henoch-Schonlein purpura, idiopathic thrombocytopenic purpura, Kawasaki disease, mixed connective tissue disease, juvenile dermatomyositis and Behcet’s disease) and pHC (n = 62) were measured by ELISA. Microtiter plates were coated with the H31–19K9me peptide conjugated to BSA. The serum samples were diluted 1:100 and tested. The KT16 anti-human IgM mAb was used as the primary Ab, and HRP-conjugated goat anti-mouse IgG was used as the secondary Ab. The horizontal bars indicate the mean values in each group. P values were calculated using the Student’s t test. doi:10.1371/journal.pone.0068520.g002
  • Figure 3. Western blot for IgG and IgM eluted from H31–19 and H31–19K9me beads. Equal volumes (1.5 ml) of pooled sera were first absorbed with the H31–19 beads and then with the H31–19K9me beads as described in the Materials and Methods. The beads were washed 5 times with 1 M NaCl, and the bound Abs were eluted with 50 ml elution buffer. 10 ml of the eluates underwent SDS-PAGE under reducing conditions and were then transferred onto nitrocellulose membranes. The membranes were cut at the 58 kDa position. IgG on the low molecular weight (,58 kDa) membrane and IgM on the high molecular weight (.58 kDa) membrane were detected. (A) Healthy adult samples; (B) aSLE patient samples. a, IgG(1–19high91x)IgM(1–19x91x); b, IgG(1– 19low91low)IgM(1–19high91high); c, IgG(1–19low91low)IgM(1–19high91low); d, IgG(1–19low91low)IgM(1–19low91high); e, IgG(1–19low91low)IgM(1– 19low91low). 1–19E, Abs eluted from the H31–19 beads; 91E, Abs eluted from the H31–19K9me beads. The percentages represent the sample proportion of each subgroup (see Table 1). The results are representative of two independent experiments. doi:10.1371/journal.pone.0068520.g003
  • Figure 4. Reactivity of purified IgG against H3 peptides. Equal volumes (1.5 ml) of the pooled serum samples from each subgroup were affinity purified using H31–19 and H31–19K9me beads in tandem (see Materials and Methods). Microtiter plates were coated with various peptides conjugated to BSA as indicated. Eluates from the H31–19 and H31–19K9me beads were separately tested for their reactivity against the peptides. KT47 anti-human IgG was used as the primary Ab and HRP-conjugated goat anti-mouse IgG was used as the secondary Ab. The samples were tested in duplicate. 1–19E, Abs eluted from the H31–19 beads; 91E, Abs eluted from the H31–19K9me beads. Data are expressed as mean+SEM. The results are representative of two independent experiments. doi:10.1371/journal.pone.0068520.g004
  • Figure 5. Reactivity of purified IgM against H3 peptides. Equal volumes (1.5 ml) of the pooled serum samples from each subgroup were affinity purified using H31–19 and H31–19K9me beads in tandem (see Materials and Methods). Microtiter plates were coated with various peptides conjugated to BSA as indicated. Eluates from the H31–19 and H31–19K9me beads were separately tested for their reactivity against the peptides. KT16 anti-human IgM was used as the primary Ab and HRP-conjugated goat anti-mouse IgG was used as the secondary Ab. The samples were tested in duplicate. 1–19E, Abs eluted from the H31–19 beads; 91E, Abs eluted from the H31–19K9me beads. Data are expressed as mean+SEM. The results are representative of two independent experiments. doi:10.1371/journal.pone.0068520.g005
  • Figure 6. Epitope analysis for IgG and IgM purified from the H31–19 or H31–19K9me beads. (A), (B) and (C) Microtiter plates were coated with H31–19, H31–19K9me, scrambled H31–19 (sH31–19) and scrambled H31–19K9me (sH31–19K9me) conjugated to BSA. IgM of healthy adults from the IgG(1–19low91low)IgM(1–19high91high) subgroup, IgM of aSLE patients from the IgG(1–19low91low)IgM(1–19high91high) subgroup and IgG of aSLE patients from the IgG(1–19high91x)IgM(1–19x91x) subgroup were tested. KT16 anti-human IgM mAb and KT47 anti-human IgG were used as the primary Abs. HRP-conjugated goat anti-mouse IgG was used as the secondary Ab. 1–19E, Abs eluted from the H31–19 beads; 91E, Abs eluted from the H31–19K9me beads. Data are expressed as mean+SEM. (D) Microtiter plates were coated with H31–19K9me or sH31–19K9me conjugated to BSA. IgM Abs of healthy adults from the IgG(1–19low91low)IgM(1–19high91high) subgroup were incubated with lysine (K) or e-amine mono-methylated lysine (Kme) at indicated concentrations for 1 h at room temperature. KT16 anti-human IgM was used as the primary Ab. HRP-conjugated goat anti-mouse IgG was used as the secondary Ab. (E) Microtiter plates were coated with H31–19, H31–19K9me and GGK(me)GGSGGSGGSG (GGKme) conjugated to BSA. IgM of healthy adults from the IgG(1–19low91low)IgM(1–19high91high) subgroup was tested. KT16 anti-human IgM was used as the primary Ab. HRP-conjugated goat anti-mouse IgG was used as the secondary Ab. The results are representative of three separate experiments. doi:10.1371/journal.pone.0068520.g006
  • Figure 7. Polyreactivity test by ELISA. dsDNA, ssDNA, LPS or insulin was coated on microtiter plates as described in Materials and Methods. Sera (1:100 dilution) and Abs purified from the H31–19 or H31–19K9me beads (1:200 dilution) were tested. KT47 anti-human IgG or KT16 anti-human IgM Ab was used as the primary Ab and HRP-conjugated goat anti-mouse IgG was used as the secondary Ab. a, IgG(1–19low91low)IgM(1–19high91high); b, IgG(1–19low91low)IgM(1–19high91low); c, IgG(1–19low91low)IgM(1–19low91high); d, IgG(1–19low91low)IgM(1–19low91low); e, IgG(1–19high91x)IgM(1–19x91x). 1–19E, Abs eluted from the H31–19 beads; 91E, Abs eluted from the H31–19K9me beads. Data are expressed as mean+SEM. The results are representative of two separate experiments. doi:10.1371/journal.pone.0068520.g007

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Guo, S., Liu, Y., Ma, Y., Zhao, Q., Zhu, L., Shao, Y., … Zhang, W. (2013). Systemic Lupus Erythematosus Patients Contain Significantly Less IgM against Mono-Methylated Lysine than Healthy Subjects. PLoS ONE, 8(7). https://doi.org/10.1371/journal.pone.0068520

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