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. 2019 Nov 15;9(11):741.
doi: 10.3390/biom9110741.

Antibodies from the Sera of Multiple Sclerosis Patients Efficiently Hydrolyze Five Histones

Svetlana V Baranova  1 , Elena V Mikheeva  1 , Valentina N Buneva  1 , Georgy A Nevinsky  1
Affiliations

Antibodies from the Sera of Multiple Sclerosis Patients Efficiently Hydrolyze Five Histones

Svetlana V Baranova et al. Biomolecules. .

Abstract

It is known that intranuclear histones can be pernicious after entering to the extracellular space. In addition, the immunization of animals with exogenous histones leads to systemic inflammatory and toxic reactions. Abzymes-autoantibodies with enzymatic activities-are the distinctive feature of autoimmune diseases and they can be especially dangerous to humans. Here, electrophoretically homogeneous IgGs were isolated from sera of patients with multiple sclerosis (MS) by chromatography on several affinity sorbents. We present evidence that sera of all MS patients contain autoantibodies against histones and 73% of IgGs purified from the sera of 59 MS patients efficiently hydrolyze from one to five histones: H1, H2a, H2b, H3, and H4. The relative average efficiency of the histones hydrolysis was ~3.9-fold higher than that for healthy donors. The relative average activity of IgGs depends on the type of MS and decreased approximately in the following order: debut of MS, secondary progressive multiple sclerosis, remitting multiple sclerosis, remittent progressive multiple sclerosis. Similar to proteolytic abzymes of patients with several autoimmune diseases, histone-hydrolyzing IgGs from MS patients were inhibited in the presence of specific inhibitors of serine and of metal-dependent proteases, but an unexpected significant inhibition of the activity by inhibitors of thiol-like and especially acidic proteases was observed. Since IgGs can efficiently hydrolyze histones, a negative role of abzymes in the development of MS cannot be excluded.

Keywords: catalytic IgGs; human blood antibodies; hydrolysis of human histones; multiple sclerosis patients.

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Conflict of interest statement

the authors declare the absence of competing financial interests.

Figures

Figure 1
Figure 1
(A) SDS-PAGE analysis of homogeneity of four individual IgGs and IgGmix (9 μg) from the sera of MS patients in a nonreducing 3–16% gradient gel (lanes 1–4 and IgGmix) followed by silver staining The arrows (lane C) indicate the positions of protein molecular mass markers; (B) SDS-PAGE analysis of the activity of several different IgGs in the hydrolysis of H1, H2a, H2b, H3, and H4 histones resulting in the formation of their fragments. The reaction mixtures was incubated for 20 h at 37 °C with 0.05 mg/mL IgGs (lanes: 1—RMS3, 2—debut of multiple sclerosis (DMS6), 3—secondary progressive multiple sclerosis (SPMS)1, 4—DMS7, 5—DMS8, 6—SPMS2, 7—RMS4); (C) Hydrolysis of recombinant H1 histone by several different IgGs leading to the formation of their fragments; lanes: 1—RMS3, 2—DMS6, 3—SPMS1, 4—DMS7, 5—DMS8, 6—RMS9 (Lanes C1 correspond to five histones (B) and H1 (C) incubated without Abs. (C) Lane C shows the position of protein markers with known molecular masses (C).
Figure 2
Figure 2
Verification of the strict criteria proving that IgGmix hydrolyze histones. Fast protein liquid gel filtration chromatography (FPLC) gel filtration of MS IgGmix on a Superdex 200 column in glycine buffer (pH 2.6) after Abs pre-incubation using the same buffer. (A) (—), Absorbance at 280 nm (A280); (■, ○, ∆, ▲) relative activities (RAs) of IgGmix in the hydrolysis of H1, H2a, H2b, and H4, respectively. A complete hydrolysis of each histone for 16 h in the presence of 5 µl of the eluates was taken for 100%. SDS-PAGE analysis of IgGmix in the hydrolysis of histones; (B) After non-reducing SDS-PAGE of MS IgGmix in 3–15% gradient gel, it was incubated using special conditions for IgGs renaturation. The RA in hydrolysis of H1, H2a, H2b, H3, and H4 histones (■, ○, ∆, □, respectively) were estimated using the extracts of many 3–4 mm fragments of one longitudinal slice of the gel; (C) The RA of IgGmix corresponding to a complete hydrolysis of every histone after 16 h of incubation with 5 μL of extracts was taken for 100%. The second control longitudinal slices of the same gels were stained with Coomassie R250; (D) Shows the position of protein with known molecular masses. The error in the RAs determination (two independent experiments) in hydrolysis of each histone did not exceed 10–20% (A,B). For details, see Materials and Methods.
Figure 3
Figure 3
Typical examples of a decrease in histones-hydrolyzing activity of two individual IgGs (DMS6 and DMS8) after their dialysis against EDTA and then the activation of dialyzed IgGs by different metal ions at fixed 2 mM concentration (A–H). A complete hydrolysis of every histone in the presence of 0.66 μM IgGs for 12 h was taken for 100%. Diagrams corresponding to various metal ions are marked in panels A–D (DMS6) and E–H (DMS6); used metal ions are listed on these panels. The differences between different groups were estimated: the significant difference (p = 0.036) was found only between the hydrolysis of H4 by DMS8 and H2b by DMS6, in other cases, p was higher than 0.05. For details, see Materials and Methods.
Figure 4
Figure 4
The pH dependence of the relative activity of three individual DMS6 (A), DMS7 (B), and DMS8 (C) preparations in the hydrolysis of the histones. The relative activity corresponding to a complete hydrolysis of every histone after 6 h of incubation in the presence of 0.33 μM IgGs was taken for 100%. The average in the initial rate determination error from two experiments did not exceed 10–25%. Significant difference (p < 0.05; 0.0001–0.041) was revealed for the following data in the hydrolysis of histones: DMS6 (H1–H4; H2a–H4; H2b–H4), DMS6–DMS7 (H2a; H4; H1–H2b; H1–H4; H2a–H2b; H2b–H4; H2a–H3; H2b–H4), and DMS6–DMS8 (H1, H2a, H2b, H1–H2a; H1–H2b;H1–H3; H1–H4; H2a–H2b; H2a–H3; H2a–H4; H2b–H3; H2b–H4). In the remaining cases, p > 0.05. See Materials and Methods for other details.
Figure 5
Figure 5
Dependencies of relative rates of H1 hydrolysis by different IgGs (0.33 μM): DMS6 (A,B), DMA7 (C,D), DMA8 (E), and SPMS1 (F) in different coordinates on the concentration of H1 histone. Determination of the Km and Vmax values for H1 histone in its hydrolysis by DMS6 (B) and DMA7 (D), and DMA8 (insert to E) was performed using the Lineweaver–Burk plot and the data corresponding to the first part of the hyperbolic curves of panels A and C, respectively; the insert to E corresponds to first part of the main dependence for DMA8. The error in the initial rate determination at each substrate concentration did not exceed 10–25%. Significance of differences between data of A and B panels, p = 0,0005. Reactions were performed as described in Materials and Methods.
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