HIV-Infected Patients: Cross Site-Specific Hydrolysis of H2a and H2b Histones and Myelin Basic Protein with Antibodies against These Three Proteins

Anti-DNA antibodies are usually produced against histone-DNA complexes appearing during cell apoptosis, while histones are known as damage-associated molecules. A myelin sheath of axons contains myelin basic protein (MBP) playing an important role in the pathogenesis of autoimmune diseases. Antibodies with enzymatic activities (abzymes) are distinctive features of some autoimmune and viral diseases. Abzymes against different proteins can usually only hydrolyze these specific proteins. Using sequential chromatographies of homogeneous IgG preparations from sera of HIV-infected patients on columns with immobilized MBP, H2a, and H2b histones, the anti-MBP, anti-H2a, and anti-H2b antibodies were obtained. It was first shown that IgGs against H2a and H2b effectively hydrolyze these histones and MBP, while anti-MBP split MBP, H2a, and H2b, but no other control proteins. Using the MALDI mass spectrometry, the cleavage sites of H2a, H2b, and MBP by abzymes against these three proteins were found. Among 14 sites of hydrolysis of H2a by IgGs against H2a and 10 sites by anti-MBP IgGs, only one site of hydrolysis was the same for these abzymes. Eleven cleavage sites of H2b with IgGs against H2b and 10 sites of its hydrolysis with antibodies against MBP were different. Anti-H2a, anti-H2b, and anti-MBP abzymes are unpredictable examples of IgGs possessing not only cross-complexation but also catalytic cross-reactivity, which may be a common phenomenon for such abzymes in patients with different autoimmune diseases. The existence of cross-reactivity of abzymes against H2a and H2b histones and MBP represent a great danger to humans since, in contrast with MBP, histones due to cell apoptosis constantly occur in human blood. Anti-H2a, anti-H2b, and anti-MBP can attack and hydrolyze myelin basic protein of the myelin sheath of axons and plays a negative role in the pathogenesis of several pathologies.


Introduction
It becomes clear during the last three decades that autoantibodies (auto-Abs) from the sera of patients with several autoimmune (AI) as well as several viral diseases similar to artificial abzymes (Abzs) against chemically stable analogs of different chemical reactions (reviewed in [1][2][3]) can possess several different enzymatic activities [3][4][5][6][7][8]. Natural IgGs, IgAs, and IgMs with protease, DNase, RNase, and amylase activities were revealed in sera of patients with several AI and viral diseases [3][4][5][6][7][8][9][10][11][12][13][14][15][16]. Some healthy humans produce Abzs with very low thyroglobulin- [9], VIP- [10], it could not be excluded that enzymatic cross-activity may not only for be abzymes against MBP and H1 but also against other histones, including H2a and H2b. Histones and their complexes with DNA appear in the blood of healthy and sick people constantly as a result of apoptosis of various cells. When cross-catalytic activity between histones and MBP exists, abzymes against histones can hydrolyze the main myelin protein of axonal envelopes of nerve tissues. This may be an internal factor in the development of disorders of the nervous system in patients with various AI diseases, including HIV-infected patients.
In this study, we revealed enzymatic cross-reactivity IgGs against H2a and H2b histones in the hydrolysis of MBP, while Abs against MBP in the hydrolysis of H2a and H2b histones. Interestingly, IgGs against H2a, H2b, and MBP did not demonstrate catalytic cross-reactivity with respect to other different control proteins. In this article, we have analyzed specific sites of the hydrolysis of H2a and H2b histones by IgGs against these proteins and against MBP using MALDI mass spectrometry.

Chemicals, Donors, and Patients
Protein G-Sepharose and Superdex 200-HR-10/30 columns were from GE Healthcare (GE Healthcare, New York, USA) and homogeneous H2a and H2b histones were provided by Sigma (St. Louis, MO, USA). Human myelin basic protein was purchased from the Molecular Diagnostics and Therapy of DBRC (Moscow, Russia). MBP-and H2a-histone-and H2b-histone-Sepharoses were obtained by immobilization of H2a and H2b histones, and MBP on BrCN-activated Sepharose according to the standard manufacturer's protocol (St. Louis, MO, USA).
Sera of 29 HIV-infected patients (18-40 y old; men and women) including 13 at the stage of pre-AIDS and 16 at the stage of generalized lymphoadenopathy according to the Center of Disease Control and Prevention classification were used in this study. The protocols of blood sampling meet the guidelines of the human ethics hospital committee (Ethics committee of Novosibirsk State Medical University, Novosibirsk, Russia, permission number 72-H). This committee approved this study in accordance with the guidelines of the Helsinki ethics committee, including a written agreement from patients to present their blood for scientific purposes.

Antibody Purification
Electrophoretically homogeneous IgGs were derived from sera of AIDS patients by chromatography of blood sera proteins using protein G-Sepharose and then by fast protein liquid chromatography (FPLC; gel filtration) in acidic buffer (pH 2.6) on Superdex 200 HR 10/30 column as in [32][33][34][35][36][37][38]. Western blotting of Abs was performed according to [33,34]. The fractions corresponding to the central parts of the IgG peak after gel filtration were concentrated for further chromatographies and assay of catalytic activities. After each step of purification, protein concentrations were measured using a known standard Bradford assay using calibration with bovine serum albumin. To protect from bacterial contamination, IgG preparations were filtered through a Millex filter (pore size 0.1 µm). After storage at +4 • C for 5-7 days for refolding after treatment with acidic buffer, the antibodies were used for their activities assays as described below.

Affinity Chromatography of IgGs on MBP-and Histone-Sepharose
Removal of anti-histone IgGs from total polyclonal Ab preparations was carried out using histone-Sepharose (immobilized equimolar mixture of five histones: H1, H2a, H2b, H3, and H4) column (7 mL) equilibrated in buffer A (20 mM Tris-HCl, pH 7.5) by analogy with [32]. After Abs loading, the column was washed with buffer A to zero optical density. The IgG fraction having no affinity for histones (eluted from histone-Sepharose at loading), was subjected to re-chromatography on MBP-Sepharose to get anti-MBP IgGs. Adsorbed anti-MBP IgGs were eluted using buffer A containing 1.0 M NaCl and finally by acidic buffer (0.1 M glycine-HCl, pH 2.6).
Anti-MBP IgGs were removed from total IgG preparations by affinity chromatography on the MBP-Sepharose column (5 mL) equilibrated in buffer A. The IgG fraction having no affinity for MBP (eluted from MBP-Sepharose at loading) was subjected to re-chromatography on H2a-or H2b-Sepharoses to get anti-H2a and anti-H2b IgGs, respectively. After column washing with buffer A to zero optical density, adsorbed anti-H2a and anti-H2b IgGs were eluted from the sorbents using buffer A containing 3 M NaCl and then with acidic buffer similar to that for MBP-Sepharose as in [32]. IgG fractions eluted from H2a-or H2b-, and MBP-Sepharoses were dialyzed against buffer A and then were used for enzyme-linked immunosorbent assay (ELISA). In addition, in order to completely remove possible anti-H2a and anti-H2b antibody impurities from the anti-MBP antibody preparations, they were passed twice through the H2a-Sepharose column and then twice through the H2b-Sepharose column (in both cases, fractions eluted from the columns upon application were collected). Anti-H2a and anti-H2b IgGs were passed through anti-MBP-Sepharose twice. All IgG preparations before and after their additional purification on alternative sorbents (anti-MBP on anti-H2a and anti-H2b Sepharose), as well as anti-H2a and anti-H2b on MBP Sepharose (see above) showed exactly the same ELISA data (see below).
The relative activity (RA) of IgGs in the hydrolysis of either MBP, H2a, H2b, or all, histones was estimated as described below.

ELISA of Anti-MBP and Anti-Histones Autoantibodies
Anti-MBP, as well as anti-H2a and anti-H2b histones IgGs concentrations were measured using homogeneous preparations of anti-MBP, anti-H2a, and anti-H2b IgGs according to [32][33][34][35][36]. The conditions that we used in this work correspond to the linear parts of the dependence of the ELISA signal on the concentrations of antigens used (40-55% from the plateau). Sodium carbonate buffer (50 µL, pH 9.6) containing 0.01 mg/mL MBP, H2a, or anti-H2b histones was added to ELISA strips for their incubation overnight at 4 • C. The solutions from the wells were removed, then they were washed three times with TBS buffer (20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Triton, and NaN 3 and 0.05%) and then twice with TBS without Triton X-100. To block the surfaces of the strips, they were treated for 2 h at 37 • C with TBS containing 0.2% egg albumin and 0.01% NaN 3 . The strips were then washed 9 times with water and then with TBS supplemented with 0.01% NaN 3 . TBS containing anti-MBP, anti-H2a, or anti-H2b IgGs, 0.2% egg albumin, 0.01% NaN 3 , and 0.05% Triton X-100 (100 µL) was added to the strips for 2 h (37 • C). After washing all strips with 100 µL water (9 times).
TBS containing egg albumin and NaN 3 was added for additional incubation for 2 h at 37 • C and washed 9 times with water. Then they were incubated with TBS (100 µL) containing 1 µg/mL conjugate of anti-human monoclonal IgGs with horseradish peroxidase at 37 • C for 30 min and washed 9 times again with water. After the addition of citric-phosphate buffer (50 µL) containing H 2 O 2 and 3,3 ,5,5 -tetramethylbenzidine, all strips were incubated for 15 min at 22 • C; the reaction was stopped by adding of 50% H 2 SO 4 (50 µL). The relative concentrations of antibodies against MBP, H2a, and H2b histones in anti-MBP, anti-H2a, and anti-H2b IgG preparations were expressed as a difference in optical density (A 450 ; an average of 3 measurements) corresponding to samples analyzed with and without the above IgGs against MBP, H2a, and H2b.
The reactions were stopped, and the efficiency of the hydrolysis of the proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 4-18% gradient gel under nonreducing conditions. The products of the hydrolysis were detected by Coomassie Blue or silver staining. The gels were first imaged and then scanning and quantified using Image Quant v5.2 software (Media Cybernetics Inc., Rockville, MD, USA). The relative activity of antibodies was evaluated from the loss of these proteins in their initial non-hydrolyzed forms.
SDS-PAGE analysis of enzymatic cross-reactivity of IgGs against MBP, H2a, and H2b was performed using MBP preparation incubation with anti-MBP, anti-H2a, and H2b IgGs.

Kinetic Analysis
The K M and k cat values were calculated from the dependencies of V versus (H2a) or (H2b) by least-squares non-linear fitting using Microcal Origin v5.0 software (Media Cybernetics Inc., Rockville, MD, USA) and presented as linear transformations using a Lineweaver-Burk plot [44]. The k cat values (calculated as V max, µM/min/(IgG), µM) are reported as mean ± standard deviation of three independent experiments for each substrate H2a and H2b in their hydrolysis by IgGs (0.04-0.05 mg/mL) against MBP, H2a, and H2b histones. Errors in the values were within 15-20%.

MALDI-TOF Analysis of Ab-Dependent Proteins Hydrolysis
H2a and H2b histones (1.0 mg/mL) were hydrolyzed by anti-H2a-IgGs, anti-H2b-IgGs, or anti-MBP-IgGs (0.005-0.1 mg/mL) for 0-24 h as described above. The analysis of products of the hydrolysis was carried out by MALDI-TOF spectrometry using the Bruker Reflex III system (Bruker Frankfurt, Germany) equipped with a 337-nm nitrogen laser (VSL-337 ND, Laser Science, Newton, MA, USA), 3 ns pulse duration. Mixtures (1 µL) of solution saturated with sinapinic acid (for analysis of proteins ≥10 kDa) or 2-cyano-3-(4-hydroxyphenyl) acrylic acid (for analysis of small peptides and proteins ≤10 kDa) in 0.1% acetonitrile and trifluoroacetic acid (1:2) with 1 µL of the reaction mixtures after hydrolysis of histones and MBP peptides were used for analysis. The final mixtures were applied on the MALDI plates, air-dried, and then used for the study. MALDI-TOF spectra calibrations were carried out using the oligopeptide and protein standards II and I (Bruker Daltonic, Germany) in the internal and external calibration mode. The analysis of histones and MBP peptides cleavage sites was carried out using Protein Calculator v3.3 (Scripps Research Institute).

Analysis of Sequence Homology
The analysis of homology between peptides and protein sequences was carried out using lalign (http://www.ch.embnet.org/software/LALIGN_form.html). This system was used to analyze possible homology between complete protein sequences of MBP with complete H2a and H2b histones.

Statistical Analysis
The results are given as the average mean ± standard deviation of 3 independent experiments for each sample of peptide, proteins, and IgG preparation.

Purification of Antibodies
We used IgGs of HIV-infected patients isolated first by affinity chromatography of serum proteins using protein G-Sepharose in conditions removing non-specifically bound proteins [32][33][34][35]. Then IgG preparations were additionally purified by FPLC gel filtration under conditions (buffer pH 2.6) destroying immune complexes as in [33,34]. It was shown recently that 100% of IgGs from the sera of 32 HIV-infected patients efficiently hydrolyze from one to five human histones [33][34][35]. In this study, for analysis of catalytic cross-reactivity of IgGs against H2a and H2b antibodies in the hydrolysis of MBP and anti-MBP antibodies in the cleavage of H2a and H2b histones were used polyclonal electrophoretically homogeneous IgG preparations of HIV-infected patients obtained and characterized as in [32]. The mixture of equimolar amounts of 29 IgG preparations (IgG mix ) with high activity in the hydrolysis of histones and MBP was used for purification of antibodies against H2a, H2b, and MBP, as described above.
It was shown earlier that the nonspecific antigens usually have 1-2 orders of magnitude lower affinity than the specific ones [17][18][19][20][21]. Abs interacting with different affinity sorbents due to cross-complexation can be eluted by 0.1-0.2 M NaCl [17][18][19][20][21]. Total Abs lacking IgGs against MBP was applied on H2a-or H2b-Sepharoses; IgGs with relatively low affinity to these two histones were eluted from the columns with buffer containing 1 M NaCl. Next, fractions of IgGs with a high affinity for H2a or H2b histones were eluted with acidic buffers, pH 2.6. These fractions were used for the analysis of H2a, H2b, and MBP hydrolysis.
To obtain Abs against MBP, the total IgGs fraction after removal of anti-histone IgGs was applied to the MBP-Sepharose column. Anti-MBP IgGs with relatively low affinity was eluted from the column using 1 M NaCl, while having high affinity with acidic buffer, pH 2.6. The fractions with high affinity for MBP were used for the analysis of catalytic cross-reactivity in the hydrolysis of histones and MBP.

ELISA of Anti-MBP and Anti-Histones Autoantibodies
Anti-MBP, as well as anti-H2a and anti-H2b histones IgGs were measured using homogeneous preparations of anti-MBP, anti-H2a, and anti-H2b Abs according to [32][33][34][35][36]. According to ELISA data, Abs against MBP gave a positive answer (A 450 units) not only against MBP (0.28 ± 0.02) but also against H2a (0.08 ± 0.009) and H2b Thus, according to ELISA data, anti-H2a IgG preparations did not contain tangible amounts of antibodies against histone H2b and vice versa. However, anti-MBP IgG preparations showed a positive response against H2a and H2b histones, while anti-H2a and anti-H2b Abs against MBP. For any case, we skipped the anti-MBP IgGs twice using columns containing immobilized H2a and H2b histones, while anti-H2a and anti-H2b IgGs Abs through a column with immobilized MBP. However, the ELISA analysis data in all cases completely coincided with that given above. The data obtained could be interpreted in two different ways. On the one hand, the possibility was not excluded that Abs against MBP could not be cleared from IgGs against H2a and H2b histones and vice versa. However, the nonspecific complexation of some proteins with Abs against other ones at ELISA and affinity chromatographies is a widely distributed phenomenon and is known as polyspecificity or polyreactivity of Abs [45][46][47][48]. Catalysis of the transformation of substrates can occur only after the formation of their complexes with abzymes and enzymes. Interestingly, non-specific cross-complexation has also been described for a large number of very different enzymes [49][50][51][52][53]. However, canonic enzymes usually catalyze only one chemical reaction and catalytic conversion by enzymes of unspecific ligands after complexes formation (catalytic cross-reactivity) is an extremely rare case [49][50][51][52][53]. All described data for abzymes against various proteins also hydrolyze only their specific globular proteins [3][4][5][6][7][8][9][10][11][12][13][14][15][19][20][21][22][32][33][34][35][36][37][38][39][40][41]. Therefore, it could be assumed that the criss-crossed complex formation revealed by ELISA could be a consequence of cross-complexation due to the homology of antigenic determinants sequences of MBP with those of H2a and H2b histones. Thus, the ELISA data could not give an answer about the possibility of the presence in each of the IgG preparations of admixtures of Abs to other antigens or implementation of the phenomenon of polyspecificity or polyreactivity. However, in the case of a presence in anti-MBP Abs preparations of IgGs against H2a or H2b abzymes and versa visa, the hydrolysis of H2a or H2b histones should occur at the same specific sites of the protein cleavage. Detection of various H2a and H2b cleavage sites with Abs against these histones, on the one hand, and IgGs against MBP, on the other, may indicate that they possess not only complexation polyreactivity, but also enzymatic cross-activity. Thus, it was very interesting to analyze a possible enzymatic cross-reactivity between Abs-abzymes against H2a and H2b histones and MBP.
Unfortunately, electrophoretically homogeneous MBP preparations are not available. Due to alternative splicing of cDNA and partial hydrolysis of protein in the brain of some humans, preparations of MBPs can contain several related protein forms (21.5, 18.5, 17.5, ≤14.0 kDa) and products of their hydrolysis, [54,55]. Line C1 of Figure 2A demonstrates the heterogeneity of the starting preparation of human MBP mainly containing 14-18.5 kDa protein forms. After incubation of this MBP for 24 h with antibodies against H2a, H2b, and MBP, all forms of protein >15 kDa disappear; the formation of smaller peptides is observed (Figure 2A). Noticeable hydrolysis by anti-H2a antibodies of H2b histone and vice versa was not observed during 10 h of the incubation. This correlates with ELISA data on the lack of interaction of anti-H2 antibodies with H2b histone and vice versa. Consequently, one can suppose that not only IgGs against MBP but also against H2a and H2b are able to hydrolyze MBP. Figure 2B demonstrates that after 24 h the H2a and H2b histones incubation with IgGs against H2a, H2b, and MBP leads to their very efficient hydrolysis. These data are evidence of the possibility of catalytic cross-reactivity of antibodies against MBP, H2a, and H2b. Nevertheless, the H2a and H2b cleavage sites with antibodies against these histones and antibodies against MBP and vice versa may be different. It is important that only the difference in the sites of histone hydrolysis by antibodies against H2a and H2b histones and against MBP can clearly indicate not only their complexation polyreactivity but also the enzymatic cross-activity of antibodies against histones and against MBP.

Analysis of Site-Specificity of H2a Histone Hydrolysis
To find sites of H2a histone hydrolysis, we analyzed its overtime hydrolysis with IgGs against H2a and MBP eluted from specific sorbents with acidic buffer (pH 2.6). Before addition of IgGs, H2a histone is nearly homogeneous demonstrating its one-(m/z = 13,981.9 Da) and two-charged ions (m/z = 6991.0 Da) ( Figure 3A). After 1 h of the incubation in the presence of anti-H2a Abs were revealed by only three very small peaks with molecular masses (MMs) of 12,629.1, 10,228.0, and 10,042.6 Da ( Figure 3B) corresponding with the following cleavage sites of H2a histone: A14-K15, R35-K36, and G37-N38, respectively ( Figure 3B). These three peaks have increased significantly during mixture incubation from 1 to 20 h, and five additional peaks appeared ( Figure 3B-D). All eight of these peaks correspond to a cluster of two major ( ) and six moderate (↓) cleavage sites. In addition, there is one cluster of seven very weak cleavage sites (♦) corresponding to the long C-terminal of H2a. After 20 h of the incubation, a lot of peptides with MMs~2.0-8.0 kDa were revealed ( Figure 3D). Most of them are additional products of the cleavage of long oligopeptides (OPs) corresponding to H2a C-terminal part. Interestingly, after H2a histone incubation for 1h with IgGs against MBP, six other average or small peaks (Da) were revealed: 8678.9 (Y50-51L), 10,609.9 (Y50-51L), 11,799.6 (R29-V30), 11,927.7 (R20-A21), 12,257.9 (R17-S18), and 13,108.4 (T120-E121) ( Figure 3E). The intensity of these peaks grows from 1 to 20 h of the reaction mixture incubation ( Figure 3E-G). Moreover, additional peaks were revealed after 3-20 h of the incubation (Da): 8082.6 (E56-Y57), 9549.4 (E56-Y57), 10,227.7 (R35-K36), 11,002.2 (R32-L33) ( Figure 3F-H). Interestingly, the four major cleavage sites of H2a with anti-MBP antibodies corresponded to approximately the same protein fragment (from 20 to 38 AAs) as in the case of anti-H2a antibodies ( Figure 4A). However, only one site of the hydrolysis (R35-K36) was the same in the case of anti-H2a and anti-MBP abzymes ( Figure 4A,B). In addition, in the case of anti-MBP IgGs, there is one cluster of two major, two average, and one weak cleavage sites in the protein zone from 41 to 64 amino acids (AAs), where there are no cleavage sites of H2a by IgGs against this protein.
A moderate T120-E121 site of the hydrolysis was also found only in the case of anti-MBP antibodies ( Figure 4B). Interestingly, the H2a cleavage sites in the case of antibodies against anti-MBP in the N-terminal zone of the histone do not form a real cluster, and all sites of cleavage correspond to the histone fragment after the R-residue: R-A, R-S, R-V, and R-K ( Figure 4B

Analysis of Site-Specificity of H2b Histone Hydrolysis with Abzymes
The initial homogeneous H2b histone demonstrates two peaks-the single-(13,780.6 Da) and the double-charged histone (6890.2 Da) ( Figure 5A). One hour of incubation leads to the formation of only three small peaks ( Figure 5B Figure 5C). After incubation of the reaction mixture during 20 h, it contains products of a deeper cleavage of long peptides with MMs~2.0-6.0 kDa ( Figure 5D). Four major (R15-S16, E35-S36, K43-V44, and R86-S87), 4 average (V44-L45, K57-A38, E71-R72, and T96-A97), and 2 weak (K15-K16 and R100-L101) sites of H2b hydrolysis by anti-MBP antibodies after 3 h of the incubation were detected ( Figure 5E). Figure 5F,G show deeper hydrolysis of H2b hydrolysis during 6 h and 20 h. Among the 11 cleavage sites of H2b with IgGs against H2b ( Figure 5E) and 10 sites of its hydrolysis with Abs against MBP, there are none of the same cleavage sites. In addition, in the case of H2b cleavage sites by Abs against this histone, there are extended cluster (P50-H63) sites of the hydrolysis, while for IgGs against MBP there are no other clusters of cleavage sites ( Figure 6A,B). Therefore, Abs against H2b and MBP proteins also possess both cross-complexation and enzymatic cross-reactivity.

Analysis of Possible Homology of H2a and H2b Histones with MBP
One of the possible reasons for the catalytic cross-hydrolysis of H2a and H2b histones and MBP by IgGs against these proteins may be a high level of homology of the protein splitting sequences. Supplementary Figure 1 shows the identity of AAs of complete sequences of H2a histone and MBP: 25.9% identity, while similarity of amino acids (non-identical AAs with highly similar physicochemical properties), 51.7%. A nearly similar situation was observed for H2b histone and MBP sequences: 25.7% identity and 53.6% similarity (Supplementary Figure S2).
Interestingly, some of the H2a sequences hydrolyzed by IgGs against H2a and MBP almost match or at least overlap, but the cleavage sites in the fragments of these sequences for anti-H2a and anti-MBP Abs are different (Figure 4). For example, anti-H2a and anti-MBP IgGs hydrolyze the same sequence, but in different sites: anti-H2a IgGs-RAKA KSRS↓SRA↓G, while anti-MBP abzymes-R AKAKSR SSRAG (Figure 4). This H2a histone sequence is homologous to the sequence of MBP (GHHAARTTHYGS): 33% identity and 75% similarity (Figure 4 and Supplementary Figure  S1). There is another fragment of H2a sequence (VHRLLRKGN) that is split by both anti-H2a and anti-MBP IgGs but also in different sites. (Figure 4 and Supplementary Figure S1). In addition, there are H2a sequences homologous with those for MBP in which there are cleavage sites only in the case of IgGs against H2a histone or only MBP (Figure 4 and Supplementary Figure S1).
To some extent, a similar situation is observed for the hydrolysis of histone H2b by IgGs against this histone and against MBP ( Figure 6 and Supplementary Figure S2). There are fragments of the sequences of H2b and MBP demonstrating increased homology. For example, MBP sequence APKRGSGKDGHHAAR is homologous to APKKGSKKAVTKAQK sequence of H2b: 43.8% identity and 68.8% similarity. This sequence of H2b was hydrolyzed by IgGs against H2b (APKK ↓GSKK↓AVTKAQK) and against MBP (APKKGSK↓KAVTKAQK), but at different specific sites ( Figure 6 and Supplementary Figure S2). However, there are only a few good matches between the sites of H2b hydrolysis by antibodies against H2b and MBP ( Figure 6, Supplementary Figure S2). Thus, abzymes against H2a and MBP, as well as H2b and MBP, demonstrate enzymatic cross-reactivity. However, cross-hydrolyzable sequences of H2a and H2b by IgGs against these histones and against MBP may overlap or be completely different (Figures 4 and 6, and Supplementary Figures S1 and S2).

Affinity of IgGs for Histones
We have estimated the K m and k cat values in the hydrolysis of H2a and H2b by anti-H2a, anti-H2b, and anti-MBP IgGs (Figure 7). The initial rate data obtained at increasing concentrations of H2a and H2b were consistent with the Michaelis-Menten kinetics. The K M and k cat values for H2a and H2b histones are given in Table 1 (Table 1). In addition, the relative rate of H2a hydrolysis (k cat = 0.33 ± 0.04 min −1 ) by antibodies against this histone is approximately 2.2-fold faster than that for the anti-MBP IgGs (0.15 ± 0.025 min −1 ). It is interesting that IgGs against H2b (0.036 ± 0.006 min −1 ) hydrolyze this histone five times slower than Abs against MBP (0.18 ± 0.03 min −1 ) (Figure 7, Table 1).  7. Determination of the K m and V max (k cat ) values for H2a and H2b substrates in their hydrolysis by IgGs against MBP ((A,B), respectively; 0.04 mg/mL) and by abzymes against H2a ((C); 0.05 mg/mL) and H2b ((D); 0.042 mg/mL) using a Lineweaver-Burk plot. The average error in the initial rate determination at each substrate concentration from two independent experiments did not exceed 10-20%.
The nonspecific complexation of some antigens with Abs against other ligands is a widely distributed phenomenon [45][46][47][48][49]. Specific for different substrates, canonic enzymes usually catalyze only one chemical reaction [49][50][51][52][53]. To date, described abzymes against various proteins can hydrolyze only their specific protein [3][4][5][6][7][8][9][10][11][12][13][14][15][19][20][21][22]. In view of this, it was difficult to anticipate that IgGs against any histones and MBP can possess catalytic cross-reactivity. The first example of catalytic cross-reactivity was anti-H1 histone and anti-MBP IgGs from sera of HIV-infected patients [32]. Therefore, in this paper, we analyzed the possible enzymatic cross-reactivity between antibodies-abzymes against H2a and H2b histones and MBP. Using several affinity chromatographies, anti-H2a, anti-H2b, and anti-MBP antibodies were obtained. However, due to the possibility of cross-complexation of IgGs against H2a and H2b histones with MBP (revealed by ELISA), this was not enough to establish enzymatic cross-reactivity. The first evidence of possible enzymatic cross-activity of Abs against two histones and MBP was obtained from the SDS-PAGE analysis of the hydrolysis of H2a, H2b, and MBP with IgGs against these substrates. However, it was obvious that only a significant difference in the hydrolysis sites of the histones by abzymes against H2a, H2b, and MBP can clearly indicate not only their complexation polyreactivity but also the IgGs enzymatic cross-activities.
Fourteen sites of the hydrolysis of H2a by Abs against H2a and 10 splitting sites by IgGs against MBP were revealed ( Figure 4). However, only one site of the hydrolysis (R35-K36) was the same in the case of anti-H2a and anti-MBP abzymes (Figure 4). In addition, among the 11 cleavage sites of H2b with IgGs against H2b and 10 sites of its hydrolysis with Abs against MBP, there are no identical cleavage sites ( Figure 5).
One of the main reasons for cross-catalysis in the case of abzymes against two histones and MBP can be the homology of their protein sequences. The identity of AAs of complete protein sequences of H2a and H2b histones and MBP is respectively, 25.9% and 25.7% identity, and 51.7% and 53.6% of similarity. Some sites of the hydrolysis of H2a with Abs against this histone and IgGs against MBP to some extent overlap and demonstrate homology with MBP sequences (Supplementary Figure S1). A similar situation was observed for splitting sites of H2a with IgGs against this histone and Abs against MBP (Supplementary Figure S2).
Abzymes against myelin basic protein hydrolyze MBP of axonal envelopes of nerve tissues and therefore are extremely harmful to humans [7]. It is believed that some autoimmune diseases may be a consequence of bacterial and viral infections [32,56,57]. First the immune system can produce antibodies against viral or parasites proteins, and then it may switch to the synthesis of autoantibodies to host antigens due to the molecular mimicry between viral or bacterial and human proteins, alteration of host antigens, abnormal expression of immunoregulatory molecules, and activation of the anti-idiotypic network.
MS is a nervous system disease resulting in the manifestation of different psychiatric and nervous disturbances [58]. However, all diseases mentioned above are also more or less related to the violation of the nervous system and mental disorders. Neuropsychiatric disturbances occur in about 50% of patients with SLE [57]. Different violations of synaptic transmission resulting in neuronal damage and serious mental dysfunction in schizophrenia [57] and HIV-infected [58,59] patients were also revealed. Thus, it cannot be excluded that the enzymatic cross-reactivity of Abs hydrolyzing H1 [32].

Conclusions
We have first shown that IgGs against H2a and H2b histones and against MBP demonstrate not only cross-complexation but also manifest catalytic cross-reactivity. H2a and H2b histones and MBP, which are able to hydrolyze the myelin basic protein of the myelin sheaths of nerve tissues, can probably play an important role in the development of neurodegenerative and neuropsychiatric diseases.

Conflicts of Interest:
The authors declare no conflict of interest.