Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, Suppl. 1, pp. S373-S400 © Pleiades Publishing, Ltd., 2025.

Russian Text © The Author(s), 2025, published in Uspekhi Biologicheskoi Khimii, 2025, Vol. 65, pp. 547-586.

S373

REVIEW

Immune Response and Production

of Abzymes in Patients with Autoimmune

and Neurodegenerative Diseases

Georgy A. Nevinsky

Institute of Chemical Biology and Fundamental Medicine,

Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia

e-mail: nevinsky@niboch.nsc.ru

Received May 16, 2024

Revised June 5, 2024

Accepted June 24, 2024

Abstract— The mechanisms of development of autoimmune, neurological, and viral diseases and the pos-

sibilities of immune response to various antigens in these pathologies still pose many questions. Human

immune system is theoretically capable of synthesizing about a million antibodies with very different prop-

erties against the same antigen. It remains unclear how many antibodies and with what properties can form

in healthy people and patients with autoimmune diseases (AIDs). The capabilities of traditional approaches,

such as enzyme immunoassay or affinity chromatography of Abs on specific sorbents, in answering these

questions and analyzing the diversity of antibodies formed against external and internal antigens, as well

as their role in the pathogenesis of various diseases, are very limited. Analysis of monoclonal antibodies in

the blood of patients with systemic lupus erythematosus (SLE) using phage display revealed that the number

of autoantibodies against DNA and myelin basic protein (MBP) can exceed 3-4 thousand, and approximately

30-40% of them are abzymes capable of hydrolyzing DNA and MBP. However, this approach does not allow

to investigate the variety of properties of such antibodies, in particular their catalytic activity. Abzymes

can play either positive or negative role in the development of various diseases. For example, in HIV-in-

fected patients, abzymes against viral polymerase and integrase cleave these proteins, thus slowing down

the development of immunodeficiency syndrome. Other antibodies play a negative role in the pathogenesis

of viral, neurological, and autoimmune diseases. Thus, antibodies capable of hydrolyzing DNA and histones

can penetrate through the cellular and nuclear membranes, stimulate cell apoptosis, and, as a result, trigger

autoimmune processes in many pathologies. Antibodies against MBP cleave this protein in the membranes

of cells in nerve tissues, leading to the development of multiple sclerosis (MS). In this case, abzymes against

individual histones were able to hydrolyze each of these histones, as well as MBP, while Abs against MBP

hydrolyzed MBP and all five histones. It has also been established that the substrate specificity of abzymes

in the hydrolysis of histones and MBP varied greatly depending on the stage of MS or SLE development.

Here, we used this example to analyze in detail the role that abzymes against various antigens play in their

expanded involvement in the pathogenesis of some AIDs. The review also describes the impact of defects in

the bone marrow stem cell differentiation characteristic of AIDs in the formation of B lymphocytes producing

harmful abzymes and summarizes for the first time the data on the exceptional diversity of autoantibod-

ies and abzymes, their unusual biological functions, and involvement in the pathogenesis of autoimmune

pathologies.

DOI: 10.1134/S0006297924604167

Keywords: autoimmune and neurological diseases, mechanisms of development, catalytic antibodies, abzymes

Abbreviations: Ab, antibody; Abz, abzyme; AGD, antigenic determinant; AID, autoimmune disease; MBP, myelin basic

protein; MOG, myelinoligodendrocyte glycoprotein; MS, multiple sclerosis; SLE, systemic lupus erythematosus.

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

INTRODUCTION

Antibodies (Abs) are traditionally described as

proteins produced by the immune system and capa-

ble of specific binding of antigens. In this classical

concept, Abs are similar to enzymes in their ability

to specifically interact with antigens, although they

are unable to catalyze chemical reactions. This state-

ment is true for most Abs; however, it has been found

unexpectedly that Abs can have a catalytic activity.

Linus Pauling (1946) suggested that the active sites

of enzymes have the maximum complementarity not

to the substrates, but rather to the transition states

of catalyzed chemical reaction. In this case, the an-

tigen recognition sites in catalytic antibodies should

be maximally adapted to the structure of the transi-

tion state [1]. However, formation of Abs against very

short-lived transition states is impossible. Later, Jencks

(1969) hypothesized that catalytically active Abs can

be obtained against stable analogs of transition states

of chemical reactions [2]. This idea was realized in ex-

perimental immunology much later– the methods for

production of enzymatically active monoclonal Abs

against chemically stable analogs were first described

in 1985  [3]. The first catalytically active monoclonal

Abs were obtained against p-nitrophenyl phosphor-

ylcholine  [4] and monoaryl phosphonate esters [5,  6]

and were named abzymes (AntiBody enZYMES; Abzs)

[4-27]. To date, about 200 artificial Abzs have been

described, most of which are specific monoclonal Abs

that had been developed as potential drugs. The de-

velopment of Abzs for medical purposes have been

described in several reviews [28-36].

Biological fluids of healthy donors contain var-

ious autoantibodies (autoAbs) at low concentrations

[37-56]. However, in patients with autoimmune diseas-

es (AIDs) [37-46], neurological pathologies, and viral

and bacterial infections [47-57], the concentrations of

these Abs are significantly higher. Beside the typical

autoAbs that are catalytically inactive, up to 30-40%

of autoAbs against specific antigens can exhibit the

enzymatic activity [58-63]. Abzs have been found in

biological fluids of patients with many AIDs, as well

as neurological, viral, and bacterial diseases [58-63].

The development of AIDs is characterized by

spontaneous or antigen-stimulated production of Abs

against peptides, proteins, DNA, RNA, their complex-

es, nucleotides, polysaccharides, etc. [28-36, 56-63].

The origin of natural Abzs against “self” molecules in

mammals is a complex process. Similar to artificial

Abzs, they can be specific to various enzyme substrates

that act as haptens [28-36, 56-63]. Some haptens can

change their conformation after forming complexes

with high-molecular components (proteins, nucleic

acids, polysaccharides, and their associations) and

simulate the transition states of respective substrates

in the enzyme-catalyzed reactions. AID development

is also accompanied by the formation of catalytically

active anti-idiotypic Abzs against the active sites of

enzymes [64-66]. Natural Abzs with various activities

identified in human and mammalian body fluids have

been described in [21-36, 56-63].

The presence of Abzs capable of hydrolyzing an-

tigens can be considered as the earliest and reliable

marker of initial stages of AIDs [58-63]. Abzs are de-

tected already at the early stages of AIDs and neuro-

logical disorders, when the typical markers of these

pathologies are yet absent and protein content in

the urine and autoAb titers are still within the range

characteristic of conditionally healthy individuals.

Detection of Abzs is a much more sensitive technique

compared to measuring autoAb titers by enzyme im-

munoassay (EIA), because catalysis results in a signif-

icant accumulation of reaction products, which allows

detection of even small amounts of Abzs with low but

reliably tested activities. For example, anti-DNA Abs

have been found at high concentrations (compared to

healthy donors) in only 17-18% patients with multiple

sclerosis (MS) and 38% patients with systemic lupus

erythematosus (SLE) [67], while DNA-hydrolyzing Abs

have been detected in ~90-95% patients with MS [68]

and SLE [69]. This is due to the fact that the titers

of autoAbs in AID patients increase only at the late

disease stages or during disease exacerbation. There-

fore, we believe that reliably detected presence of

Abzs can be considered as an indicator of early stages

of pathologies associated with the impaired immune

status. At the same time, a dramatic increase in the

Abzs activity occurs only after reaching severe stages

of AIDs [58-63].

Conditionally healthy people typically lack Abzs

[58-63]. However, it was found that the blood of some

conditionally healthy donors contained low-active

autoAbzs that hydrolyzed polysaccharides  [70], thy-

roglobulin  [71-73], and vasoactive neuropeptide  [74].

DNA-hydrolyzing Abzs were detected in patients with

SLE [68,  69,  75,  76], MS  [68,  69], Hashimoto’s thyroid-

itis  [77], diabetes mellitus  [78], viral hepatitis  [79],

schizophrenia [80], and HIV (human immunodefi-

ciency virus) infection  [81]. The blood of patients

with SLE  [68, 69, 82, 83], MS  [69, 82], viral hepatitis

[78], and schizophrenia  [84] contained Abzs capa-

ble of cleaving RNA and microRNAs. IgGs efficiently

hydrolyzing myelin basic protein (MBP) were found

in the blood of patients with SLE  [85], MS  [86,  87],

schizophrenia  [88], and HIV infection  [89]. The blood

of patients with sepsis contained Abs with a broad

proteolytic activity  [90]; Abzs with the aldolase activ-

ity were found in some AID patients  [91]; Abzs in pa-

tients with hemophilia A hydrolyzed factor VIII [92];

Abzs from human milk efficiently cleaved DNA and

proteins [93].

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

Abs capable of cleaving all five histones (H1, H2A,

H2B, H3, and H4) were identified in the blood of pa-

tients with MS [94], schizophrenia [95], and HIV infec-

tion [96]. The blood of healthy humans and animals

contained Abzs with the redox functions [97-102];

however, the activity of such antibodies in patients

with AIDs and neurological disorders was found to be

significantly higher [103].

MECHANISMS OF PROGRESSION

OF AUTOIMMUNE DISEASES

To develop new approaches to treating patients

with autoimmune and neurological diseases, it is im-

portant to understand the mechanisms and causes

underlying the emergence and development of such

disorders. As has been shown in [58-63, 104-109], the

major trigger of autoimmune processes in AIDs, neu-

rological disorders, and viral and bacterial infections

is impaired differentiation of bone marrow stem cells

(BMSCs).

According to the unitarian theory of hematopoie-

sis postulated by A.  A.  Maximov, there are four class-

es of hematopoietic cells [110]. All blood cells origi-

nate from the polypotent stem cells of bone marrow.

A stem cell divides with the formation of two new

cells, one of which maintains the properties of a stem

cell and the other can differentiate into any blood cell

without exception. The four major types of hemopoi-

etic precursors of blood cells are:

1. Early erythroid colonies (BFU-Es, erythroid

burst-forming units);

2. Granulocytic–macrophagic colonies (CFU-GMs,

granulocytic–macrophagic colony-forming units);

3. Later erythroid colonies (CFU-Es, erythroid

burst-forming units);

4. Granulocytic–erythroid–megakaryocytic–macro-

phagic colonies (CFU-GEMMs, granulocytic–erythroid–

megakaryocytic–macrophagic colony-forming units).

Bone marrow also contains T and B cells, which

are the products of differentiation of hemopoietic cell.

In healthy people, the four types of hemopoietic

cells, as well as T and B cells, are formed at a certain

ratio. During AID progression, various “non-self” and

“self” antigens penetrate through the blood–brain bar-

rier, resulting in the disturbance in the ratio between

the six types of cells, i.e., in the impaired differentia-

tion profile of stem cells [58-63, 104-109]. This leads to

the formation of new types of cells, including B cells

producing antibodies with various properties, in par-

ticular, pathological Abzs. Cell differentiation occurs

in several stages, so some B cells acquire the ability

to synthesize Abzs already in the cerebrospinal fluid,

while other cells undergo further differentiation in

the blood and organs of humans and animals.

The synthesis of Abzs by specific B cells occurs

already in the bone marrow [58-63, 104-109, 111-113].

Abzs from the cerebrospinal fluid of MS patients hy-

drolyzed DNA, MBP, and oligosaccharides ~30-60 times

more efficiently than Abzs from the blood of the same

patients [111-113]. Four types of bone marrow cells

enter the blood and transform into the cells of hu-

man blood and various organs, where cells that have

not finally differentiated in the cerebrospinal fluid

can undergo further differentiation when exposed to

exogenous and endogenous antigens. Impaired differ-

entiation of BMSCs leads to a dramatic activation of

lymphocyte proliferation in various organs, including

blood, bone marrow, spleen, thymus, and lymph nodes

[58-63, 104-109], where additional synthesis of Abzs

with various catalytic functions and properties takes

place.

B lymphocytes synthesizing harmful Abz can be

produced in healthy individuals [114], but these cells

are eliminated by apoptosis. In AID patients, apoptosis

of lymphocytes is inhibited [58-63, 104-109], resulting

in increased proliferation of cells producing harmful

antibodies, including Abzs.

Therefore, progression of AIDs is determined

mainly by the impaired differentiation of BMSCs, ac-

tivation of lymphocyte proliferation in organs, and

suppression of apoptosis. An important role in AID

development belongs to Abzs hydrolyzing blood, cell,

and tissue components.

When analyzing the mechanisms of AID devel-

opment, one should take into account another very

important factor. If canonical enzymes are synthesized

according to the “one gene – one enzyme” principle,

Abs are formed by an absolutely different mechanism,

namely, the V(D)J recombination that produces unique

DNA sequences encoding variable domains of Abs.

Thevariable regions of heavy  (H) and light  (L) chains

are encoded by the locus divided into several gene

segments designated as V  (variable), D  (diverse), and

J  (joining) [115]. Activation by an antigen results in

rapid proliferation of B cells. Loci encoding hypervari-

able domains of heavy and light chains are character-

ized by an increased frequency of point mutations and

deletions (the so-called somatic hypermutation). As a

result, the daughter cells formed by cell division will

produce antibodies with different variable domains.

Therefore, somatic hypermutation is another mecha-

nism that increases the diversity of Abs, thus influ-

encing the affinity of these Abs to antigens [116, 117].

The number of B cells synthesizing Abs with different

properties, including Abzs against the same antigen,

can be very large. Theoretically, human immune sys-

tem can produce about a million of B lymphocytes

secreting antibodies with different properties against

the same antigen [118]. In view of this, the potential

diversity of the Abz active site structure compared

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

toclassical enzymes is of great interest. It would also

be interesting to know how many different Abs, in-

cluding Abzs, can be formed in mammals with AIDs

and neurological disorders and to decipher the biolog-

ical functions of these Abs.

DIVERSITY OF ABZYMES

WITH THE DNase AND RNase ACTIVITIES

An exceptional diversity of polyclonal Abzs with

the DNase and RNase activities in the blood of pa-

tients with AIDs and neurological pathologies ([39,

58-63, 68, 69, 77-84] and references within), AID-prone

mice [119], and healthy animals immunized with DNA,

RNA, RNase  A, DNase  I, and DNase  II ([58-63, 68] and

references within) has been demonstrated using af-

finity chromatography of polyclonal IgGs on DNA-cel-

lulose in a NaCl concentration gradient (0.05-3.0  M).

IgGs with the DNase activity were distributed over the

entire chromatographic profile, including Abs eluted

with 3.0  M  MgCl

and acidic buffer (pH  2.6), which

destroys strong immune complexes [39, 58-63, 68, 69,

77-84, 119]. It is known that classical DNases typically

have only one pH optimum [120, 121]. The fractions

with different affinity to DNA-cellulose demonstrated

several pH optima and isoelectric points (pI from 4.5

to 9.0), as well as dependence (or lack of it) on differ-

ent metal ions, such as Mg

, Mn

, Ca

, Co

, Ni

([39,

58-63, 68, 69, 77-84] and references within).

It has been shown that the active sites of Abzs are

located in the variable regions of light chains, while

variable regions of heavy chains increase the affinity

of Abs for antigens [58-63, 94-96, 106-109].

A potential number of Abzs with the DNase ac-

tivity in the blood of SLE patients was estimated for

the kappa-type Ab monoclonal light chains (MLCs)

that were obtained using a phage library contain-

ing 10

variants of light chains [122, 123]. The pool

of phage particles was divided into 11 peaks, all of

which contained MLCs that efficiently hydrolyzed

DNA. Individual colonies were obtained using phage

particles eluted from DNA cellulose with 0.5  M  NaCl

and acidic buffer (pH 2.6). Forty-five colonies were

randomly selected from 451 individual colonies elut-

ed with 0.5  M  NaCl, and 15 of them (~33%) efficiently

hydrolyzed DNA [122]. Among 33 colonies selected out

of 687 individual colonies eluted with the acidic buffer

(peak 11), 19 (58%) exhibited the DNase activity [123].

The resulting 34 MLC preparations efficiently cleaved

DNA, but differed in the relative activity, dependence

on metal ions, and optimal pH values [122, 123].

The affinity of obtained MLCs for DNA was approxi-

mately 2 to 3 orders higher than the affinity of DNase  I

, 46-58  µM) [124]. The average content of Abzs with

the DNase activity among 78 colonies isolated from

two out of 11 analyzed peaks was 43.6%. Out of 1138

colonies grown on two Petri dishes, approximately

496 colonies contained cells producing Abzs with the

DNase activity. The total number of different MLCs

with the DNase activity in 11 peaks was estimated

as ~2000-3000 [122, 123]. It was shown that anti-DNA

Abzs from the sera of patients with SLE can have ab-

solutely different properties corresponding to canon-

ical DNase I and DNase II: they can either depend or

not on DNA sequences ([39,  58-63,  68,  69,  77-84] and

references within).

IgGs with the RNase activity capable of hydro-

lyzing homopolynucleotides, cCMP, and yeast RNA

were identified in the sera of patients with SLE  [69,

82,  83], MS  [68], Hashimoto’s thyreoiditis, polyarthri-

tis  [82], schizophrenia [84], and different types of hep-

atitis [79]. Similar to DNA-cleaving Abs, Abs with the

RNase activity were separated into a large number

of fractions by affinity chromatography. The fractions

differed in the pH optimum values, specificity toward

RNA and microRNAs, and dependence (or lack of it)

on metal ions [58-63, 69, 79, 82-84]. The activity of

the obtained Abzs strongly depended on the substrate

used and disease under study; the highest RNase ac-

tivity was observed for Abzs from patients with MS

and SLE [58-63, 68, 69, 82, 83]. Some IgGs were found

to hydrolyze various microRNAs in a site-specific

manner. The average activity of IgGs in patients with

SLE, MS, and schizophrenia was significantly higher

(22.3 to 84.8-fold) than in conditionally healthy donors

[82-84].

DIVERSITY OF ABZYMES

WITH THE PROTEOLYTIC ACTIVITY

Abzs with the proteolytic activity have been found

in patients with some autoimmune, neurological, and

viral diseases. An exceptional diversity of these Abzs

has been demonstrated by the affinity chromatography

of polyclonal Abs on sorbents with the corresponding

immobilized proteins [85, 125, 126]. Chromatography

on MBP–Sepharose showed that catalytically active

Abs distributed over the entire elution profile [85, 125,

126]. IgGs of all four subclasses (IgG1-4) were able to

hydrolyze MBP [85, 126]. The contribution of Abs of

each subclass to the total activity was individual for

each patient and disease, as well as for the type of

proteolytic activity. Proteolytically active total IgG and

IgM preparations mainly contained Abs with the ser-

ine proteases-like activity [58-63].

HIV is a causative agent of acquired immunode-

ficiency syndrome (AIDS), one of the most dangerous

human diseases. Retroviruses (including HIV) cause

chronic infections in humans. During the virus life

cycle, viral DNA is integrated into the host genome

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

with the involvement of HIV enzyme integrase [127,

128]. Abzs from the blood serum of HIV-infected pa-

tients demonstrated an extreme diversity in the hy-

drolysis of viral reverse transcriptase and integrase

[58-63].

The first discovered Abs with the serine prote-

ase-like and metal-dependent proteolytic activities

were IgGs from patients with MS [86, 129] and SLE

[130]. Quite unexpected results were obtained in the

analysis of proteolytic activity of integrase-hydrolyz-

ing IgGs and IgMs from HIV-infected patients ([58-63,

131] and references within). Along with inhibitors of

serine and metal-dependent proteases, the activity

of these Abzs was strongly suppressed by inhibitors

of acidic and thiol proteases. However, for five IgG

preparations and seven IgM preparations, the total ef-

fect of specific inhibitors against these four classes of

proteases was over 100%, indicating that the immune

system of HIV-infected patients produced anti-inte-

grase Abzs with active sites that contained amino acid

residues typical of four different classes of proteases:

acidic, thiol, serine, and metalloproteases [58-63, 131].

All IgG preparations isolated from the serum of 32

HIV-infected patients cleaved all five human histones

(H1, H2A, H2B, H3, and H4) [132], but their significant-

ly varied between IgGs from different patients. IgGs

from the blood of 40% healthy donors also hydrolyzed

five histones, although ~16 to 20 times less efficiently

compared to Abzs from the blood of HIV-infected pa-

tients [132]. The ability of Abs to cleave histones was

suppressed by specific inhibitors of metal-dependent

and serine proteases and, unexpectedly, by inhibitors

of thiol proteases. These findings suggested that the

immune system of HIV-infected patients synthesized

a variety of antibodies with different catalytic prop-

erties, which was confirmed by the analysis of mono-

clonal Abzs with the proteolytic activity.

Using the same library of kappa-type light chains,

MLCs capable of hydrolyzing MBP were obtained

and homogeneous MLC samples were analyzed [133-

136] using the same approaches as in the analysis of

MLCs with the DNase activity [122, 123]. The pool of

phage particles was separated into ten fractions with

different affinities for MBP. All fractions efficiently

hydrolyzed MBP. The fraction eluted from MBP–Sep-

harose with 0.5  M  NaCl solution was used to isolate

individual colonies [133-136]. Seventy-two colonies

were randomly selected out of 440 individual colonies;

22 out of the selected colonies (~30%) demonstrated

the MBP-hydrolyzing activity. These MLC preparations

were purified by chromatography on a Ni

-HiTrapTM

sorbent, followed by high-performance gel filtration.

All 22 isolated MLC preparations hydrolyzed MBP

with different efficiency [133-136]. Unexpectedly,

12 out of 22 MLC preparations exhibited a metallopro-

tease activity, which was inhibited only by ethylene-

diaminetetraacetate (EDTA). Four MLC preparations

had the serine protease-like activity, as they were in-

hibited by phenylmethylsulfonyl fluoride (PMSF) only.

For three MLC preparations, the effects of PMSF and

EDTA were comparable (~40% and 60%, respectively).

The activity of three other MLCs was strongly sup-

pressed by iodoacetamide, a specific inhibitor of thi-

ol proteases. Interestingly, iodoacetamide significantly

inhibited the activity of MLCs that were also inhibited

by EDTA, which suggests that two MLCs were chime-

ric antibodies with their active sites typical of thiol

proteases and metalloproteases. Very unexpectedly,

the activity of one MLC preparation was inhibited

by EDTA, PMSF, and iodoacetamide, the total effect

of three inhibitors being 154%, i.e., active site of this

MLC combined amino acid residues characteristic of

three types of classical proteases. All described MLCs

differed in the optimal pH values and ability to be

activated by metal ions (Ca

, Mg

, Co

, Zn

, Ni

and Mn

Unexpected results were also obtained in the

analysis of three MLC preparations: NGTA1-Me-pro,

NGTA2-Me-pro-Tr, and NGTA3-pro-DNase [133-136].

DNA sequences encoding these three MLCs were ana-

lyzed and proved to be very similar (88-100% identity)

to the embryonic lines of IgLV8 light chain genes of

several antibodies described in literature [133-136].

NGTA1-Me-pro was a metalloprotease inhibited

only by EDTA [134]. In the presence of different metal

ions, its activity toward MBP decreased in the follow-

ing order: Ca

>Mg

>Ni

≈Zn

≈Co

≈Mn

>Cu

NGTA1-Me-pro demonstrated two different pH op-

tima: 6.0 and 8.5. The optimal concentration of CaCl

was 6.0  mM at pH  6.0 and 1.0  mM at pH  8.5. The

value for MBP (20.0  ±  2.0  µM) and k

cat

at pH  6.0

(0.22  ±  0.02  min

−1

) were different from those at pH  8.5

(40.0  ±  3.0  µM and 0.07 ± 0.005  min

−1

, respectively),

which clearly indicates that NGTA1-Me-pro had two

different metal-dependent activities within the same

active site [134].

NGTA2-Me-pro-Tr was specifically inhibited by

PMSF (42%) and EDTA (58%). i.e., exhibited the proper-

ties of chimeric protease with serine and metal-depen-

dent activities [135]. After the treatment with PMSF,

the pH optimum for its metalloprotease activity was

6.5-6.6; after dialysis vs. EDTA, the pH optimum for

its serine protease-like activity was 7.4-7.5. At pH  7.5,

the K

value was 9.0  ±  1.0  µM (for MBP) and k

cat

was

8.0  ±  0.6  min

−1

; at pH 6.5 (in the presence of CaCl

the affinity for MBP decreased (K

, 24.0  ±  2.0  µM)

and the reaction rate increased (k

cat

, 15.2  ±  1.1  min

−1

)

[135]. NGTA2-Me-pro-Tr was the first example of Abzs

with the active site combining serine protease-like and

metalloprotease activities [135].

It should be emphasized that all recombinant

MLCs were obtained by the same technique [133-136].

NEVINSKYS378

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

Fig. 1. Arrangement of amino acid residues in the catalytic triad (Asp–His–Ser) responsible for the serine protease-like

activity, metal ion-chelating cluster, MBP recognition site, Glu-containing active site similar to that of Zn

-dependent metal-

loproteases, and active site responsible for the DNase activity of NGTA3-pro-DNase [136].

Taking this into account, analysis of enzymatic activity

of NGTA3-pro-DNase yielded quite unexpected results

[136]. Out of 25 recombinant MLCs, only NGTA3-pro-

DNase cleaved both MBP and DNA. Incubation of

NGTA3-pro-DNase with both PMSF (67%) and EDTA

(36%) resulted in the inhibition of its protease activity.

Metal ions activated proteolytic activity of NGTA3-pro-

DNase in the following order: Ca

≥Ni

>Co

≈Mn

≥

≥Cu

≈Zn

≥Mg

, with CaCl

(3.0  mM) being the best

activator. NGTA3-pro-DNase displayed two pH optima

in MBP hydrolysis: after incubation with PMSF, the

metalloprotease activity of this MLC reached the maxi-

mum at pH8.6; in the presence of EDTA, the optimum

of its serine protease-like activity was pH  7.0 [136].

NGTA3-pro-DNase treated with PMSF in the presence

of 3.0 mM CaCl

(pH  7.0) displayed a higher affinity

to MBP (K

, 15.0  ±  1.1  µM) than in the presence of

EDTA at pH 8.6 (K

, 45  ±  1.1  µM). At the same time,

the rate of MBP hydrolysis by the serine protease ac-

tive site (k

cat

, 0.40  ±  0.03  min

−1

) was about twice as

high compared to the metalloprotease active site (k

cat

0.2  ±  0.04  min

−1

) [136].

Magnesium ion (10 mM) has been shown to be

the optimal cofactor for DNase  I, while other metal

ions activated this enzyme very weakly [120,  121].

The optimal concentrations of Mn

, Mg

, and Ni

for NGTA3-pro-DNase were lower (4-5  mM); the op-

timal concentration of Ca

and Zn

was 2  mM, and

the optimal concentration of Co

and Cu

was

10  mM. The DNase activity of NGTA3-pro-DNase

in the presence of metal ions decreased in the fol-

lowing order: Mn

≈Co

≥Mg

>Cu

≈Ni

≥Ca

>Zn

which makes this MLC very different from canon-

ical DNases. Under the optimal conditions, NGTA3-

pro-DNase had the pH optimum at 6.5-6.6 and dis-

played a very high affinity to DNA [K

  =  2.0  ±  0.2  nM;

cat

=(1.1  ±  0.1)  ×  10

−3

min

−1

] [136], which was approx-

imately 3.5 orders of magnitude higher than DNase I

, 46-58  µM) [124]. Therefore, NGTA3-pro-DNase is

the first example of Abz with the active site combining

three activities: serine protease-like, metalloprotease,

and DNase [136].

How can we explain formation of NGTA3-pro-

DNase active site with the three types of catalyt-

ic activities? As has been shown previously, MBP

forms strong complexes with DNA in the blood [62,

63]. This can lead to the formation of new anti-

genic determinants (AGDs) at the junction between

MBP and DNA molecules that would include frag-

ments of both MBP and DNA sequences. In addition,

MBP–DNA complexes can bind one or several metal

ions. The process of V(D)J recombination can result

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in the formation of unique variable domains specific

against both DNA and MBP.

The homology between the protein sequences of

NGTA1-Me-pro [133], NGTA2-Me-pro-Tr [134, 135], and

NGTA3-pro-DNase [136] and sequences of known hu-

man serine proteases and Zn

- and Ca

-dependent

proteases has been analyzed and fragments of these

MLCs responsible for the specific binding of MBP and

chelation of metal ions, as well as amino acid residues

directly involved in catalysis, were identified. NGTA3-

pro-DNase was found to contain sequences similar to

the sequences of DNase  I responsible for its DNase

activity. The arrangement of amino acid residues re-

sponsible for the three activities of NGTA3-pro-DNase

is shown in Fig. 1 [136].

Therefore, in contrast to classical enzymes, the

active sites of Abzs can include structural elements

of enzymes with different activities.

VARIATIONS IN THE PROPERTIES OF ABZYMES

WITH THE CATALASE, PHOSPHATASE,

AMYLASE, AND microRNase ACTIVITIES

Of particular interest is how changes in the

BMSC differentiation profile during AID progression

are related to the production of various Abzs at dif-

ferent stages of pathology development. C57BL/6 mice

typically demonstrate a relatively slow spontaneous

development of experimental autoimmune encepha-

lomyelitis (EAE), which can be significantly acceler-

ated by myelin oligodendrocyte glycoprotein (MOG)

and DNA–histone complexes [58-63, 104-109, 111-112].

Spontaneous development of EAE in mice starts during

the 3rd month of life.

Abzs with the amylase activity have been found

in the cerebrospinal fluid and blood of MS patients

[111] and in the blood of patients with AIDs [70] and

autoimmune MRL-lpr/lpr mice [104, 105].

Abzs with the phosphatase activity were detected

in the blood of rabbits immunized with DNA, RNA,

DNase  I, DNase  II, and RNase  A [137-139]. Abs hydro-

lyzing RNA and microRNAs were isolated from the

blood of patients with AIDs [58-63, 79, 82, 83]. Abs

with the oxidoreductase (including catalase) activity

were isolated from human and animal blood [58-63,

97-103].

These data demonstrate that Abzs with the amy-

lase, catalase, phosphatase, and microRNA-hydrolyzing

activities can be formed as a result of immunization

of humans and animals with specific autoantigens.

It was interesting to find out how the catalytic

activities of Abzs can change at different stages of EAE

in C57BL/6 mice starting from postnatal day 90.

The hydrolyzing activity of Abzs toward DNA,

MBP, MOG, and histones demonstrated a relatively

steady increase within 90-150 days after the start

of the experiment [58-63, 118-125]. There was also

a steady increase in the catalase, amylase, phospha-

tase, and microRNA-hydrolyzing activities of Abs

in C57BL/6 mice beginning at the age of 3 months

[140-143].

It should be noted that the catalytic properties

of Abzs with different enzymatic activities varied

significantly at different stages of spontaneous EAE,

development, as well as during progression of EAE

induced by MOG and DNA–histone complex. The pro-

file of BMSC differentiation changed after appearance

of B cells synthesizing Abzs with different pH opti-

ma and dependence (or lack of it) on metal ions. This

could be seen most clearly for Abzs with the phospha-

tase activity. Figure  2 shows changes in the pH opti-

mum of the phosphatase activity of IgGs during dif-

ferent stages in the development of spontaneous EAE

Fig.  2. pH dependence of the phosphatase activity (hydrolysis of p-nitrophenyl phosphate) of four Ab preparations corre-

sponding to different periods of spontaneous EAE development: postnatal days 50 (Spont-50d), 90 (Spont-90d), 110 (Spont-

110s), and 150 (Spont-150d) (a) and after immunization of 3-month-old mice with MOG (MOG20; 20 days) or DNA–histone

complex: DNA20 (20 days) and DNA60 (60 days) (b) [143].

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and after immunization of mice with MOG and DNA–

histone complex.

During spontaneous development of EAE, the pH

optimum for p-nitrophenyl phosphate hydrolysis by

Abzs varied from 4 to 10. However, most IgG sub-

fractions exhibited the maximal activity at pH 6.5-7.0

(Fig. 2a).

Immunization of mice with the DNA–histone com-

plex and MOG resulted in the formation of Abzs the

maximal phosphatase activity at pH 7.5 to 10.0 (with

the peak activity at 8.5-9.5) (Fig. 2b). Similar to Abzs

formed during spontaneous EAE development, these

Abzs were able to hydrolyze p-nitrophenyl phosphate

at pH 6.5-7.0, but their activity in this pH region was

low and corresponded to the shoulders of pH depen-

dence curves (Fig. 2b).

Canonical phosphatases are magnesium-depen-

dent enzymes [144-146], The phosphatase activity of

the obtained Abzs also depended on Mg

[143]. How-

ever, in contrast to Abzs formed during spontaneous

EAE development, Abzs produced after immunization

of mice with MOG and DNA–histone complex demon-

strated a two-phase dependence on the Mg

concen-

tration, which suggested the presence of different

monoclonal Ab subfractions. Abs can significantly

vary in the affinity for magnesium ions. Immunization

with MOG led to a ~1060-fold increase in the phos-

phatase activity at pH 9.0 compared to Abs emerging

during spontaneous EAE development. Immunization

with the DNA–histone complex increased the phospha-

tase activity 570-fold (DNA60) and 310-fold (DNA20).

Similar changes in the pH optima and metal ion

dependency were observed for the amylase, catalase,

and microRNA-hydrolyzing activities of the formed

Abzs. Immunization of mice with MOG and DNA–his-

tone complex also resulted in the appearance of Abzs

that hydrolyzed microRNAs at different sites.

Therefore, different stages of spontaneous and

antigen-induced development of EAE can be accom-

panied by the activation of production of B cells se-

creting Abzs with different enzymatic properties. The

most interesting and unexpected results were ob-

tained for the cleavage of five histones and MBP by

Abzs formed during spontaneous and antigen-induced

development of EAE.

CHANGES IN THE SUBSTRATE SPECIFICITY

OF ABZYMES WITH THE PROTEASE

ACTIVITY AGAINST H1 HISTONE

DURING PROGRESSION OF EAE

The most unexpected results demonstrating an

extreme diversity and variation in the properties of

Abzs at different stages of EAE progression were ob-

tained in the studies of Abzs hydrolyzing five histones

and MBP. As mentioned above, IgGs from the blood

of HIV-infected patients against each of the five in-

dividual histones efficiently hydrolyzed all histones

and MBP, and vice versa; anti-MBP Abs cleaved both

MBP and each of the histones [131, 132]. These data

demonstrated that Abzs against histones and MBP are

characterized not only by the polyreactivity in the for-

mation of complexes with the antigens [126-129], but

also by the catalytic cross-reactivity, as discovered for

the first time for Abzs from HIV-infected patients [89,

96, 132].

Analogous results were obtained in the detailed

investigation of Abzs against five histones and MBP

that were isolated from the serum of C57BL/6 mice at

different stages of EAE development [147-150]. First,

electrophoretically homogeneous and free from the

admixtures of canonical proteases polyclonal IgGs

against five individual histones and MBP were ob-

tained from mice with EAE [147-150]. All procedures

were performed with IgG preparations correspond-

ing to different stages of spontaneous EAE develop-

ment (designated as Spont) and those obtained af-

ter immunization of mice with MOG (designated as

MOG) or DNA–histone complex (designated as DNA)

(Table 1).

All obtained IgG preparations were assessed for

the hydrolysis of H1, H2A, H2B, H3, and H4 histones.

IgGs obtained against each individual histone hydro-

lyzed all five histones and MBP and, vice versa, the

anti-MBP antibodies hydrolyzed MBP and each of the

five histones [150-153]. IgG fractions with a high affin-

ity to five individual histones and MBP were used to

detect the corresponding cleavage sites in histones us-

ing matrix-activated laser desorption/ionization mass

spectrometry (MALDI–MS) [147-150]. Although it is im-

possible to present the total data on the hydrolysis sites

in this review, in order to understand what happens

to the production of Abs against different histones at

different stages of EAE development, it is necessary

to describe the features of immune response in EAE

mice using at least one histone as an example. Below,

we present detailed analysis of H1 histone hydrolysis

by Abs against all five histones and MBP.

First, we determined the sites cleaved in H1 his-

tone by IgGs against 5 individual histones and MBP

that were isolated at the start of the experiment from

3-month-old mice and then after spontaneous EAE de-

velopment for 60 days (Fig. 3).

Immediately after addition of IgG antibodies, the

preparation of H1 histone was almost homogeneous,

demonstrating two signals corresponding to the single-

(m/z 20719.1) and double-charged (m/z 10359.6) ions.

The MALDI-MS spectra of the products of H1 histone

hydrolysis were obtained for all 30 IgG preparations

listed in Table1 (8 to 10 spectra for each preparation;

see Fig. 3 for the representative spectra).

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Table  1. IgG preparations against individual histones

(H1, H2A, H2B, H3, H4) and MBP corresponding to dif-

ferent stages of EAE progression [147-150]

Total IgG no. Preparation

Control (Cont); day 0

(start of the experiment);

IgGs against 5 histones

and MBP

1 Cont-aH1-0d

2 Cont-aH2A-0d

3 Cont-aH2B-0d

4 Cont-aH3-0d

5 Cont-aH4-0d

6 Cont-MBP-0d

Spontaneous (Spont) EAE

development, day 60;

IgGs against 5 histones

and MBP

7 Spont-aH1-60d

8 Spont-aH2A-60d

9 Spont-aH2B-60d

10 Spont-aH3-60d

11 Spont-aH4-60d

12 Spont-aMBP-60d

Immunization

with MOG; day 20;

IgGs against 5 histones

and MBP

13 MOG20-aH1

14 MOG20-aH2A

15 MOG20-aH2B

16 MOG20-aH3

17 MOG20-aH4

18 MOG20-aMBP

Immunization

with DNA–histone

complex; day 20;

IgGs against 5 histones

and MBP

19 DNA20-aH1

20 DNA20-aH2A

21 DNA20-aH2B

22 DNA20-aH3

23 DNA20-aH4

24 DNA20-aMBP

Immunization

with DNA–histone

complex; day 60;

IgGs against 5 histones

and MBP

25 DNA60-aH1

26 DNA60-aH2A

27 DNA60-aH2B

28 DNA60-aH3

29 DNA60-aH4

30 DNA60-aMBP

The cleavage sites and relative efficiency of H1

histone hydrolysis by IgG preparations were estab-

lished by averaging the data of 8-10 independent

spectra [150] (see Fig. 4 for the data on H1 histone

hydrolysis by the antibodies against H1, H2A, H2B, H3,

and H4 histones formed after immunization of mice

with MOG). All 30 IgG preparations hydrolyzed H1 his-

tone at multiple sites that varied between different

preparations.

To simplify the comparison of different sites in

H1 histone cleaved by the Abs against different his-

tones, we summarized them in the tables. As an exam-

ple, Table 2 presents the data on the cleavage of H1

histone by IgG preparations against H1, H2A, and H2B

histones at zero time and after 60 days of spontaneous

EAE development (the tables for H1 histone cleavage

by all 30 preparations were given in [150]).

The anti-H1 IgGs of 3-month mice (Cont-aH1-0d)

hydrolyze this histone at 16 sites, while after the

60-day spontaneous development of EAE (Spont-aH1-

60d) it is hydrolyzed at only 15 sites. For anti-H1 –

abtibodies hydrolyzing H1 histone at 15 and 16 sites

only 9 sites were different (Table 2).

During spontaneous development of EAE for 60-day,

the number of sites in H1 histone cleaved by IgGs

against H2A histone increased from 15 (Cont-aH2A-0d)

to 25 (Spont-aH2A-60d), including 11 new major and

medium sites (Table 2). The number of sites hydro-

lyzed by Cont-aH2B-0d was only 4 and then increased

to 8 by day 60 of the experiment (Spont-aH2B-60d).

The pattern was slightly different in the case of

H1 histone hydrolysis by IgGs against H3 and H4 his-

tones. The number of cleavage sites for Spont-aH3-60d

decreased to 10 from 17 (Cont-aH3-0d), and only one

site was common for both IgG preparations (Table2).

The maximum number of cleavage sites (23) at

zero time was found for Cont-aH4-0d, but this num-

ber decreased to 17 by day 60 of spontaneous EAE

development (Spont-aH4-60d). Both IgG preparations

cleaved 5 major sites each, and these sites did not

overlap (Table 2).

Immunization of mice with MOG led to a very

dramatic change in the BMSC differentiation profile

compared to profiles at zero time and after sponta-

neous EAE development [106-109, 147-150], including

appearance of B lymphocytes secreting anti-H1 his-

tone antibodies with different properties. Immuniza-

tion with MOG increased the number of sites cleaved

by anti-H1 IgGs from 16 (Cont-aH1-0d) to 26 [150]:

4 major and several minor new sites were found for

MOG20-aH1. Only 5 out of 26 sites hydrolyzed by

MOG20-aH1 were the same as 5 out of 15 sites cleaved

by Abs formed by day 60 of spontaneous EAE devel-

opment (Spont-aH1-60d).

Interestingly, immunization of mice with MOG

led to a significant decrease in the number of

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Fig.  3. MALDI-MS spectra of products of H1 histone hydrolysis in the absence (a) and presence of anti-histone IgGs: Cont-

aH1-0d (b), Cont-aH2A-0d (c), Spont-aH2A-60d (d), Cont-aH2B-0d (e), and Spont-aH2B-60d (f). All designations of IgGs and

mass/charge ratio (m/z) values are from [150].

hydrolyzed sites from 15 (Cont-aH2A-0d) to 4 (MOG20-

aH2A). These Abzs shared only one common major

cleavage site (F106-K107). One minor site hydrolyzed

by MOG20-aH2A was the same as one medium site

cleaved by Spont-aH1-60d.

The number of sites hydrolyzed MOG20-aH2B

increased to 5 from 4 (Cont-aH2B-0d). All four sites

cleaved by Cont-aH2B-0d were minor, and four out

of 5 sites cleaved by MOG20-aH2B were major.

Immunization with MOG decreased the number of

hydrolyzed sites from 17 (Cont-aH2B-0d) to 7 (MOG20-

aH3). Only one minor hydrolysis site (R93-L94) was

common for these two IgG preparations. MOG20-aH3

and Spont-aH3-60d shared two common sites.

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Fig.  4. Cleavage sites in H1 histone hydrolyzed by IgGs against five histones formed by day 20 after immunization of mice

with MOG: MOG20-aH1 (a), MOG20-aH2A (b), MOG20-aH2B (c), MOG20-aH3 (d), and MOG20-aH4 (e). Major, medium, and

minor cleavage sites are indicated with stars (★), arrows (↓), and asterisks (*), respectively [150].

The number of sites hydrolyzed by MOG20-aH4

decreased to 17 from 24 (Cont-aH4-0d). Only three

sites were the same for the two preparations, but they

were hydrolyzed with different efficiency.

In general, immunization of mice with MOG led

to a dramatic increase in the number of sites only for

the IgGs against H1 histone. For all other Abzs, with

the exception of anti-H2B histone IgGs, the number

of cleavage sites decreased. However, in all the cas-

es, the cleavage sites in H1 histone at zero time and

after 60 days of spontaneous EAE development were

significantly different from those identified after im-

munization with MOG [150].

The development of EAE in mice can also be

accelerated by immunization with the DNA–histone

complex [146], although the profile of BMSC differ-

entiation in this case differed from the profiles ob-

served during spontaneous EAE development and

after immunization with MOG. As a result, Abzs

against individual histones hydrolyzed H1 histone at

different sites compared to Abzs formed during spon-

taneous EAE development and after immunization

with MOG.

Twenty days after immunization of mice with

the DNA–histone complex, the number of sites in H1

histone hydrolyzed by DNA20-aH1 was the same (16)

as at zero time (Cont-aH1-0d), unlike during sponta-

neous EAE development. However, only 7 out of 16

sites were identical for the two Abz preparations. The

number of sites hydrolyzed 20 days after immuniza-

tion with MOG (MOG20-aH1) was 26 [150], and only 4

sites were identical for MOG20-aH1 and DNA20-aH1.

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Table  2. Cleavage sites in H1 histone hydrolyzed by IgGs against H1, H2A, and H2B histones at zero time and after 60 days of spontaneous EAE

development (from [150])

Number and type of sites

Cont-aH1-0d Spont-aH1-60d Cont-aH2A-0d Spont-aH2A-60d Cont-aH2B-0d Spont-aH2B-60d Cont-aH3-0d Spont-aH3-60d Cont-aH4-0d Spont-aH4-60d

16 15 15 25 4 8 17 10 23 17

K11-P12*– – – – – – – – –

–– –R14-A15* –––––R14-A15

–– – – – – – –D23-H24 D23-H24*

A33-A36 –– – – –––––

F33-A34 –– – – –––––

–– –S44-S45 ––––––

–– – – – – –Y52-I53 ––

–– – – – – – –S55-H56 S55-H56

Y57-K58 Y57-K58 ––– ––Y57-K58 ––

–– – – – –V59-G60 – V59-G60 V59-G60

–– -G60-E61 – G60-E61 G60-E61 –––

––N62-A63 N62-A63 ––––––

–– – –D64-S65 –––––

–– – – – –I67-K68 – I67-K68 –

–– – – –K68-L69 –– – –

–– – – – – – –I71-K72 I71-K72

–– –K72-R73 – – – R73-L74 – –

R73-L74 R73-L74 R73-L74 R73-L74 ––––––

–– – – – – – –L74-V75 L74-V75

–– – – – –V75-T76 – V75-T76 V75-T76

T76-T77 T76-T77 ––– –––––

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Table 2 (cont.)

Number and type of sites

Cont-aH1-0d Spont-aH1-60d Cont-aH2A-0d Spont-aH2A-60d Cont-aH2B-0d Spont-aH2B-60d Cont-aH3-0d Spont-aH3-60d Cont-aH4-0d Spont-aH4-60d

–– –T77-G78 – T77-G78 –– – –

–– –V79-L80 – V79-L80 –– – –

L80-K81 L80-K81 L80-K81 – – –––––

–– –K81-Q82 ––––––

– Q82-T83 Q82-T83 Q82-T83 ––Q82-T83 - Q82-T83 Q82-T83

T83-K84 T83-K84 ––– –––––

K84-G85 K84-G85 K84-G85 K84-G85 K84-G85 K84-G85 –– – –

–– – – – –G85-V86 –––

V86-G87 –– – – –––––

–– –A88-S89 –––A88-S89 ––

– F92-R93 ––– –––––

– – ––– –R93-L94 – R93-L94 -

– L94-A95 ––– –––––

–– –D98-E99 ––D98-E99 – D98-E99 –

–– – – – – – –E99-P100 –

–– –K101-K102 ––K101-K102 – K101-K102 –

– – – K102-S103 – – – – –

–– – – – –S103-V104 – S103-V104 S103-V104

–– – – – –V104-A105 – V104-A105

– A105-F106 ––– –A105-F106 – A105-F106 A105-F106

F106-K107 F106-K107 F106-K107 – F106-K107 F106-K107 – F106-K107 ––

K107-K108 K107-K108 – K107-K108 – K107-K108 – K107-K108 ––

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Table 2 (cont.)

Number and type of sites

Cont-aH1-0d Spont-aH1-60d Cont-aH2A-0d Spont-aH2A-60d Cont-aH2B-0d Spont-aH2B-60d Cont-aH3-0d Spont-aH3-60d Cont-aH4-0d Spont-aH4-60d

K108-T109 K108-T109 K108-T109 – K108-T109 ––K108-T109 ––

–– – – – – – –K110-K111 –

–– – – – –E112-I113 E112-I113 E112-I113 –

–– – – – – – – –I113-K114

––K114-K115 K114-K115 – K114-K115 –– – –

–– – – – –K115-V116 –––

K120-K121 K120-K121 K120-K121 K120-K121 ––––K120-K121 –

–– –A122-S123 –––S123-K124 ––

–– – – – – – -K126-K127 K126-K127

–– – – – –A128-A129 - A128-A129 A128-A129

–– –K131-A132 ––––––

K138-A139 K138-A139 - K138-A139 ––––––

–– – – – –A139-T140 A139-T140 A139-T140

–– – – – –K147-K148– – –

K148-L149 ––K148-L149 ––––––

– – – A150-A151 – – – – A150-A151 A150-A151

–––-–––A151-T152 A151-T152 –

–– –K170-A171 ––––––

–– – – ––––K181-A182 K181-A182

–– –S184-S185 ––––––

Note. * Molecular weights of H1 histone cleavage products and cleavage sites were determined based on the data from 8 to 10 spectra. ** Major, medium, and minor

sites are shown in bold, standard, and underlined italic fonts, respectively. The absence of histone cleavage at a particular site is indicated with a dash (–).

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MOG20-aH1 efficiently cleaved H1 histone at the C-ter-

minus (K120-A186), while no cleavage sites for DNA20-

aH1 were identified in this region. The number of

sites hydrolyzed by DNA20-aH2A decreased from to

13 to 15, and only one major cleavage site (F106-K107)

was common for Cont-aH2A-0d and DNA20-aH2A.

Immunization of mice with MOG decreased the

number of sites cleaved by anti-H2A Abzs from 15 to

4, and only one (Y57-K58) out of these four cleavage

sites was hydrolyzed by DNA20-aH2A [150].

The number of sites hydrolyzed by DNA20-aH2B

increased from to 5 from 4 compared to zero time

(Cont-aH2B-0d); 4 of these sites were common for both

preparations. However, only 2 sites were common for

DNA20-aH2B and MOG20-aH2B. Therefore, immuniza-

tion of mice with the DNA–histone complex led to a

decrease in the number of anti-H2B IgG preparations

capable of hydrolyzing H1 histone.

Twenty days after immunization with the DNA–

histone complex, the number of sites hydrolyzed by

anti-H3 IgGs decreased from 17 to 9. Interestingly, all

sites cleaved by DNA20-aH3 were completely different

from the sites identified at zero time (Cont-aH3-0d).

After immunization of mice with MOG, the number

of sites decreased to 7 (MOG20-aH3) from 17, and 3

out of these 7 sites were the same as for DNA20-aH3.

The maximal number of cleavage sites in H1

histone (23) at zero time was found for the anti-H4

antibodies (Cont0aH4-0d); however, it decreased to 10

after immunization with the DNA–histone complex

(DNA20-aH4) and to 17 after immunization with MOG

(MOG20-aH4). Out of 10 sites found for DNA20-aH4,

7 were identical to those for MOG20-aH4 and only

two sites were shared with Cont-aH4-0d.

BMSC differentiation profile could change several

times during spontaneous EAE development and after

immunization with MOG or DNA–histone complex. In

the case of immunization with MOG, there were only

three well-pronounced stages: the onset of accelerat-

ed EAE development (7-8 days after immunization),

acute phase (days 20-21) characterized by the maxi-

mum activity of Abzs, and remission (after ~30 days)

accompanied by a slow decrease in the Abz activity.

The same three stages were observed after immuni-

zation with the DNA–histone complex; however, they

were followed by another acute phase after ~50-60

days that was characterized by a dramatic increase

in the activity of Abzs, especially those with the DNase

activity [107]. In view of the above, we analyzed the

activity of Abzs 60 days after immunization with the

DNA–histone complex.

Interestingly, compared to the spontaneous EAE

development, immunization of mice with the DNA–

histone complex led to the increase in the number

of cleaved sites in H1 histone by DNA60-aH2B and

DNA60-aH3 60 days after immunization. However, the

number of sites hydrolyzed by IgGs against the other

three histones (H1, H2A, and H4) decreased.

Among 8 sites hydrolyzed by DNA60-aH1, only

3 were shared with Spont-aH1-60d [150], and none

were identical to 16 cleavage sites observed for Cont-

aH1-0d. In contrast to the immunization with the DNA–

histone complex, immunization with MOG increased

the number of cleaved sites from 16 (Cont-aH1-0d) to

26 (MOG20-aH1), i.e., the difference in the number

sites cleaved by MOG20-aH1 and DNA60-aH1 was 18.

Spontaneous EAE development for 60 days re-

sulted in the production of Abs that hydrolyzed H1

histone at 25 sites (Spont-aH2A-60d), whereas immu-

nization with the DNA–histone complex decreased the

number of sites to 15 (DNA60-aH2A), and only 3 sites

were shared by both Abz preparations. The number

of sites hydrolyzed by Cont-aH2A-0d and DNA60-aH2A

was comparable (16 and 15, respectively), but only 1

weak site was common for these preparations. Among

the 4 cleavage sites found for MOG20-aH2A, 2 sites

where the same as 2 sites (out of 8) hydrolyzed by

Spont-aH2A-60d.

Spont-aH2B-60d and DNA60-aH2B hydrolyzed H1

histone at 8 and 12 sites, respectively. Cont-aH2B-0d

cleaved H1 histone at only 4 sites; 2 of them were

shared with DNA60-aH2B. Out of the 5 sites cleaved by

MOG20-aH2B, 3 sites were common with DNA60-aH2B.

Spontaneous development of EAE decreased the

number of sites hydrolyzed by anti-H3 histone Abs

from 17 (Cont-aH3-0d) to 10 (Spont-aH3-60d), where-

as DNA60-aH2B cleaved H1 histone at 16 sites. Spont-

aH3-60d and DNA60-aH2B shared only 3 cleavage sites.

Among the 7 sites hydrolyzed by MOG20-aH3, 4 were

common with Spont-aH3-60d (16 cleavage sites).

Spont-aH4-60d hydrolyzed H1 histone at 17 sites,

while DNA60-aH4 cleaved it only at 6 sites. Moreover,

in the latter case, formed B lymphocytes produced

different types of Abs, as none of these 6 sites were

identical to the sites identified for Spont-aH4-60d, and

only 4 sites were shared with Cont-aH4-0d. Five sites

were common with MOG20-aH4 (17 sites), and 5 sites

were shared with DNA60-aH4 (6 sites).

The data on changes in the number of cleavage

sites in H1 histone at different stages of spontaneous

EAE development (Table  3) are of particular interest.

Depending on the specificity of antibodies (anti-H1,

H2A, H2B, H3, or H4), the number of cleavage sites

after 60 days of spontaneous EAE development either

remained almost unchanged, increased, or decreased:

H1 (16→15), H2A (15→25), H2B (4→8), H3 (17→10), and

H4 (23→17). Immunization of mice with the DNA–his-

tone complex led the increase in the number of sites

cleaved by the anti-H3 histone Abs from 9 (DNA20-

aH3) to 16 (DNA60-aH3). during the period from day

20 to day 60. Significant changes in the number and

type of hydrolyzed sites were observed for the Abs

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

Table  3. Changes in the number of cleavage sites in five individual histones hydrolyzed by Abs against these

histones and MBP at different stages of EAE development

Antibody preparation*

Cont-

aH1-0d

Spont-

aH1-60d

Cont-

aH2A-0d

Spont-

aH2A-60d

Cont-

aH2B-0d

Spont-

aH2B-60d

Cont-

aH3-0d

Spont-

aH3-60d

Cont-

aH4-0d

Spont-

aH4-60d

Number of cleavage sites in H1 histone

16 15 15 25 4 8 17 10 23 17

Number of cleavage sites in H2A histone

7284 17 8 271512 8 7

Number of cleavage sites in H2B histone

4289 6 6106156 7

Number of cleavage sites in H3 histone

15 21 16 11 13 13 11 12 18 6

Preparations of antibodies after immunization with the DNA–histone complex

DNA20-

aH1

DNA20-

aH2A

DNA20-

aH2B

DNA20-

aH3

DNA20-

aH4

DNA60-

aH1

DNA60-

aH2A

DNA60-

aH2B

DNA60-

aH3

DNA60-

aH4

Number of cleavage sites in H1 histone

16 13 5 9 10 8 16 12 16 6

Number of cleavage sites in H2A histone

11 27 19 11 23 11 22 35 23 24

Number of cleavage sites in H2B histone

7810 5 1533311106

Number of cleavage sites in H3 histone

20126 9 8797135

Note. * Abs preparations are described in Table 1 [147-150].

against other histones during the same time period

of time: anti-H1 (16→8), anti-H2A (13→16), anti-H2B

(5→12), and anti-H4 (10→6). In the case of sponta-

neous EAE development, there was only a weak over-

lap of cleavage sites on days 20 and 60. Therefore,

both spontaneous and antigen-induced development

of EAE was accompanied by changes in the BMSC

differentiation profile that resulted in the production

of new lymphocytes secreting Abzs that hydrolyzed

H1 histone at different number of sites and different

types of sites.

Data on the sites of histones hydrolysis after im-

munization of mice with MOG or DNA–histone com-

plex are presented in Table 4.

As mentioned above, Abs against all five histones

hydrolyzed not only all individual histones, but also

MBP, and, vice versa, anti-MBP Abs efficiently hydro-

lyzed all five histones.

Anti-MBP antibodies at zero time (Cont-aMBP)

hydrolyzed H1 histone only at 5 sites; 20 days after

immunization with MOG and DNA–histone complex,

the number of cleavage sites decreased to 3 and 2,

respectively; 60 days after the immunization with the

DNA–histone complex, the number of sites increased

to 8 (DNA60-aMBP) (Table 4) Therefore, the number

of sites in H1 histone hydrolyzed by anti-MBP Abs

was less than for the Abs against five individual

histones. In addition, only few sites of hydrolyzed

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

Table  4. Changes in the number of cleavage sites in five individual histones hydrolyzed by Abs against these

histones and MBP after immunization of mice with MOG or DNA-histones complex

Preparations of antibodies after immunization of mice with MOG and \DNA–histones complex*

MOG20-

aH1

MOG20-

aH2A

MOG20-

aH2B

MOG20-

aH3

MOG20-

aH4

Cont-

aMBP

MOG20-

aMBP

DNA20-

aMBP

DNA60-

aMBP

Number of cleavage sites in H1 histone

26457175328

Number of cleavage sites in H2A histone

15 11 7 6 11 19 21 22 18

Number of cleavage sites in H2B histone

26 26 7 6 11 11 14 9 4

Number of cleavage sites in H3 histone

15 12 16 8 20 14 12 9 8

Note. * Abs preparations are described in Table 1 [147-150].

by anti-MBP Abzs coincided with the sites cleaved by

anti-histone Abs [150].

The above detailed analysis of H1 histone hydro-

lysis by Abs against all five histones and MBP formed

at different stages of EAE development was performed

to demonstrate very complex changes in the BMSC

differentiation profile resulting in the formation of

B lymphocytes producing Abs with very different

properties.

CHANGES IN THE SUBSTRATE SPECIFICITY

OF ABZYMES AGAINST H2A, H2B, H3,

AND H4 HISTONES

Similar detailed analysis was performed for the

hydrolysis of other histones (H2A, H2B, H3, and H4) by

Abs against all five histones and MBP (see [147-150]).

As can be seen from Tables 3 and 4, all Ab prepara-

tions hydrolyzed histones at different number of sites.

The minimal number of cleavage sites (2 and 3)

was found for H1 histone hydrolysis by DNA20-aMBP

and MOG20-aMBP. Three antibody preparations hydro-

lyzed different histones at 4 sites, and two of them

at 5 sites (Table 4). The maximal number of cleavage

sites was found for DNA60-aH2B and DNA60-aH1 in

the hydrolysis of H2A (35 sites) and H2B (33 sites)

histones, respectively.

Anti-H1 Abs at zero time (Cont-aH2A-0d) hydro-

lyzed H2A histone at 7 sites. After 60 days of spon-

taneous EAE development, the number of cleaved

sites increased to 28 (Spont-aH2A-60d), and only 5 of

them were common for the two preparations [148].

The number of sites in H2A histone hydrolyzed by

anti-H2B antibodies at zero time (Cont-aH2B-0d) was

8, but increased to 27 after 60 days of spontaneous

EAE development (Spont-aH2B-60d). Each of these

preparations had 4 major cleavage sites that did not

overlap. The number of sites cleaved by Spont-aH3-

60d preparation decreased to 12 from 15 (Cont-aH3-

0d), and no identical sites have been identified for

these two preparations. After 60 days of spontaneous

EAE development, the number of cleavage sites in

H2A histone anti-H4 histone Abs decreased from 8

(Cont-aH4-0d) to 7 (Spont-aH4-60d), and all hydrolysis

sites turned out to be different. Similar changes in the

number and type of sites hydrolyzed in H2A histone

was found for all preparations obtained after sponta-

neous development of EAE and immunization of mice

with MOG and DNA–histone complex [148], as well as

for hydrolysis of all five histones by Abs against each

of the five histones [147-150].

Number of sites for antibodies against H1 histone

in H2B hydrolysis corresponding to 60 days after im-

munization of mice with DNA–histone complex is 29.

Eleven out of 29 antibody preparations hydrolyzed

different histones at 23-28 sites (Table 3). These data

demonstrate that EAE development, either sponta-

neous or induced by MOG and DNA–histone complex,

was accompanied by specific changes in the BMSC

differentiation profiles, resulting in the appearance of

B lymphocytes that synthesized antibodies with high-

ly different properties. These changes led not only to

the production of antibodies hydrolyzing five histones

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

at different number of sites, but also cleaving differ-

ent sites and with different efficiency.

The obtained data provide a lot of examples for

changes in the cleavage sites in histones, even when

the number of sites did not change dramatically (see

above for H1 histone hydrolysis by different antibod-

ies).

The number of sites in H2A histone hydrolyzed

by anti-H2B antibodies at zero time (Cont-aH2B-0d)

was 8, but increased to 27 after 60 days of sponta-

neous EAE development (Spont-aH2B-60d). Each of

these preparations had 4 major cleavage sites that did

not overlap. The number of sites cleaved by Spont-

aH3-60d preparation decreased to 12 from 15 (Cont-

aH3-0d), and no identical sites have been identified

for these two preparations. After 60 days of sponta-

neous EAE development, the number of cleavage sites

in H2A histone anti-H4 histone Abs decreased from 8

(Cont-aH4-0d) to 7 (Spont-aH4-60d), and all hydrolysis

sites turned out to be different. Similar changes in the

number and type of sites hydrolyzed in H2A histone

was found for all preparations obtained after sponta-

neous development of EAE and immunization of mice

with MOG and DNA–histone complex [148], as well

as for hydrolysis of all five histones by Abs against

each of the five histones [147-150].

During spontaneous development of EAE, the

number of sites in H2B histone hydrolyzed by an-

ti-H2A antibodies decreases from 9 (Cont-aH2A-0d)

to 4 (Spont-aH2A-60d); only two sites were identical

[149]. The number of sites cleaved by Cont-aH2B-0d

was only 6, but increases to 10 by day 60 of sponta-

neous EAE development of (Spont-aH2B-60d); again,

only 2 sites were common for the both preparations.

The number of sites hydrolyzed by Abs against H4 his-

tone increased from 6 (Cont-aH4-0d) to 7 (Spont-aH4-

60d); however, all sites, except for one, were different.

Spontaneous EAE development led to the increase

in the number of sites in H3 histone hydrolyzed by

anti-H1 histone IgGs from 15 (Cont-aH1-0d) to 21

(Spont-aH1-60d), with the formation of 9 major and

medium sites. Immunization of mice with MOG had

no effect on the number of hydrolysis sites compared

to zero time, but only 7 out of 15 sites for Cont-aH1-

0d and MOG20-aH1 were identical. The number of

sites in H3 histone cleaved by anti-H2A histone IgGs

was maximal (16) at zero time (Cont-aH2A-0d), but

then decreases to: 11 (Spont-aH2A-60d), 12 (MOG20-

aH2A and DNA20-aH2A), and 10 sites (DNA60-aH2A).

Interestingly, there was not a single common site for

all five of the anti-H2A histone Ab preparations. The

development of EAE from day 20 to 60 after immu-

nization with the DNA–histone complex occurred in

such a way that DNA20-aH2A and DNA60-aH2A shared

only two cleavage sites in H3 histone. An interesting

feature of anti-H2A Abs was that the sites recognized

by Cont-aH2A-0d and Spont-aH2A-60d were identified

vary rarely for the other Ab preparations.

When analyzing such data, it is necessary to

take into account that changes in the BMSC differenti-

ation profile results in the synthesis of antibodies by B

cells in the cerebrospinal fluid [111-113]. Abs isolated

from the cerebrospinal fluid from MS patients were

30-60-fold more active in the hydrolysis of DNA, MBP,

and oligosaccharides than Abs isolated from the sera

of the same patients [111-113]. In addition, different

stages of spontaneous development of SLE and EAE

were accompanied by several changes in the in the

BMSC differentiation profile [104-109].

Analysis of catalytic activity of Abs has shown for

the first time that autoAbs, 30-40% of which are Abzs,

can be very different in their properties even at the

early stages of AID development. In view of the above,

the question about the causes of exceptional diversity

of Abs and Abzs in patients with autoimmune and

neurological diseases is of particular interest.

DISCUSSION

There are still many questions about immune

response to various antigens in autoimmune, neu-

rological, and some viral diseases. Theoretically, hu-

man immune system is capable of synthesizing about

a million Abs with very different properties against

the same antigen [118]. Yet, it remains unclear how

many types of Abs and with what properties can be

formed in healthy people and patients with autoim-

mune, neurological, and viral diseases. Unfortunately,

these questions cannot be answered using such com-

mon techniques as EIA or affinity chromatography of

Abs on specific sorbents, as the possibilities of these

methods in the analysis of diversity of Abs formed

in response to exogenous and endogenous antigens

and of the role of these Abs in the pathogenesis of

various diseases are very limited [58-63]. To estimate

a probable number of Abs in the blood of SLE pa-

tients, researchers analyzed monoclonal Abs obtained

by the phage display technique [122, 123, 133-136].

It was found that the number of anti- DNA and anti-

MBP Abs in the blood of SLE patients can be over

3-4 thousands, and approximately 30-40% of them are

Abzs capable of hydrolyzing DNA and MBP. However,

this approach also cannot provide full understanding

of the diversity of produced antibodies.

One of the methods for a more detailed analysis

of the properties of Abs is evaluation of their catalytic

activity in the hydrolysis of various exo- and endog-

enous antigens. Catalytic Abs can play either positive

or negative role in disease development. For exam-

ple, HIV-infected patients produce Abs against viral

reverse transcriptase and integrase that can cleave

IMMUNE RESPONSE AND PRODUCTION OF ABZYMES S391

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

these two proteins, thus slowing down the develop-

ment of the acquired immunodeficiency syndrome

[58-63, 131]. Other Abs play a negative role in the

pathogenesis of viral, autoimmune, and neurological

diseases. For example, Abs capable of hydrolyzing

DNA and histones can penetrate through the cellular

and nuclear membranes, cleave DNA and histones

in chromatin, and stimulate cell apoptosis [34, 58-63,

75], which “fuels” autoimmunity in many pathological

states. The cleavage of MBP in the myelin sheath of

neurons by anti-MBP antibodies can lead to the de-

velopment of MS [58-63]. The fact that Abzs against

five histones hydrolyzed each of these histones and

MBP, while Abs against MBP hydrolyzed MBP and all

five histones, deserves special attention [62, 63, 89,

106-109, 147-149] and can be used for a more detailed

analysis of potential role of Abzs against different an-

tigens in the pathogenesis of AIDs.

As mentioned above, the autoimmune processes

in patients with AIDs and some neurological and vi-

ral diseases are associated with changes in the BMSC

differentiation profile and expansion of autoreactive

B cell clones [62, 63, 89, 106-109, 147-149]. Howev-

er, during spontaneous development of EAE in mice,

such changes occurred in several stages. Moreover, the

BMSC differentiation profile in the case of accelerat-

ed EAE development triggered by immunization with

MOG or DNA–histone complex was absolutely differ-

ent compared to profile characteristic of spontaneous

EAE development [26-28, 140-143]. So, the important

question is how changes in the BMSC differentiation

profile influence production of B cells synthesizing

autoAbs and Abzs harmful for humans and animals.

Some studies have shown that the ratio between

catalytically active and inactive autoAbs varies dra-

matically at the early stages of EAE development in

mice [26-28, 140-143]. However, Abzs catalyzing reac-

tions corresponding to different EAE stages can vary

greatly in their activity, pH optimum, dependence

(or lack of it) on mono- and divalent metal ions, af-

finity to antigens (substrates), isoelectric point, ther-

mostability, etc. [58-63, 106-109, 147-150]. The most

pronounced changes in the BMSC differentiation pro-

file at different EAE stages were found for hydroly-

sis of histones and MBP [147-150]. As shown above,

the cleavage patterns for H1, H2A, H2B, H3, and H4

histones observed for Abs against these histones at

different stages of EAE development accelerated by

immunization with MOG or DNA–histone complex

significantly differed in the number and types of hy-

drolyzed sites. For example, the number of cleavage

sites in H1 histone varied from 2 to 26 depending on

the IgG preparation [150]. The same was observed for

hydrolysis of other four histones [147-150]. However,

even if when the number of cleaved sites was signif-

icant, the overlapping of these sites for any pair out

of 30 Ab preparations was minimal. These data have

posed a series of new questions on the possibility of

formation of Abs and Abzs with absolutely different

properties at different stages of spontaneous EAE de-

velopment and after immunization with MOG and

DNA–histone complex. This could be due to several

factors.

First, human MS is an autoimmune pathology

whose development includes at least two or even

three stages [151]. The cascade of reactions at the

first stage of inflammation is very complex and in-

volves chemokines, cytokines, proteins, enzymes, mac-

rophages, and cells producing the NO radical [151].

The coordinated action of B and T cells, inflammation

mediators, complement system, and autoAbs results

in the formation of demyelination foci and impaired

axonal conduction. The following neurodegenerative

stage of the disease is directly associated with the

destruction of nervous tissue [151]. Therefore, medi-

cal, immunological, and biochemical indicators of MS

should be analyzed with regard to the features of each

particular disease stage, including changes in system-

ic metabolism, immunoregulation, and depletion of

adaptive and compensatory mechanisms [151].

It is believed that the development of AIDs, in-

cluding MS, can be stimulated by foreign (bacterial

or viral) antigens [152-156]. Molecular mimicry asso-

ciated with the homology between the molecules of

human body, bacteria, and viruses (e.g., Epstein–Barr,

measles, hepatitis B, herpes simplex, and influenza

viruses, papillomaviruses) can trigger autoimmune

processes in MS and other AIDs [58-63, 152-156].

Some viral and bacterial antigens are able to pene-

trate through the blood–brain barrier and stimulate

specific changes in the bone marrow, which results in

the production of antibodies against these antigens. In

the case of a long-term disease, due to the molecular

mimicry, immune system may “switch” to the produc-

tion of B cells synthesizing Abs against “self” antigens

[58-63, 152-156].

However, the development of MS in humans

and EAE in mice is not necessarily associated with

viral and bacterial infections. Ongoing apoptosis of

cells in a mammalian body causes an increase in

the concentration of DNA–histone complexes in the

blood [157]. In addition, human blood contains free

molecules of MBP and peptides from proteins of my-

elin sheath that envelopes nerve cells. At the early

stages of MS and EAE, these molecules can penetrate

through the blood–brain barrier and trigger local in-

flammatory processes, including induction of propa-

gation of B cell clones producing autoreactive Abzs.

Such changes in the BMSC differentiation profile can

affect the repertoire of B cells synthesizing antibodies

with different properties against these antigens. AIDs

are known to be accompanied by the impairments

NEVINSKYS392

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

of the blood–brain barrier, which becomes permeable

for various molecules, including large ones [158, 159].

MBP, histones, their fragments and complexes

with DNA can bind to a variety of other proteins,

nucleic acids, lipids, polysaccharides, cells, etc., at

different stages of spontaneous and antigen-induced

EAE development. At the same time, each stage of MS

is characterized by the presence of different compo-

nents in the blood. With regard to the above, differ-

ent complexes of histones and MBP with DNA and

other blood components can penetrate into the bone

marrow liquor at each EAE stage, which must lead

to changes in the BMSC differentiation profile and

appearance of completely or incompletely differenti-

ated B cells producing autoAbs and Abzs with new

properties in the blood and organs. As it has been

shown in [111-113], the activity of Abs hydrolyzing

DNA, MBP, and oligosaccharides from the cerebrospi-

nal fluid of MS patients was 30-60-fold higher than the

activity of Abs from the blood of the same patients,

which demonstrated that some lymphocytes fully dif-

ferentiated already in the bone marrow. However,

not all lymphocytes undergo complete differentiation

in the cerebrospinal fluid; some of them are trans-

ported to organs, where they further differentiate

under the influence of blood components and cells.

These lymphocytes can also produce Abzs with prop-

erties other than the properties of Abzs in the cere-

brospinal fluid.

Another important question is the mechanisms

of formation of Abzs with the cross-catalytic activity

and potential role of these Abzs in the pathogenesis

of AIDs. It should be noted that Abs against various

antigens can form “semispecific” complexes with com-

pounds containing elements of structure of specific an-

tigens. This widespread phenomenon has been named

polyreactivity (or polyspecificity) of antibody complex

formation [160-166]. As has been shown previously,

amino acid sequences of all five histones and MBP

and, especially, their antigenic determinants show a

high level of homology [58-63, 106-109, 147-150]. Also,

all histones and MBP contain many positively charged

lysine and arginine residues. As a result, autoAbs and

Abzs against five histones and MBP can form com-

plexes with any of these proteins, thus demonstrat-

ing polyreactivity in the formation of “semispecific”

complexes.

Natural Abzs are formed against specific struc-

tures of molecules that simulate transition states of

chemical reactions [1-12, 58-63]. In principle, anti-pro-

tein Abzs can be formed against AGD or protein se-

quences simulating transition states of the peptide

bond hydrolysis reaction. In proteins, sites cleaved by

Abzs are located mainly in the AGDs [58-63]. There are

3 to 11 different AGDs identified for each histone [167-

170], while MBP has 4 AGDs [171]. The efficiency of

formation of Abzs against different AGDs of histones

and MBP significantly varies and may depend on the

immunogenicity of either AGDs themselves or their

complexes with other molecules [58-63]. It has been

shown that the major antigens in the formation of Abs

against histones and DNA are DNA-histone complexes

that appear in the blood as a result of apoptosis [157].

However, MBP also effacingly forms complexes with

DNA [164]. Therefore, different autoAbs and Abzs can

be formed against individual histones or protein com-

plexes, as well as their associates with DNA and oth-

er molecules. Abzs against MBP and MBP–DNA com-

plexes can also differ. According to the data of some

studies, AGDs of histones and MBP can significantly

vary in the efficiency of formation of Abzs against

their protein sequences [58-63, 146-150]. At the same

time, due to the formation of complexes of individual

histones and histone complexes with different blood

molecules typical of different EAE stages, some AGDs

can be less immunogenic, while other AGDs or even

other histone and MBP sequences can become more

accessible and immunogenic, i.e., can better simulate

transition states of peptide bond hydrolysis reaction.

Recently, it has been shown that new AGDs can form

at the interfaces between histones and DNA, so that

the immune response against such complexes can re-

sult in the formation of Abzs hydrolyzing both DNA

and histones [136].

Therefore, at different EAE stages, all histones,

MBP, and their complexes can provide different frag-

ments of protein sequences that would simulate tran-

sition state of peptide bond hydrolysis for further Abz

formation. As a result, anti-histone or anti-MBP Abzs

formed at different EAE stages can target different se-

quences of these proteins and their complexes, which

might be the major cause of significant differences

in the number and type of hydrolyzed sites for Abzs

generated during spontaneous and antigen-induced

development of EAE.

The high frequency of occurrence of catalytic an-

tibodies is due to several reasons. Immunization of

AID-prone mice with antigens results in a significant

expansion of Abz repertoire compared to non-auto-

immune mice [165, 166]. This is due to the fact that

immunization causes changes in the BMSC differenti-

ation profile with the expansion of autoreactive B cell

clones producing Abzs only in mice prone to AIDs [38-

63, 104, 105], but not in normal mice [104, 105]. The

appearance of Abzs with a lower activity in the blood

of normal mice might be associated with the differ-

entiation and enhanced proliferation of lymphocytes

in the blood, thymus, lymph nodes, spleen, and other

organs [104, 105]. The appearance of Abzs with the

cross-enzymatic activity, e.g., capable of hydrolyzing

both histones and MBP, is caused by a high level of

sequence homology of these proteins.

IMMUNE RESPONSE AND PRODUCTION OF ABZYMES S393

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

CONCLUSIONS

At present, many questions remain about the

mechanisms of progression of autoimmune and neuro-

logical diseases and possibilities of immune response

to different antigens in these pathologies. Theoretical-

ly, human immune system is capable of synthesizing

about a million types of Abs with different proper-

ties against the same antigen. It is still unknown how

many Abs and with what properties can be formed

in healthy people and patients with various diseases.

Analysis of monoclonal Abs obtained by the phage

display technique has shown that the number of Abs

against DNA and proteins in the blood of patients with

SLE can be more than 3-4 thousand, and approximate-

ly 30-40% of them are Abzs hydrolyzing DNA and MBP.

This review presents the first analysis of the role of

the BMSC differentiation in the progression of multiple

sclerosis and SLE and formation of B cells producing

Abzs harmful for mammals. It has been shown that

Abzs against five histones hydrolyzed each of these

histones and MBP, while anti-MBP Abzs hydrolyzed

MBP and all five histones. Itwas also established that

the substrate specificity of Abzs in the hydrolysis of

histones and MBP significantly varied depending on

the MS or SLE stage. The review presented for the

first time the data on the exceptional diversity of au-

toAbs and Abzs and their biological functions, as well

as discussed their role in the pathogenesis of AIDs.

Funding. The research was supported by the Rus-

sian Science Foundation (project no. 22-15-00103).

Ethics approval and consent to participate.

This work does not contain studies involving human

and animal subjects.

Conflict of interest. The author of this work de-

clares that he has no conflict of interest.

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