Vol.2, No.10, 1204-1207 (2010) Health
doi:10.4236/health.2010.210177
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Histone H1/MBP hydrolysing antibodies - novel potential
marker in diagnosis of disease severity in systematic
lupus erythematosus patients
Valentyna Chopyak1,2, Yaroslav Tolstiak1,2, Iryna Magoryvska3, Rostyslav Bilyy3,
Natalya Korniy4, Yuriy Kit3*, Rostyslav Stoika3,4
1Lviv Medical Institute, Lviv, Ukraine
2Danylo Halytsky Lviv National Medical University, Pekarska St., Lviv, Ukraine
3Institute of Cell Biology, Lviv, Ukraine; *Corresponding Author:
4Ivan Franko National University, Lviv, Ukraine
Received 30 July 2010; revised 5 August 2010; accepted 9 August 2010.
ABSTRACT
Recently we have shown the presence of cata-
lytically active IgGs, capable to cleave histone
H1 and bovine myelin basic protein (MBP), in
blood serum of SLE patients. Here we present
data that demonstrate the correlation between a)
proteolytic activity towards histone H1 and MBP
of IgG-antibodies from blood serum of SLE pa-
tients and b) disease severity level in these pa-
tients. IgGs were isolated from blood serum by
chromatography on protein G-sepharose. Com-
mercial preparations of bovine myelin basic
proteins (MBP) and calf thymus histone H1 were
used as substrates. Analysis of the proteolytic
activity showed that 16 of 38 lgG-preparations
(42,1%) obtained from blood serum of SLE pa-
tients were capable of cleaving both histone H1
and MBP with different efficiency. It was reve-
aled that the presence in blood serum of lgGs
possessing proteolytic activity towards both hi-
stone H1 and bMBP closely correlates with man-
ifestation of the disease severity in SLE patients.
Keywords: System Lupus Erythematosus;
Proteolytic Activity; IgG-Antibodies; Disease
Severity
1. INTRODUCTION
Systematic Lupus Erythematosus (SLE) is a chronic au-
toimmune disease characterized by dysfunction of T and
B-lymphocytes and the dendritic cells, by production of
the autoantibodies (auto-AB) to specific nuclear con-
stituents. Its clinical manifestations are accompanied by
the affection of connective tissues and small blood ves-
sels of vital organs such as kidney, lung, heart and brain.
For the past 30 years, great improvement in SLE prog-
nosis was achieved, however the mortality and disability
of SLE patients stays rather high [1,2]. A number of
investigations revealed negative prognostic factors –
contracting SLE in childhood or humans after 50, tardi-
ness of active therapy and development of such clinical
manifestations as the affection of kidney, and central
nervous system, complications in cardiovascular and
respiratory systems, as well as high general severity of
the disease [1,3-5]. The presence of auto-AB in blood
serum of SLE patients is an important diagnostic factor
[6]. Hereby, antinuclear auto-AB serves as molecular
markers for the disease severity prognosis, as well as for
its treatment efficiency [6,7]. Among antinuclear auto-
AB especially attract attention anti-DNA AB and anti-
histone AB [8-10]. Possessing a cross reactivity to dif-
ferent protein autoantigens, these auto-AB can be in-
volved in various mechanisms of pathogenesis, apopto-
sis of including immunocompetent cells, which is a de-
cisive factor in the development of autoimmune syn-
drome and the disease in general. It is known that anti-
DNA AB of IgG classes of blood serum of SLE patients
are also capable of hydrolyzing DNA and different types
of RNA (having DNase and RNase activity) [10,11]. It
was also found that DNA/RNA hydrolyzing AB (DNA-
abzymes) possess high cytotoxicity and their level in the
blood serum of SLE patients correlates with the disease
severity [12,13]. In contrast to anti-DNA AB, the role of
anti-histone AB in the development of autoimmune
processes in SLE patients is poorly studied. Recently, we
have shown for the first time that in blood serum of SLE
patients there are catalytic active IgG AB capable of
hydrolyzing both histone H1 and MBP [14]. Antibodies

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possessing similar proteolytic activity were found in
blood serum of multiple myeloma and multiple sclerosis
patients [15,16]. We have shown that these proteolyti-
cally active AB can be classified as anti-histone auto-AB
[17]. We also hypothesized that such AB could be re-
sponsible for the development of health disorders in SLE
patients. We aimed to establish a correlation between the
proteolytic activity of IgG-antibodies of blood serum
towards histone H1 and myelin basic protein and the
disease runing in SLE patients.
2. EXPERIMENTAL
2.1. Patients
Blood serum of 38 patients with SLE of different sever-
ity was used. SLE was diagnosed by criteria of the Am-
erican Collegium of Rheumatologists (ACR, 1997) and
stated in accordance with the classification recomm-
ended by the Association of Rheumatologists of Ukraine
(2002). According to the ACR classification criteria, the
disease activity was divided in the following way: I deg-
ree – 28.9% (11 patients); II degree - 50% (19 patients);
III degree - 23% (8 patients). 25 patients (65,8%) were
in the acute stage, among them 7 (63,6%) – with the first
degree of activity, 14 (73,6) – with the II degree of ac-
tivity, and 5 (55%) – with the III degree of activity. 13
patients (34,2%) were in the stage of remission. The ave-
rage age of the patients was 34 ± 9, 8, and the average
length of the disease was 5-8 years. Number of women
and men was 34 (89,5%) and 4 (10,5%), respectively.
All patients were tested on the presence of LE-cells,
ANA-autoantibodies and anti-dsDNA auto-AB.
2.2. Blood Serum Preparation
All blood samples were collected and utilized under ap-
proved protocols of the Institutional Review Board and
with the informed consent. Blood samples were withd-
rawn using sterile conditions and allowed to clot at room
temperature for minimum 10 min. Blood serum was iso-
lated by centrifugation at 4000 rpm for 10 min, divided
among several vials, and kept at −20℃ until use.
2.3. Antibody Purification
Isolation of IgG from blood serum was performed, as
described earlier [17]. 2 ml of blood serum were precip-
itated with ammonium sulfate (50% saturation). The pe-
llet was dissolved in 0.15 M NaCl, 20 mM Tris-HCl, pH
7.5 and dialyzed against the same buffer. Then, IgGs
were purified by chromatography on protein G-Sepha-
rose and eluted from the column with 0, 1 M Glu-HCl,
pH 2,6 and immediately neutralized with 1.5 M Tris-HCl,
pH 8.8. AB was dialyzed against 20 mM Tris-HCl buffer,
pH 7.5 for 18 hours. Protein concentration was measure-
ed on the NanoDrop ND 1000 spectrophotometer (Nano-
Drop Technologies, USA) using extinction coefficient of
IgG, preloaded in the device and the AB were tested for
the proteolytic activity.
2.4. Proteolytic Activity Assay
Protease activity of IgG preparations was tested as de-
scribed [17]. Commercial bovine basic protein (Sigma,
USA) and calf thymus histone H1 (Axxora, Germany)
were used as substrates for proteolysis. The hydrolysis
reaction lasted 3 hours at 37℃ in 20 µl of the incubation
medium containing 20 mM Tris-HCl, pH 7.5, 3 µg of
AB and 5 µg of protein. The reaction was stopped by
adding 5 µl of denaturing buffer (0.2 M Tris-HCl, pH
6.8, 4% Ds-Na, 8% 2-mercaptoethanol, 20% glycerol).
The reaction mixture was heated at 100℃ for 3 min and
hydrolysis products were separated by electrophoresis in
12% PAGE in the presence of 0.1% Ds-Na [18]. The
gels were stained with Coomassie G-250. Quantitative
analysis was done by using Gel-Pro program.
3. RESULTS AND DISCUSSION
Recently, we have revealed a novel hydrolytic activity of
the IgG-antibodies towards histone H1 and MBP in bl-
ood serum of patients with clinically diagnosed SLE [14,
17]. At the same time, proteolytic activity was absent in
the sera-derived antibodies of 18 healthy donors under
control [17]. IgGs were isolated by chromatography on
Protein G-Sepharose, and 4 of 10 SLE patients were
found to possess IgGs that were capable of cleaving both
histone H1 and MBP. Such activity was confirmed to be
an intrinsic property of the IgG molecule, since it was
preserved after gel filtration at alkaline and acidic pH.
From the proteolytically active IgG preparations by the
affinity chromatography on histone H1-Sepharose were
purified anti-histone IgGs and have shown they capabil-
ity of hydrolyzing histone H1 and MBP. Summarizing,
we have shown an existence in blood serum of SLE pa-
tients of anti-histone H1 IgGs with previously unknown
proteolytic activity towards both histone H1 and MBP
[17]. We suggest that these protelytically active AB cou-
ld be associated with specific disorders appearing in SLE
patients. To clarify this suggestion, 38 patients with diff-
erent SLE severity were inquired. IgGs were purified by
chromatography on the Protein G-Sepharose column and
assayed for proteolytical activity towards histone H1 and
MBP. Typical data of such assay are present on Figure 1,
and also summarized in Table 1 and Table 2. It was
found that 15 of 38 IgG preparations obtained from SLE
patients are capable of hydrolyzing histone H1 and MBP
with different efficiency. 10 of 15 protease active IgG

V. Chopyak et al. / Health 2 (2010) 1204-1207
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
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Figure 1. Proteolytic activity of polyclonal IgGs preparations,
purified by chromatography on Protein G-sepharose from blo-
od serum of SLE patients. Lines 1-4 – IgG preparations incu-
bated with histone H1; Lines 1’-4’ – IgGs preparations incu-
bated with bMBP. C-histone H1 before incubation with IgGs
(control). C’ – bMBP before incubation with IgGs (control).
Position of heavy (H) and light (L) chains of IgG, and also
position of histone H1 (his H1) and bovine myeline basic pro-
tein (bMBP) are shown by arrows. M - protein molecular mass
markers (17, 34, 43, 55, 72, 95, 130, 170 kDa, respectively).
Table 1. Demographical and clinical characteristics of SLE pa-
tients.
Parameters М ± SD Proteolytical
activity of IgG
Gender:
Woman:
Men:
34 (89.5%)
4 (10.5%)
H(3);M(1);HM(10 )
H(0); M(1); HM(0)
Disease activity:
І degree
ІІ degree
ІІІ degree
11 (28,9%)
19 (50%)
8 (21.1%)
H(3);M(1);HM(1)
H(1); M(0);HM(7)
H(0); M(0);HM(2)
Clinical features:
Joints affection (arthralgia)
Skin affection (erythema “butterfly”)
nervous system affection:
а) headache
б) epileptic seizures
в) sleep disturbance
26 (68.4%)
23 (60.5 %)
12 (31.5%)
1 (0.026%)
4 (10.5%)
H(3);M(2); HM(9)
H(3); M(2); HM(6)
H(0);M(0);HM(6)
H(0);M(0);HM(0)
H(0);M(0);HM(5)
Heart affection (carditis) 4 (10.5% ) H(0);M(0);HM(5)
Nephritis 15 (39.4%) H(0);M(2);HM(7)
Active Epstein-Barr viruses infection 5 (13.1%) H(0);M(0);HM(2)
Body temperature increase 19 (50%) H(2);M(0);HM(9)
H, M, HM - proteolytic activity of IgGs towards histone H1, myelin basic
protein, and both histone H1 and myelin basic protein, respectively. In brac-
kets are shown a number of patients.
Table 2. Correlation between disease severities and proteolytic
activity of IgGs.
Disease
activity Stage of disease Histone Н1/MBP – hydrolyzing activity
І Acute 7 (63.3%)
Remission 4 (36.7%)
4 (57.1%)
1 (25%)
ІІ Acute 14 (73.6%)
Remission 5 (26.4%)
8 (57.1%)
0
ІІІ Acute 4 (50%)
Remission 4 (50%)
2 (50%)
0
preparations hydrolyzed both histone H1 and MBP, 3
preparations were capable of cleaving only histone H1
and 2 of them – only MBP. Protease activity assay rev-
ealed that AB of 3 of 10 patients with diagnosed SLE
disease for the first time was capable of cleaving only
histone H1. None of 10 seropositive (ANA + dsDNA +)
systematically treated SLE patients for year possessed of
IgG with catalytical activity. Among 25 patients in acute
stage of the disease, 2 showed a hydrolysing activity on-
ly towards histone H1; 1 patient (age 23) possessed a
low activity towards MBP, and IgGs of 12 patients were
capable of effectively cleaving both proteins. IgG pre-
paration purified from blood serum of 6 patients with
nervous system affection (headache or sleep disturbance)
showed high catalytic activity towards both histone H1
and MBP proteins. Among 6 children of the puberty pe-
riod (age 13-16) suffering from SLE, 4 were in the acute
stage and contained IgG-antibodies capable of hydro-
lyzing both histone H1 and MBP. The catalytic activity
was not observed in 12 patients in the remission stage as
well as in 3 patients with other systematic disoders (an-
kylosing spondylitis, systemic vasculitis, mixed disorder
of the conjuctive tissue).
4. CONCLUSIONS
Appearance of proteolytic activity of IgG in blood serum
of SLE patients towards both histone H1 and myelin
basic protein tightly correlates with the disease severity
in these patients.
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Appendix
List of Abbreviations:
SLE - systemic lupus erythematosus; MBP - myelin ba-
sic protein; AB – antibodies; auto-AB - autoantibodies