Proteolytic activity of igGs from blood serum of wistar rats at exPerimental rheumatoid arthritis

The aim of this work was to study the proteolytic activity of IgGs purified from blood serum of Wistar rats at experimental rheumatoid arthritis (ErA) induced by an injection of bovine collagen of type II. twenty rats were immunized with a preparation of bovine collagen II (Sigma-Aldrich, USA) in the presence of complete Freund's adjuvant. ERA development was determined by inflammation in limbs of treated animals. IgG preparations were isolated from blood serum of immunized and non-immunized animals by precipitation of antibodies with 33% ammonium sulfate followed by chromatography on the protein G-Sepharose column. Human histone H1, bovine collagen II, calf thymus histones, myelin basic protein (MBp), bovine serum albumin (BSA), and bovine casein were used as substrates of the proteolytic activity of IgGs. It was found that IgG preparations from blood serum of rats with ErA were capable of cleaving histone H1 and MBp, however, they were catalytically inactive towards collagen II, casein, BSA, and core histones. IgGs from blood serum of non-immunized rats were proteolytically inactive towards all used protein substrates. thus, we demonstrated that immunization of rats with bovine collagen II induced IgG-antibodies possessing the proteolytic activity towards histone H1 and MBP. This activity might be associated with the development of inflammatory processes in the immunized rats.

The aim of this work was to study the proteolytic activity of IgGs purified from blood serum of Wistar rats at experimental rheumatoid arthritis (ErA) induced by an injection of bovine collagen of type II.twenty rats were immunized with a preparation of bovine collagen II (Sigma-Aldrich, USA) in the presence of complete Freund's adjuvant.ERA development was determined by inflammation in limbs of treated animals.IgG preparations were isolated from blood serum of immunized and non-immunized animals by precipitation of antibodies with 33% ammonium sulfate followed by chromatography on the protein G-Sepharose column.Human histone H1, bovine collagen II, calf thymus histones, myelin basic protein (MBp), bovine serum albumin (BSA), and bovine casein were used as substrates of the proteolytic activity of IgGs.It was found that IgG preparations from blood serum of rats with ErA were capable of cleaving histone H1 and MBp, however, they were catalytically inactive towards collagen II, casein, BSA, and core histones.IgGs from blood serum of non-immunized rats were proteolytically inactive towards all used protein substrates.thus, we demonstrated that immunization of rats with bovine collagen II induced IgG-antibodies possessing the proteolytic activity towards histone H1 and MBP.This activity might be associated with the development of inflammatory processes in the immunized rats.

K e y w o r d s: antibodies, proteolytic activity, substrate specificity, rheumatoid arthritis, Wistar rats, bovine
collagen II, immunization.

I
nteraction of antibodies with antigens may cause not only binding, but also a destruction or modification of these antigens.Some proteins, nucleic acids and oligosaccharides play a role of substrates during catalysis with participation of antibodies.Antibodies possessing the catalytic activity are named abzymes [1][2][3][4], and abzymes with the proteolytic activity are named protabzymes [2,4].The protabzymes of IgG sub-class that are capable of hydrolyzing intestinal vasoactive peptide (VIP), have been detected in blood serum of asthma patients [5].It was found that destruction of autoantigens by the protabzymes is tightly associated with development of some autoimmune diseases.For instance, in patients with the acute allergic thyroiditis, the anti-thyreoglobulin auto-АBs are capable of hy-drolyzing thyreoglobulin [6], while in patients with autoimmune myocarditis, the auto-Abs are capable of hydrolyzing myosin [7].In patients with multiple sclerosis, the auto-ABs were capable of hydrolyzing myelin basic protein (MBP) [8].The proteolytically active ABs have been also detected in blood serum of patients with some cancer and inheritable diseases [9,10], as well as in blood serum of some clinically healthy people [3,[11][12][13][14].It was shown that in norm protabzymes may participate in destruction and removal from the organism of some pathogenic proteins involved in virus infections [3] and neurodegenerative diseases [11][12][13].These data demonstrate that, depending on substrate specificity, protabzymes may possess the pathogenic or defending function in human organism.
We have found for the first time that IgG-protabzymes capable of hydrolyzing histone Н1 are present in blood serum of patients with multiple sclerosis, systematic lupus erythematosus (SLE), multiple myeloma, as well as in colostrum of healthy women [11,15,16].These protabzymes have not been detected in blood serum of healthy donors.Comparative studies of blood serum of SLE patients and colostrum of healthy mothers have shown that IgG-and sIgA-protabzymes with affinity for histone H1 are capable of cleaving two different substrates -histone H1 and MBP [17,18].Their level in blood serum positively correlated with heaviness of multiple sclerosis [19,20].We suggested that the appearance of IgG-protabzymes in blood serum may be caused by an inflammatory process linked with development of the autoimmune diseases.To check this presumption, experimental model of the rheumatoid arthritis (RA) has been used.The inflammatory processes in joints and subtend tissues are major features of the RA.[21].The development of experimental RA-similar pathology may be induced in rats by the injection of complete Freund adjuvant (adjuvant arthritis) [22], or bovine collagen ІІ type in a complex with incomplete Freund adjuvant (collageninduced arthritis) [22,23].In this work, we aimed at a search of proteolytically active IgG in blood serum of rats with experimental collagen-induced arthritis.

materials and methods
Blood was obtained from 20 immunized and 20 non-immunized (control) Wistar rats of different sex, aged 2 months, weighing 160-200 g.Animals were treated in compliance with the Council of Europe Convention on protection of vertebrate animals which are used for scientific purposes.
Immunization of rats.Іmmunization was performed according to [22].Rats were injected once with 400 μg of bovine collagen II type (Sigma-Aldrich) in 15 mM acetic acid in the incomplete Freund adjuvant (Calbiochem-Вehring) into right hind leg.The animals were observed for 36 days after the immunization, and arthritis development was determined by monitoring inflammation in tissue of hind legs [22][23][24].Animals were decapitated in order to obtain blood of the immunized and control animals.

results and discussion
Inflammation of tissues in the hind legs of immunized rats, but not in control animals, were observed (Fig. 1).IgG isolated from blood serum of both immunized and control animals was checked for the presence of the proteolytic activity towards possible protein antigens.Typical results of lacking proteolytic activity towards bovine collagen II of the IgGs purified from blood serum of the immunized and control rats are presented on Fig. 2, A. On the other hand, IgGs purified from rats with ERA were capable of cleaving histone H1 contained in the preparation of total calf thymus histones (Fig. 2, B, lane 2).IgG isolated from the non-immunized animals was lacking such activity (Fig. 2, B, lane 1).The destruction of core histones by the IgG of immunized, as well as control animals, was not detected.
At the next step, IgG-preparations capable of cleaving histone H1 were tested for hydrolyzing activity towards other protein substrates.It was found that IgGs from blood serum of rats with ERA cleaved histone H1 (Fig. 3, lanes 4, 4', 5, 5').The amount of peptide products of the proteolytic reaction suggests that the efficiency of hydrolysis of MBP was much higher than the hydrolysis of histone H1.At the same time, the IgGs under study were inactive towards bovine serum albumin and casein (Fig. 3, lanes 2, 2', 3, 3').Similar substrate specific-ity towards hydrolysis of histone H1 and MBP was demonstrated for the protabzymes which we earlier purified from blood serum of systemic lupus erythematosis patients [16][17][18][19][20].

Fig. 2. Elecrtophoretic pattern of IgGs possessing proteolytic activity and purified from blood serum of immunized and non-immunized rats towards collagen ІІ (А) and histones of calf thymus (B). Lane 1 -nonimmunized animals. Lane 2 -animals 36 days after immunization. Lane 3 -control (collagen ІІ (А) and total histones (B) without addition of IgGs). On the left, the positions of collagen ІІ on the gel (coll ІІ), heavy (H) and light (L) IgG chains are shown. on the right, the position of linker histone H1 and core histones of calf thymus (core his) are shown
Recently, it was shown that IgG-and IgM-antibodies present in blood serum of RA patients, are capable of hydrolyzing synthetic Pro-Phe-Arg-4-methyl -cumarinimid peptide [26].Antibodies possessing similar proteolytic activity have been found in blood serum of sepsis patients [28].It was detected that increasing of level of protabzymes in blood serum positively correlated with the development of that pathology.The obtained results allow one to suggest that revealed protabzymes possess either positive or negative action in human organism; however, this assumption should be studied additionally.
Summarizing, we have shown that ERA induced by the immunization of rats with bovine collagen type ІІ was accompanied by an appearance of the catalytically active antibodies capable of hydrolyzing histone Н1 and MBP in blood serum.That might be connected with the development of inflammatory processes in the immunized animals.Further studies to prove this assumption are in progress.к л ю ч е в ы е с л о в а: антитела, протеолитическая активность, субстратная специфичность, ревматоидний артрит, крысы линии Wistar, коллаген быка ІІ, иммунизация.