Affinity and catalytic heterogeneity and metal‐dependence of polyclonal myelin basic protein‐hydrolyzing IgGs from sera of patients with systemic lupus erythematosus

Abstract

It was shown using enzyme-linked immunosorbent assay (ELISA) that titers of antibodies against human myelin basic protein (hMBP) in systemic lupus erythematosus (SLE) patients 4.2-fold higher than in healthy individuals, but 2.1-fold lower than in patients with multiple sclerosis (MS). Approximately 86% electrophoretically and immunologically homogeneous SLE immunoglobulin Gs (IgGs) purified using several affinity resins including Sepharose with immobilized hMBP specifically hydrolyze only hMBP but not many other tested proteins. Several rigid criteria were applied to show that the hMBP-hydrolyzing activity is an intrinsic property of SLE IgGs but not from healthy donors. In contrast to MS IgGs, abzymes from SLE patients are more sensitive to ethylenediaminetetraacetic acid and less sensitive to specific inhibitors of serine-like proteases. We present the first evidence demonstrating a significant diversity of different fractions of SLE IgGs in their affinity for hMBP-Sepharose, the ability of IgGs to hydrolyze hMBP at different optimal pHs (5-10) and be activated by different metal ions: Ca(2+) > Mg(2+) ≥ Co(2+) ≥ Fe(2+) ≥ Ni(2+) ≥ Zn(2+) ≥ Cu(2+) ≥ Mn(2+) . Combinations of Ca(2+) + Mg(2+) and Ca(2+) + Co(2) lead to a significant increase in the antibody proteolytic activity as compared with Ca(2+) , Co(2+) , or Mg(2+) ions taken separately. Our findings suggest that the immune systems of individual SLE similar to MS patients can generate a variety of anti-hMBP abzymes with different catalytic properties, which can attack hMBP of myelin-proteolipid shell of axons and play an important role in pathogenesis not only MS but also SLE patients.

Multiple sites of the cleavage of 17- and 19-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP) by specific anti-MBP antibodies from patients with systemic lupus erythematosus

“…In this work, electrophoretically and immunologically homogeneous polyclonal IgGs (pIgGs) were purified from the sera SLE and MS patients as well as healthy donors by sequential chromatography of the serum proteins on Protein G-Sepharose under conditions that remove non-specifically bound proteins, followed by gel filtration under the conditions that destroy immune complexes as in [9]. Electrophoretical and immunological homogeneity of the pIgGs was confirmed respectively by SDS-PAGE with silver staining and by Western blotting similarly to [9,[27][28][29][30].…”

“…Electrophoretical and immunological homogeneity of the pIgGs was confirmed respectively by SDS-PAGE with silver staining and by Western blotting similarly to [9,[27][28][29][30]. To analyze an ''average'' situation, we have prepared a mixture of equal amounts of homogeneous pIgGs from the sera of ten SLE (sle-IgG mix ), ten MS (ms-IgG mix ) patients, and ten healthy donors (hd-IgG mix ).…”

“…To exclude possible artefacts due to traces of contaminating proteases, these fractions were separated by SDS-PAGE and their proteolytic activity was detected after the extraction of proteins from the excised gel slices as in [9]; only sle-IgG mix and ms-IgG mix preparations were active, when hdIgG mix was catalytically inactive. The detection of MBP-hydrolyzing activity of these Abs similarly to [9] in the gel region corresponding only to IgGs (150 kDa) together with the absence of any other band of the activity or protein, provided a direct evidence that all pIgG preparations used are not contaminated with canonical proteases. In addition, similarly to [9] it was shown that, in contrast to canonical proteases, the SLE and MS IgG mix purified on MBP-Sepharose specifically hydrolyzed only MBP but not many other tested proteins.…”

“…Natural abzymes hydrolyzing DNA, RNA, polysaccharides, oligopeptides (OPs), and proteins are described from the sera of patients with several autoimmune (systemic lupus erythematosus, Hashimoto's thyroiditis, polyarthritis, multiple sclerosis, asthma, rheumatoid arthritis, etc.) and viral diseases with a pronounced immune system disturbance (viral hepatitis, AIDS, and tick-borne encephalitis) (reviewed in [2][3][4][5][6][7][8][9][10]). Abzymes may play a significant positive and/ or negative role in broadening Ab properties, forming specific pathogenic patterns and clinical settings in different autoimmune conditions [1][2][3][4][5][6][7][8][9][10].…”

“…At the same time, the similarity in some immunological indexes between MS and SLE can speak in favour of that anti-MBP Abs with proteolytic activity can occur in SLE patients. Recently, it was shown that electrophoretically and immunologically homogeneous IgGs (approximately 86% of SLE patients) purified using several affinity resins including Sepharose with immobilized MBP (MBP-Sepharose) specifically hydrolyze only MBP but not many other tested proteins [9]. Several rigid criteria were applied to show that the MBPhydrolyzing activity is an intrinsic property of SLE IgGs but not from healthy donors.…”