Antibodies from the sera of HIV‐infected patients efficiently hydrolyze all human histones

Abstract

Histones and their post-translational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecular pattern molecules when they are released into the extracellular space. Administration of exogenous histones to animals leads to systemic inflammatory and toxic responses through activating Toll-like receptors and inflammasome pathways. Here, using ELISA it was shown that sera of HIV-infected patients and healthy donors contain autoantibodies against histones. Autoantibodies with enzymic activities (abzymes) are a distinctive feature of autoimmune diseases. It was interesting whether antibodies from sera of HIV-infected patients can hydrolyze human histones. Electrophoretically and immunologically homogeneous IgGs were isolated from sera of HIV-infected patients by chromatography on several affinity sorbents. We present first evidence showing that 100% of IgGs purified from the sera of 32 HIV-infected patients efficiently hydrolyze from one to five human histones. Several rigid criteria have been applied to show that the histone-hydrolyzing activity is an intrinsic property of IgGs of HIV-infected patients. The relative efficiency of hydrolysis of histones (H1, H2a, H2b, H3, and H4) significantly varied for IgGs of different patients. IgGs from the sera of 40% of healthy donors also hydrolyze histones but with an average efficiency approximately 16-fold lower than that of HIV-infected patients. Similar to proteolytic abzymes from the sera of patients with several autoimmune diseases, histone-hydrolyzing IgGs from HIV-infected patients were inhibited by specific inhibitors of serine and of metal-dependent proteases, but an unexpected significant inhibition of the activity by specific inhibitor of thiol-like proteases was also observed. Because IgGs can efficiently hydrolyze histones, a negative role of abzymes in development of acquired immune deficiency syndrome cannot be excluded. Copyright © 2016 John Wiley & Sons, Ltd.

Antibodies to H1 histone from the sera of HIV‐infected patients recognize and catalyze site‐specific degradation of this histone

“…Three of 8 fractions of IgG mix with different affinity to immobilized human histones, eluted by 0.3 (marked below as IgG(fr.1) fraction) and 3.0M (IgG(fr.2)) NaCl or 3.0M MgCl 2 , (IgG(fr.3)) and non‐fractionated preparation of IgG mix were used for analysis of H1 histone hydrolysis. We have shown recently that pIgGs from sera of HIV‐infected patients are inhibited by specific inhibitors of serine, thiol‐like, and metal‐dependent proteases . The same situation was observed for 3 fractions fr.1 to fr.3 of polyclonal IgG mix after their separation on histone‐Sepharose: They possess to some extent comparable serine‐, thiol‐like, and metalloprotease activities.…”

“…In this work, we have used previously described electrophoretically and immunologically homogeneous IgGs from the sera of HIV‐infected patients purified by sequential chromatography of the serum proteins on Protein G–Sepharose under conditions that remove nonspecifically bound proteins, followed by gel filtration under conditions destroying immune complexes as in the work of Baranova et al To analyze the “average” site‐specific proteolytic activity of IgGs hydrolyzing H1 histone, we have prepared mixture of equal amounts of 10 IgGs (IgG mix ) with different relatively high activities in the degradation of H1 histone. To exclude possible artifacts due to traces of contaminating proteases, the IgG mix was separated by SDS‐PAGE, and its MBP‐hydrolyzing activity was detected after proteins extraction from gel slices as in the work of Baranova et al The detection of H1‐hydrolyzing activity only in the gel region corresponding to approximately 150 kDa IgG mix , together with the absence of any other bands of protein or activity, guaranteed a direct evidence in the absence of traces of canonical proteases . Then, we have analyzed the relative affinity of IgG mix for 5 histones (H1, H2a, H2b, H3, and H4) using chromatography on histone‐Sepharose (Figure ).…”

“…Electrophoretically and immunologically homogeneous IgGs were obtained from AIDS patients by sequential affinity chromatography of the serum proteins on Protein G–Sepharose and gel filtration on a Superdex 200 HR 10/30 column as in the work of Baranova et al The type of Abs (IgA, IgG, or IgM) in the fractions during different chromatographies was determined by type‐specific Western blotting as described previously . The fractions corresponding to the central parts of the IgGs peak were concentrated and used in further purification and catalytic activity assay.…”

“…After 1 week of storage at 4°C for refolding, the IgG preparations were used in assay relative activity as described below. To exclude artifacts due to possible traces of contaminating canonical enzymes, the IgGs were analyzed using a sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) assay of proteolytic activity and the strict specificity of H1 histone hydrolysis …”

“…We have suggested that, because of apoptosis of HIV‐infected patients cells, the blood can contain not only complexes of DNA and/or RNA with histones but also free histones, which can be antigens stimulating production of different auto‐Abs, including abzymes hydrolyzing histones. Therefore, using ELISA the titers of Abs against 5 histones (H1, H2a, H2b, H3, and H4) in the sera of HIV‐infected patients were recently analyzed . It was shown that the titers of Abs against histones in the case of HIV‐infected patients are statistically significantly higher than those in healthy donors.…”