G. A. Nevinsky

Lactoferrin and its biological functions.

Lactoferrin, a component of mammalian milk, is a member of the transferrin family. These glycoproteins transfer Fe3+ ions. Lactoferrin is a unique polyfunctional protein that influences cell proliferation and differentiation. It can regulate granulopoiesis and DNA synthesis in some cells. Lactoferrin inhibits prostaglandin synthesis in human milk macrophages and activates the nonspecific immune response by stimulating phagocytosis and complement. It can interact with DNA, RNA, proteins, polysaccharides, heparin-like polyanions, etc.; in some of its effects, lactoferrin is found in complexes with ligands. It was recently demonstrated that lactoferrin also possesses ribonuclease activity and is a transcription factor. The list of known biological activities of lactoferrin is constantly increasing. This review analyzes possible mechanisms of its polyfunctionality.

Catalytic antibodies in healthy humans and patients with autoimmune and viral diseases.

Antibodies have been first characterized as proteins produced by the immune system solely for binding other molecules, called antigens, with the goal of eliciting immune response. In this classical conception, antibodies act similarly to enzymes in specific binding to different molecules but cannot catalyze their chemical conversion. However, in 1986 the first monoclonal catalytic antibodies against a chemically stable analog of the transition state of a reaction were obtained and termed abzymes (Abzs). At present, artificial monoclonal Abzs catalyzing more than 100 distinct chemical reactions have been obtained. The discovery of IgG specifically hydrolyzing intestinal vasoactive peptide in the blood serum of asthma patients stimulated studies of natural Abzs. Numerous Abzs discovered afterwards in sera of patients with various autoimmune diseases, viral disorders, or in the milk of healthy mothers, are capable of hydrolyzing proteins, DNA, RNA, polysaccharides, or nucleotides, as well as to phosphorylate proteins and lipids. The phenomenon of catalysis by auto‐Abzs is more and more in research focus. In this review we summarize new data on Abzs applications in basic science, medicine and biotechnology.

Heavy metal ions affect the activity of DNA glycosylases of the Fpg family

Prokaryotic enzymes formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei) and their eukaryotic homologs NEIL1, NEIL2, and NEIL3 define the Fpg family of DNA glycosylases, which initiate the process of repair of oxidized DNA bases. The repair of oxidative DNA lesions is known to be impaired in vivo in the presence of ions of some heavy metals. We have studied the effect of salts of several alkaline earth and transition metals on the activity of Fpg-family DNA glycosylases in the reaction of excision of 5,6-dihydrouracil, a typical DNA oxidation product. The reaction catalyzed by NEIL1 was characterized by values Km = 150 nM and kcat = 1.2 min−1, which were in the range of these constants for excision of other damaged bases by this enzyme. NEIL1 was inhibited by Al3+, Ni2+, Co2+, Cd2+, Cu2+, Zn2+, and Fe2+ in Tris-HCl buffer and by Cd2+, Zn2+, Cu2+, and Fe2+ in potassium phosphate buffer. Fpg and Nei, the prokaryotic homologs of NEIL1, were inhibited by the same metal ions as NEIL1. The values of I50 for NEIL1 inhibition were 7 µM for Cd2+, 16 µM for Zn2+, and 400 µM for Cu2+. The inhibition of NEIL1 by Cd2+, Zn2+, and Cu2+ was at least partly due to the formation of metal-DNA complexes. In the case of Cd2+ and Cu2+, which preferentially bind to DNA bases rather than phosphates, the presence of metal ions caused the enzyme to lose the ability for preferential binding to damaged DNA. Therefore, the inhibition of NEIL1 activity in removal of oxidative lesions by heavy metal ions may be a reason for their comutagenicity under oxidative stress.

Age-associated changes in oxidative damage and the activity of antioxidant enzymes in rats with inherited overgeneration of free radicals

Reactive oxygen species have been hypothesized to play an important role in the process of aging. To investigate the correlation between oxidative stress and accumulation of protein and DNA damage, we have compared the age‐dependent levels of protein carbonyl groups and the activities of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase in cytosol and mitochondrial extracts from liver cells of Wistar and OXYS rats. The latter strain is characterized by increased sensitivity to free radicals. Faster age‐dependent increase in the level of protein carbonyl groups was found in OXYS as compared with Wistar rats. A complicated enzyme‐specific pattern of age‐dependent changes in the activities of antioxidant enzymes was observed. Long‐term uptake of dietary supplements Mirtilene forte (extract from the fruits of Vaccinium myrtillus L.) or Adrusen zinco (vitamin E complex with zinc, copper, selenium and ω‐3 polyunsaturated fatty acids) sharply decreased the level of protein oxidation in cytosol and mitochondrial extracts of hepatocytes of Wistar and of OXYS rats. Both dietary supplements increased the activity of catalase in the liver mitochondria of OXYS rats. Our results are in agreement with the shorter life‐span of OXYS and with the mitochondrial theory of aging, which postulates that accumulation of DNA and protein lesions leads to mitochondrial dysfunction and accelerates the process of aging.

Dynamics of Antibody Nuclease Activity in Blood of Women during Pregnancy and Lactation

In human milk we previously found catalytic antibodies (abzymes) catalyzing hydrolysis of DNA, RNA, NMP, NDP, and NTP and also phosphorylation of proteins and lipids. In the present study we have analyzed nuclease activities of antibodies in blood of women during pregnancy and lactation. Blood of healthy male and female volunteers lacked catalytically active antibodies, whereas antibodies from blood of pregnant women hydrolyzed DNA and RNA and their relative activity varied over a wide range. Relative blood abzyme activities significantly increased after delivery and at the beginning of lactation. The highest abzyme activity was observed in blood of parturient women. Although the dynamics of changes in antibody DNase activity during pregnancy was rather individual for each woman, there was a common trend in the increase in antibody activity in the first and/or third trimester of the pregnancy. The DNase activity of IgG and IgM from blood of healthy pregnant women was 4-5 times less than that from pregnant women with pronounced autoimmune thyroiditis.

DNA-Hydrolyzing Antibodies from Sera of Autoimmune-Prone MRL/MpJ-lpr Mice

Catalytically active antibodies (abzymes) hydrolyzing proteins, polysaccharides, ATP, DNA, and RNA have been detected in the sera of patients with various autoimmune and some viral diseases, but abzymes from the sera of animals are practically unstudied. The development of lupus-like autoimmune disease of MRL/MpJ-lpr mice is an experimental model for study of autoimmune pathologies and immunopathogenesis. In this work, homogeneous IgG preparations were isolated from the sera of MRL/MpJ-lpr mice. These antibodies (Abs), their Fab-fragments, and isolated light chains were shown to possess catalytic activity in DNA hydrolysis, whereas Abs from the sera of control healthy mice did not hydrolyze DNA. The data demonstrate that DNA hydrolyzing activity is an intrinsic property of Abs from MRL/MpJ-lpr mice. It was shown that various markers of autoimmune pathologies (level of total protein concentration in urea (proteinuria), Abs titers to native and denatured DNA, and DNA-hydrolyzing activity of IgG) increased in animals with aging, but they noticeably increased (2-22 times) only after appearance of obvious indicators of pathology independently of age. The highest increase in proteinuria (25-fold), anti-DNA Abs titers (12-19-fold), and abzyme activity (120-fold) was found in mice after their immunization with DNA–protein complex.

Antibodies against DNA hydrolyze DNA and RNA

In this work, rabbits were immunized with a high polymer DNA complexed with methylated BSA (mBSA) and by mBSA. It is shown that electrophoretically homogeneous preparations of polyclonal antibodies (Ab) from non-immunized rabbits and animals immunized by mBSA do not exhibit catalytic activity. Ab from the blood of rabbits immunized with the DNA-mBSA complex hydrolyzed poly(C) and different RNAs with efficiency exceeding that towards DNA by approximately 3–4 orders of magnitude. Affinity chromatography of the IgG on DNA cellulose separated the Ab into fractions hydrolyzing both RNA and DNA, and for the first time fractions that hydrolyze only RNA were found. Kinetic parameters that characterize the RNA and DNA hydrolysis by initial Ab preparations and their fractions obtained by separation on an affinity sorbent are compared.

Lactoferrin Is the Major Deoxyribonuclease of Human Milk

Lactoferrin is the major iron-transferring protein of human barrier fluids such as blood and milk. It is a polyfunctional protein capable of binding DNA exposed on the surface of various cells. Electrophoretically homogenous lactoferrin was prepared by sequential chromatography of human milk proteins on DEAE-cellulose, heparin-Sepharose, and Sepharose containing immobilized anti-lactoferrin antibodies. By subsequent chromatography on Blue Sepharose the resulting lactoferrin was fractionated into several subfractions with different affinity for the sorbent, and this was associated with separation of additional lactoferrin peaks with DNase activity from the main peak. By various techniques, in particular, by in situ testing the DNase activity of lactoferrin in a DNA-containing gel after SDS-electrophoresis, hydrolysis of DNA was for the first time shown to be an intrinsic property of lactoferrin. The substrate specificity of lactoferrin in hydrolysis of DNA was different from specificities of known human DNases. Hydrolysis of DNA was activated by bivalent metal ions and also by ATP and NAD. Unlike the main fraction of lactoferrin with the highest affinity for Blue Sepharose, all protein subfractions with DNase activity were cytotoxic and suppressed growth of human and mouse tumor cell lines.

Novel DNA Glycosylases from Mycobacterium tuberculosis

Oxidized bases are removed from DNA of Escherichia coli by enzymes formamidopyrimidine DNA glycosylase (Eco-Fpg) and endonuclease VIII (Eco-Nei) of the same structural family Fpg/Nei. New homologs of these enzymes not characterized earlier have been found in genomes of Actinobacteria. We have cloned and expressed two paralogs (Mtu-Nei2 and Mtu-Fpg2) from 36KAZ and KHA94 isolates of Mycobacterium tuberculosis and studied their ability to participate in DNA repair. Under heterologous expression in E. coli, Mtu-Nei2 decreased the rate of spontaneous mutagenesis in the rpoB gene, whereas Mtu-Fpg2 moderately increased it, possibly due to absence of residues crucially important for catalysis in this protein. Mtu-Nei2 was highly active toward double-stranded DNA substrates containing dihydrouracil residues and apurine-apyrimidine sites and was less efficient in cleavage of substrates containing 8-oxoguanine and uracil residues. These lesions, as well as 8-oxoadenine residues, were also recognized and removed by the enzyme from single-stranded DNA. Fpg and Nei homologs from M. tuberculosis can play an important role in protection of bacteria against genotoxic stress caused by oxidative burst in macrophages.

Natural antibodies to nucleic acids.

Blood of healthy donors contains low concentrations of autoantibodies to its own components, including DNA and RNA. Increased concentrations of antibodies to DNA and RNA have been found in blood of people and animals with autoimmune diseases and viral and bacterial infections. Detection of different antibodies with catalytic activities, including abzymes with DNase and RNase activities, is the earliest indicator of the development of some autoimmune diseases. This review reveals possible mechanisms of generation of anti-DNA and anti-RNA antibodies without catalytic activities and abzymes in normal organisms and in organisms with different pathologies. A possible role of these autoantibodies and the reasons of their exceptional diversity in normal organisms and in organisms with different autoimmune diseases are discussed.