Blood-Derived RNA- and microRNA-Hydrolyzing IgG Antibodies in Schizophrenia Patients.

Abzymes with various catalytic activities are the earliest statistically significant markers of existing and developing autoimmune diseases (AIDs). Currently, schizophrenia (SCZD) is not considered to be a typical AID. It was demonstrated recently that antibodies from SCZD patients efficiently hydrolyze DNA and myelin basic protein. Here, we showed for the first time that autoantibodies from 35 SCZD patients efficiently hydrolyze RNA (cCMP > poly(C) > poly(A) > yeast RNA) and analyzed site-specific hydrolysis of microRNAs involved in the regulation of several genes in SCZD (miR-137, miR-9-5p, miR-219-2-3p, and miR-219a-5p). All four microRNAs were cleaved by IgG preparations (n = 21) from SCZD patients in a site-specific manner. The RNase activity of the abzymes correlated with SCZD clinical parameters. The data obtained showed that SCZD patients might display signs of typical autoimmune processes associated with impaired functioning of microRNAs resulting from their hydrolysis by the abzymes.

[Exceptional Diversity of Catalytic Antibodies with Varying Activity in the Blood of Autoimmune and Viral Disease Patients].

Antibodies (ABs) that target autoantigens were more abundant in the blood of humans and animals suffering from certain autoimmune and viral diseases than in the blood of healthy donors. The emergence of ABs with diverse types of catalytic activity is among the earliest manifestations of certain autoimmune diseases. The putative mechanisms that underlie the accumulation of autoantibodies and abzymes in different autoimmune diseases are addressed in the present review. The extraordinary diversity of abzymes with various types of catalytic activity is discussed.

Features of hydrolysis of specific and nonspecific globular proteins and oligopeptides by antibodies against viral integrase from blood of HIV-infected patients.

It was shown previously that, as differentiated from canonical proteases, abzymes against myelin basic protein (MBP) from blood of patients with multiple sclerosis and systemic lupus erythematosus effectively cleaved only MBP, while antibodies (ABs) against integrase (IN) from blood of HIV-infected patients specifically hydrolyzed only IN. In this work, all sites of effective hydrolysis by anti-IN antibodies (IgG and IgM) of 25-mer oligopeptide (OP25) corresponding to MBP were identified using reversed-phase and thin-layer chromatographies and MALDI mass spectrometry. It was found that amino acid sequences of OP25 and other oligopeptides hydrolyzed by anti-MBP abzymes were partially homologous to some fragments of the full sequence of IN. Sequences of IN oligopeptides cleavable by anti-IN abzymes were homologous to some fragments of MBP, but anti-MBP abzymes could not effectively hydrolyze OPs corresponding to IN. The common features of the cleavage sites of OP25 and other oligopeptides hydrolyzed by anti-MBP and anti-IN abzymes were revealed. The literature data on hydrolysis of specific and nonspecific proteins and oligopeptides by abzymes against different protein antigens were analyzed. Overall, the literature data suggest that short OPs, including OP25, mainly interact with light chains of polyclonal ABs, which had lower affinity and specificity to the substrate than intact ABs. However, it seems that anti-IN ABs are the only one example of abzymes capable of hydrolyzing various oligopeptides with high efficiency (within some hours but not days). Possible reasons for the efficient hydrolysis of foreign oligopeptides by anti-IN abzymes from HIV-infected patients are discussed.

Polyreactivity of natural antibodies: exchange by HL-fragments.

The polyreactivity of binding (formation of antibody (AB) complexes not only with specific but also with foreign antigens) is a widespread phenomenon that in some cases can be caused by a conformational lability of the antigen-binding sites of antibodies (which increases upon treatment with various destabilizing agents) and leads to AB binding with very different antigens. Some ABs exist as dimers of the initial ABs and their idiotypes (or anti-idiotypes) capable of producing intramolecular cyclic complexes with features of polyreactants. Another mechanism of binding polyreactivity is an exchange in blood by halves of IgG4 molecules (HL-fragments) against various antigens. Also, for the first time catalytic polyfunctionality of human milk ABs has been detected, which is caused by an exchange by HL-fragments between molecules of λ- and κ-IgG (IgG1-IgG4) and also by λ- and κ-sIgA against different antigens with formation of very different chimeric antibodies. This review considers all possible pathways of formation of polyspecific immunoglobulins and their biological functions described in the literature, as well as mechanisms of binding polyreactivity and catalytic polyfunctionality of natural antibodies.

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.

DNase, RNase, and phosphatase activities of antibodies formed upon immunization by DNA, DNase I, and DNase II.

Relative DNase, RNase (efficiency of hydrolysis of ribo- and deoxyribooligonucleotides (ON)), and phosphatase (removal of the ON 5' terminal phosphate) catalytic activities of antibodies (AB) obtained after rabbit immunization by DNA, DNase I, and DNase II were compared. It is shown that electrophoretically homogeneous preparations of polyclonal AB from non-immunized rabbits did not exhibit such activities. Immunization of rabbits by DNA, DNase I, and DNase II results in generation of IgG abzymes that exhibit high activity in the ON hydrolysis reaction and even higher activity in cleavage of 5' terminal phosphate of ON. In this case K(m) values for supercoiled plasmid DNA and ON found in reactions of their AB-dependent nuclease hydrolysis and phosphatase cleavage of 5' terminal phosphate differ by 2-4 orders of magnitude. This shows that nuclease and phosphatase activities belong to different abzyme fractions within polyclonal AB. Thus, in this work data indicative of the possibility of a formation of antibodies exhibiting phosphatase activity after immunization of animals with DNA, DNase I, and DNase II, were obtained for the first time. Possible reasons for production of AB with phosphatase activity after immunization of rabbits with these immunogens are discussed.

Diversity of integrase-hydrolyzing IgGs and IgMs from sera of HIV-infected patients.

It was previously shown that small fractions of IgGs and IgMs from the sera of AIDS patients specifically hydrolyze only HIV integrase (IN) but not many other tested proteins. Here we present evidence showing that these IgGs and IgMs are extreme catalytically heterogeneous. Affinity chromatography on IN-Sepharose using elution of IgGs (or IgMs) with different concentration of NaCl and acidic buffer separated catalytic antibodies (ABs) into many AB subfractions demonstrating different values of K(m) for IN and k(cat). Nonfractionated IgGs and IgMs possess serine-, thiol-, acidic-like, and metal-dependent proteolytic activity. Metal-dependent activity of abzymes increases in the presence of ions of different metals. In contrast to canonical proteases having one pH optimum, initial nonfractionated IgGs and IgMs demonstrate several optima at pH from 3 to 10. The data obtained show that IN-hydrolyzing polyclonal IgG and IgM of HIV-infected patients are cocktails of anti-IN ABs with different structure of the active centers possessing various affinity to IN, pH optima, and relative rates of the specific substrate hydrolysis.

Peculiarities of abzymes from sera and milk of healthy donors and patients with autoimmune and viral diseases.

The detection of catalytic activity of antibodies is the earliest indicator of development of autoimmune diseases (AID). In early stages of AID, the repertoire of abzymes with various properties is relatively small, but it is greatly increased during their development. Catalytic diversity of the abzymes includes DNase, RNase, ATPase, and oxidoreductase activities; there are antibodies phosphorylating proteins, lipids, and polysaccharides. This review summarizes new data on abzyme heterogeneity and possible reasons for this phenomenon. A possible role of abzymes and their exceptional multiplicity in the pathogenesis of different AID is discussed.

[DNA and oligosaccharides stimulate oligomerization of human milk lactoferrin].

Lactoferrin (LF), the glycoprotein transferring Fe+ ions, is contained in barrier liquids, human blood and milk. LF is an acute phase protein and one of the most important factors of nonspecific defense. The protein has unique set of biological functions. Using the methods of small-angle X-ray scattering and light-scattering it was shown for the first time that addition of DNA and oligosaccharides to LF with different level of initial oligomerization leads to an enhance in the oligomerization rate. 1M NaCl stimulates almost a complete dissociation of LF oligomeric complexes obtained in the pesence of DNA, oligosaccharides, or early founded oligomerization effectors (nucleotides). It was shown that LF oligomeric complexes obtained in the presence of different oligomerization effectors have different stability. Incubation with 50 mM MgCl2 leads to complete destruction of the protein complexes formed by ATP and oligosaccharide but partially dissociate the complexes with following formation of new in the case of AMP- and d(pT)10-dependent associates, which possess higher stability i n presence of the salt. A possible role of LF oligomerization for different biological functions of the protein is discussed.

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.

[Interactions of pro- and eukaryotic DNA repair enzymes with oligodeoxyribonucleotides containing clustered lesions].

Interactions of oxoGuanine-DNA glycosylases from Escherichia coli (Fpg) and human (OGG1) and abasic site endonucleases from yeast (Apnl) and E. coli (Nfo) with oligodeoxyribonucleotides containing oxoGuanine (oxoG) and tetrahydrofuran (F, a stable analog of an abasic site) separated by various numbers of nucleotides have been studied. Inhibitor analysis has shown that the affinity of Fpg for single-stranded ligands does not depend on the relative positions of oxoG and F lesions. KM and kcat values have been determined for all double-stranded substrates and all enzymes under study. The effect of introducing the second lesion was strongly dependent on the relative positions of the lesions and the nature of the enzyme. The highest drop in the affinity (1.6-148-fold) and the reaction rate (4.8-58-fold) has been observed with Fpg and OGG1 for substrates containing F immediately 5' or 3' adjacent to oxoG. Introduction of the second lesion barely changed the KM values for Apnl and Nfo substrates. At the same time, the reaction rates were 5-10-fold lower for substrates containing two adjacent lesions. For all enzymes studied, increasing the distance between two lesions in duplex DNA reduced the effect of the lesion in KM and kcat values.

[Interactions of human 8-oxoguanine-DNA glycosylase with single- and double-stranded DNA].

Interactions of human 8-oxoguanine-DNA glycosylase (hOGG1) with single- and double-stranded oligodeoxyribonucleotides (ODN) have been studied by the method of stepwise increase in ligand complexity. The ODNs have been found to inhibit the glycosylase-catalyzed reaction competitively. The K1 values have been determined for a set of ODNs. All units of non-specific DNA within the enzyme footprint have been shown to interact with the protein globule in an additive manner. An increase in the d(pN)n length (n) by one unit caused a monotonous 1.4-1.5-fold increase in their affinity for the glycosylase ODN until n = 10, mostly due to weak nonspecific contacts of the enzyme and the sugar-phosphate backbone. The weak nonspecific additive interactions contributed about five orders of magnitude in the affinity of hOGG1 for specific DNA (Kd approximately 10(-5) M), whereas introduction of a 8-oxoguanine residue added about three orders of magnitude to this affinity (Kd approximately 10(-8) M). Quantitative features of recognition of specific DNA by the enzyme are analyzed.

[The effect of nucleotides on oligomeric state of human lactoferrin].

A polyfunctional protein lactoferrin (LF) which is present in human barrier fluids, blood and milk and this protein of acute phase is responsible for nonspecific cells defense against microbial and viral infection and cancer diseases. Using the methods of small-angle X-ray scattering and light-scattering it was shown that LF in solution exists in oligomeric state. The level of LF oligomerization depends upon its concentration and time of keeping of no frozen neutral protein solutions. At the concentrations comparable with those in human milk (1-6 mg/ml) the average inertial radius values (Rg) of LF can reach 100-450 angstroms, while Rg for monomer LF form is 26.7 angstroms. LF was shown to complex with different nucleotides and hydrolyze them. The addition of ATP and AMP to LF demonstrating any level of oligomerization leads to increase of oligomerization processes and enhancement of the Rg values up to 600-700 angstroms According to different models of LF monomer association to its oligomeric forms (sphere, plate, cylinder) the oligomeric complexes demonstrate high Rg values which can contain from several tens up to several thousands of LF monomers. A possible role of LF oligomerization for different biological functions of the protein is discussed.

[DNA-hydrolyzing IgG antibodies from the blood of patients with acquired immune deficiency syndrome].

DNAase activity of 110 samples of IgG from the blood of AIDS patients was analyzed. It was shown that the relative activity of preparations varies very much from patient to patient, but 96% preparations show detectable level of DNAase activity. Several rigid criteria were applied and it was shown that DNAase activity is an intrinsic property of antibodies from AIDS patients. It was shown that catalytic activity could posses not only intact IgG, but also separated light chains of polyclonal antibodies. The abzymes catalyze DNA hydrolysis effectively in a wild range of pH (5.0-9.5). K(M) and V(MaKC) values of antibody-dependent hydrolysis of DNA was estimated.

[Human milk lactoferrin hydrolyzes nucleoside-5'-triphosphates].

Lactoferrin (LF) is a main iron-transfering glycoprotein of human barrier body fluids, blood and milk. LF, a protein of the acute phase, is responsible for nonspecific cells defense against microbial and viral infection and cancer diseases. LF is an important component of the passive immunity of newborns system. LF, an extremely polyfunctional protein, is the object of intensive investigations. In this work electrophoretically homogeneous LF from human milk was prepared. Affinity chromatography of LF on Blue Sepharose separated the protein into several distinct isoforms with different affinities to this resin. Two of this isoforms possess nucleoside-5'-triphosphate-hydrolyzing activity. Using several methods including in-gel ATPase activity assays, we show that ATP (and others NTP) hydrolysis is an intrinsic property of LF, and that LF is the major ATPase of human milk. It was shown that ATP-hydrolyzing site is located in C-lobe of LF.

DNA-hydrolyzing activity of the light chain of IgG antibodies from milk of healthy human mothers.

Various catalytically active antibodies or abzymes have been detected recently in the sera of patients with several autoimmune pathologies, where their presence is most probably associated with autoimmunization. Normal humans are generally considered to have no abzymes, since no obvious immunizing factors are present. Recently we have shown that IgG (its Fab and F(ab)2 fragments) from the milk of normal humans possesses DNase activity. Here we demonstrate for the first time that the light chain of IgG catalyzes the reaction of DNA hydrolysis. These findings speak in favor of the generation of abzymes in the tissue of healthy mothers, and since a mother's breast milk protects her infant from infections until the immune system is developed, they raise the possibility that these abzymes may contribute to this protective role.

High affinity interaction of mammalian DNA topoisomerase I with short single- and double-stranded oligonucleotides.

The interaction of DNA topoisomerase I (topo I) with a set of single- and double-stranded oligonucleotides containing 5-27 mononucleotides was investigated. All single- and double-stranded oligonucleotides were found to inhibit competitively the supercoiled DNA relaxation reaction catalyzed by topo I. The enzyme affinity for specific sequence pentanucleotides of the scissile (GACTT, Ki = 2 microM) and non-cleaved chain (AAGTC, Ki = 110 microM) is about 2-4 orders of magnitude higher than that for non-specific oligonucleotides. This specific sequence affinity increases in several cases; lengthening of single-stranded oligonucleotides, formation of stable duplexes between complementary oligonucleotides and preincubation of the enzyme with ligands before addition of supercoiled DNA. We assume that oligonucleotides having a high affinity to the enzyme can offer a unique opportunity for rational design of topoisomerase-targeting drugs.

Dynamic aspects of affinity labelling as revealed by alkylation and phosphorylation of pancreatic ribonuclease with reactive deoxyribodinucleotide derivatives.

Affinity labelling of pancreatic RNase with 4-(N-2-chloroethyl-N-methylamino)benzylamide and (N----P) N-methylimidazolide of d(pTpA) results in the formation of monomodified enzyme derivatives retaining partially enzymatic activity. These data together with some cases described in the literature are considered as suggesting the dynamic nature of the enzyme-reagent complex represented by a set of states differing in the probability of intra-complex reaction. In particular, modification may proceed in a low probability state with an especially favorable mutual orientation of reagent and some protein residue remote from the active site of the enzyme resulting in the removal of the covalently attached reagent moiety from the active center.

Human milk lactoferrin binds two DNA molecules with different affinities.

Evidence is presented that lactoferrin (LF), an Fe3+-binding glycoprotein, possesses two DNA-binding sites with different affinities for specific oligonucleotides (ODNs) (Kdl = 8 nM; Kd2 approximately 0.1 mM). The high affinity site became labeled after incubation with affinity probes for DNA-binding sites; like the antibacterial and polyanion-binding sites, this site was shown to be located in the N-terminal domain of LF. Interaction of heparin with the polyanion-binding site inhibits the binding of ODNs to both sites. These data suggest that the DNA-binding sites of LF coincide or overlap with the known polyanion and antimicrobial domains of the protein.

High affinity interaction of mouse DNA topoisomerase I with di- and trinucleotides corresponding to specific sequences of supercoiled DNA cleaved chain.

Recently mouse DNA topoisomerase I (topo) was shown to possess high affinity for a single-stranded AAGACTTAG nonanucleotide (K(i) = 2.0 microM) corresponding to the scissile strand of the minimal DNA duplex, which is necessary for cleavage of supercoiled DNA. In order to determine the most important part of the above sequence for the DNA recognition by topo, the interactions of the enzyme with a set of extremely short (2-5 nucleotides in length) oligonucleotides corresponding to different parts of the nonanucleotide have been investigated. The affinities of different oligonucleotides corresponding to the CTTAG part of the sequence (K(i) = 0.13-0.92 mM) were shown to be significantly lower than that for the AAGA tetranucleotide (K(i) = 9.0 microM). Topo effectively recognized even short oligonucleotides containing only two or three bases (AGA and pAG, K(i) = 20 and 50 microM). We suppose that oligonucleotides having a high afffinity to the enzyme can offer a unique opportunity for the rational design of topoisomerase-targeting drugs.