Casein-hydrolyzing activity of sIgA antibodies from human milk

During pregnancy and immediately after delivery (i.e. at the beginning of lactation), the female organism is frequently characterized by an immune status similar to that of patients with autoimmune diseases. In addition, lactation is associated with an appearance of catalytically active antibodies or abzymes (Abzs) with DNAse, RNase, ATPase, amylolitic, protein kinase and lipid kinase activities in breast milk. However, until now there were no examples of human milk Abzs with a proteolytic activity. We present the first evidence that electrophoretically and immunologically homogeneous human milk sIgAs possess a beta-casein-hydrolyzing activity different from known proteases. Abzs specifically hydrolyze both human and bovine beta-caseins but not many other proteins tested. Using different methods including in situ analysis of proteolytic activity in a gel after SDS-PAGE it was shown that the observed proteolytic activity is an intrinsic property of human milk polyclonal sIgAs. Specific inhibitors of acidic and thiol proteases demonstrated a weak effect on proteolytic activity of Abzs, while a specific inhibitor of serine proteases (AEBSF) significantly inhibited the proteolytic activity of the abzymes. The K(M) value for human casein as a substrate was estimated (7.3 microM). Our findings suggest that the immune system of clinically healthy mothers can generate IgAs with a beta-casein-specific serine protease-like activity.     

Lipid kinase activity of antibodies from milk of clinically healthy human mothers

We have shown recently that polyclonal human milk sIgA contains a subfraction of antibodies (Abs) tightly bound to unusual minor milk lipids containing sialic acid. Here, we show that a small subfraction of milk IgG is tightly bound to the similar or the same minor lipids. The ability of small fractions of sIgA and IgG from human milk to phosphorylate selectively two minor lipids in the presence of [gamma-(32)P]nucleoside triphosphates was shown here for the first time to be an intrinsic property of these antibodies. In contrast to known kinases, antibodies with lipid kinase activity can transfer phosphoryl group to lipids not only from ATP but also from other different nucleotides (dATP, GTP, dGTP, UTP, TTP) with comparable efficiencies (30-100%). To our knowledge, there are no examples of enzymes using orthophosphate as a substrate of phosphorylation reactions. An extremely unusual property of lipid kinase Abs is their high affinity for orthophosphate (K(m)=1.6-5.6 microM) and capability to phosphorylate minor lipids using [(32)P]orthophosphate as donor of phosphate group. The relative specific activity and affinity of abzymes for orthophosphate and ATP depend significantly on donor milk. However, the levels of Ab-dependent phosphorylation of lipids for all Abs in the case of ATP (100%) and orthophosphate (60-80%) as substrates are comparable. The first example of natural abzymes with synthetic activity was milk sIgA with protein kinase activity. Most probably, lipid kinase sIgA and IgG of human milk are the second example of Abs with synthetic activity.     

Polysaccharide kinase activity of human milk IgG antibodies

A small fraction of human milk IgG antibodies is shown to possess polysaccharide kinase activity for the first time. Unlike all known kinases, IgG antibodies can use as phosphate donor not only [gamma-(32)P]ATP, but also directly [(32)P]ortho-phosphate. Human milk IgGs therefore possess high affinity to ortho-phosphate (K(m) = 9-71 microM), which is a more effective substrate than ATP. IgG antibodies possessing polysaccharide kinase activity are yet another example of natural abzymes possessing not hydrolytic, but synthetic enzymatic activity.     

HIV-1 integrase-hydrolyzing antibodies from sera of HIV-infected patients

Autoantibodies with enzymic activities (abzymes) are a distinctive feature of autoimmune diseases. It was interesting whether Abs from patients with viral diseases can hydrolyze viral proteins. Electrophoretically and immunologically homogeneous IgGs were isolated from sera of AIDS patients by chromatography on several affinity sorbents. We present evidence showing that 89.5% IgGs purified from the sera of HIV-infected patients using several affinity resins including Sepharose with immobilized integrase specifically hydrolyze only HIV integrase (IN) but not many other tested proteins. Several rigid criteria have been applied to show that the IN-hydrolyzing activity is an intrinsic property of AIDS IgGs but not from healthy donors. Similar to autoimmune proteolytic abzymes, IN-hydrolyzing IgGs from some patients were inhibited by specific inhibitors of serine and metal-dependent proteases but a significant inhibition of the activity by specific inhibitors of acidic- and thiol-like proteases was observed for the first time. Although HIV infection leads to formation of Abs to many viral and human antigens, no possible biological role for most of them is known. Since anti-IN IgG can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded. In addition, detection of IN-hydrolyzing activity can be useful for diagnostic purposes and for estimation of the immune status in AIDS patients.     

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.     

Isolation and partial characterization of catalytic antibodies with oligonuclease activity from bovine colostrum

Catalytic antibodies (abzymes) which hydrolyze RNA and DNA were isolated from bovine colostrum by sequential chromatography on Protein A Sepharose, denaturated DNA-cellulose, Mono Q, and gel permeation chromatography on Superose 12 at pH 2.3 after acidic shock. Metachromatic agar containing toluidine blue and yeast RNA was used to measure RNase activity. Electrophoresis in agarose showed DNase activity on plasmid DNA from Escherichia coli and DNA from calf thymus in fractions from all 4 purification steps. Gel permeation chromatography showed that the abzymes hydrolysed both a single-stranded polyadenylic acid (Poly A) and single-stranded polycitidylic acid (Poly C), while partially purified RNase from the colostrum hydrolysed Poly (C), but not Poly (A). Electrophoresis of purified abzymes under denaturing conditions showed protein bands of molecular mass corresponding to heavy and light chains of IgG. The abzymes immunoreacted with anti-bovine IgG. The RNase activity of the purified abzymes represented 0.022% of total RNase activity in the colostrum; acid shock and gel filtration at low pH reduced the specific RNase activity of abzymes 3.6-fold. The RNase activity of abzymes at pH 6.6 was reduced by 90% by heat treatment at 75 degrees C for 52 min.     

工程抗体酶的研究进展

本文评述了工程抗体酶的研究现状和抗体酶在防化医学研究中的应用。抗体库技术的出现使抗体酶的研究呈现了新的生机。不仅使不具备细胞工程实验条件的实验室能够制备抗体酶,而且实验周期大大缩短,比杂交瘤技术提供多20倍的结合性抗体作为潜在的抗体酶,使抗体酶更易获得。抗体库技术能够容易地评价编码抗体酶的基因,因而可以在分子水平上认识和改造抗体酶。噬菌体抗体库可以不经免疫即可制备抗体的特点,使通过人源性噬菌体抗体库生产的抗体酶能够直接用于临床治疗。随着工程抗体酶研究的不断深入,新颖实用的抗体酶将会被应用于包括军事医学在内的多种领域中。     

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.     

Comparison of Enzymatic Properties of DNA‐Abzymes and Human DNAses

DNA‐hydrolyzing antibodies (DNA‐abzymes, Abz) were shown to be good biochemical markers of some autoimmune diseases such as systemic lupus erythematosus (SLE) and multiple sclerosis (MS). To better understand mechanisms of abzyme generation, one needs to know optimal conditions for DNA hydrolysis by DNA‐abzymes, as well as their enzymatic properties in comparison with those of enzymes possessing the same activity. In contrast to human urine deoxyribonucleases, DNA‐hydrolyzing antibodies efficiently digested both single‐ and double‐strand DNA. It was shown that polyclonal antibodies (Abs) in MS may contain up to several types of DNase activities, either activated by metal ions or not.     

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.     

Hydrolysis of myelin basic protein by polyclonal catalytic IgGs from the sera of patients with multiple sclerosis

Various catalytic 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. Recently we have shown that DNase, RNase, and polysaccharide-hydrolyzing activities are associated with IgGs from the sera of patients with multiple sclerosis (MS). Here we present evidence demonstrating that highly purified MS IgGs (but not Igs from the sera of healthy individuals) catalyze specifically hydrolysis of human myelin basic protein (hMBP). In contrast to many known proteases, IgGs do not hydrolyze many other different proteins. Specific inhibitors of acidic and thiol proteases have no remarkable effect on proteolytic activity of IgGs. However, specific inhibitor of serine (PMSF, AEBSF, and benzamidin) and metal-dependent (EDTA) proteases significantly inhibit activity of proteolytic abzymes. Interestingly, the ratio of serine-like and metal-dependent activities of MS IgGs varied very much from patient to patient. The findings speak in favor of the generation by the immune systems of individual MS patients of a variety of polyclonal anti-MBP IgGs with different catalytic properties.     

Selection and characterization of lipase abzyme from phage displayed antibody libraries

Antibodies with enzymatic activity were named abzymes or catalytic antibodies. In the present study, the lipolytic abzymes were selected from the phage displayed antibody libraries against a transition state analog (TSA) of lipases/esterases. After three rounds of selection, four monoclonal phage particles capable of binding significantly with the TSA were obtained. The soluble scFv antibody fragments were further expressed and obtained using Escherichia coli strain HB2151. The binding capabilities and the apparent enzymatic activities of the purified antibody proteins were measured. The 3D structures of the expressed antibodies were also predicted through homology modeling and binding-site prediction algorithm. The present method demonstrates that selection from phage displayed antibody libraries is an efficient and convenient means to find new abzymes.     

Why specific anti‐integrase antibodies from HIV‐infected patients can efficiently hydrolyze 21‐mer oligopeptide corresponding to antigenic determinant of human myelin basic protein

Human immunodeficiency virus‐infected patients possess anti‐integrase (IN) catalytic IgGs and IgMs (abzymes), which, unlike canonical proteases, specifically hydrolyze only intact globular IN. Anti‐myelin MBP abzymes from patients with multiple sclerosis and systemic lupus erythematosus efficiently hydrolyze only intact MBP. Anti‐MBP and anti‐IN abzymes do not hydrolyze several other tested control globular proteins. Here, we show that anti‐IN abzymes efficiently hydrolyze a 21‐mer oligopeptide (OP21) corresponding to one antigenic determinant (AGD) of MBP, whereas anti‐MBP abzymes extremely poorly cleave oligopeptides corresponding to AGDs of IN. All sites of IgG‐mediated and IgM‐mediated proteolysis of OP21 by anti‐IN abzymes were found for the first time by a combination of reverse phase and thin layer chromatography and mass spectrometry. Several clustered sites of OP21 cleavage were revealed and compared with the cleavage sites within the complete IN. Several fragments of OP21 had good homology with many fragments of the IN sequence. The active sites of anti‐IN abzymes are known to be located on their light chains, whereas heavy chains are responsible for the affinity for protein substrates. Interactions of intact IN with both light and heavy chains of the abzymes provide high affinity for IN and the specificity of its hydrolysis. Our data suggest that OP21 interacts mainly with the light chains of polyclonal anti‐IN abzymes, which possess lower affinity and specificity for substrate. The hydrolysis of the non‐cognate OP21 oligopeptide may be also less specific than the hydrolysis of the globular IN because in contrast to previously described serine protease‐like abzymes against different proteins, anti‐IN abzymes possess serine, thiol, acidic, and metal‐dependent protease activities. Copyright © 2013 John Wiley & Sons, Ltd.     

Colostral Trypsin-Inhibitor Capacity in Different Animal Species

Colostral trypsin-inhibitor capacity was monitored during the first two weeks from parturition. The colostrum of the mare, sow, cow and ewe showed high antitrypsin activity at parturition, decreasing to about one hundredth during the first week. Canine milk remained on a relatively high antitrypsin level, and human milk was poor in antitrypsin from childbirth. The antitrypsin content seems to parallel the known changes in the colostral immunoglobulin levels of different species. The role of antitrypsin in protection of immunoglobulins from proteolytic damage during passive transfer of immunity to the newborn is obvious.     

Enzymes phosphorylating lipids and polysaccharides

Phosphorylation plays an important role in regulation of living functions of organisms; phosphorylation may significantly alter chemical properties of proteins, lipids, and carbohydrates. Canonical kinases catalyze transfer of terminal phosphate group from ATP (or other NTPs) to specific nucleophilic groups of proteins, lipids, and polysaccharides. Recently, unique kinases, catalytically active antibodies (abzymes) phosphorylating proteins, lipids, and polysaccharides have also been discovered. This review highlights biological functions and enzymatic characteristics of canonical kinases and abzymes phosphorylating lipids and polysaccharides.     

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.     

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

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.