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.     

Antibodies with amylase activity from the sera of autoimmune-prone MRL/MpJ-lpr mice

Animals spontaneously developing lupus-like autoimmune pathology (SLE) are very promising models to study the mechanisms of natural abzymes (Abzs) generation and their role in etiology and pathogenesis of autoimmune diseases, but Abzs from the sera of animals remain virtually unstudied. In this work, electrophoretically homogeneous IgGs were isolated from the sera of MRL/MpJ-lpr mice. It was shown for the first time that amylase activity is an intrinsic property of antibodies (Abs) and their isolated heavy and light chains. Various markers of SLE pathology (proteinuria, enhanced concentration of anti-DNA Abs) increased with spontaneous development of SLE and especially after animal immunization, correlating with the increase in Abz relative amylase activity. The highest amylase activity was found in the sera Abs of healthy mice after delivery and at the beginning of lactation; this was not correlated with markers of mouse SLE but supports the idea that pregnancy could "activate" or "trigger" autoimmune-like manifestations and Abzs production in healthy mammals. The possible differences in mechanisms of Abzs production in lactating mice and animals developing SLE are discussed.     

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.     

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.     

Specific anti‐integrase abzymes from HIV‐infected patients: a comparison of the cleavage sites of intact globular HIV integrase and two 20‐mer oligopeptides corresponding to its antigenic determinants

HIV‐infected patients possess anti‐integrase (IN) IgGs and IgMs that, after isolation by chromatography on IN‐Sepharose, unlike canonical proteases, specifically hydrolyze only IN but not many other tested proteins. Hydrolysis of intact globular IN first leads to formation of many long fragments of protein, while its long incubation with anti‐IN antibodies, especially in the case of abzymes (Abzs) with a high proteolytic activity, results in the formation of short and very short oligopeptides (OPs). To identify all sites of IgG‐mediated proteolysis corresponding to known AGDs of integrase, we have used a combination of reverse‐phase chromatography, matrix‐assisted laser desorption/ionization spectrometry, and thin‐layer chromatography to analyze the cleavage products of two 20‐mer OPs corresponding to these AGDs. Both OPs contained 9–10 mainly clustered major, medium, and minor sites of cleavage. The main superficial cleavage sites of the AGDs in the intact IN and sites of partial or deep hydrolysis of the peptides analyzed do not coincide. The active sites of anti‐IN Abzs are localized on their light chains, whereas the heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of Abzs provide high specificity of IN hydrolysis. The affinity of anti‐IN Abzs for intact integrase was ~1000‐fold higher than for the OPs. The data suggest that both OPs interact mainly with the light chains of different monoclonal Abzs of the total pool of IgGs, which possesses lower affinity for substrates; and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific and remarkably different in comparison with the cleavage of intact globular IN. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Metal dependent hydrolysis of β‐casein by sIgA antibodies from human milk

We present the first evidence that electrophoretically and immunologically homogeneous sIgAs purified from milk of healthy human mothers by chromatography on Protein A‐Sepharose and FPLC gel filtration contain intrinsically bound metal ions (Ca > Mg ≥ Al > Fe ≈ Zn ≥ Ni ≥ Cu ≥ Mn), the removal of which by a dialysis against ethylenediamine tetraacetic acid (EDTA) leads to a significant decrease in the β‐casein‐hydrolyzing activity of these antibodies (Abs). An affinity chromatography of total sIgAs on benzamidine‐Sepharose interacting with canonical serine proteases separates a small metalloprotease sIgA fraction (6.8 ± 2.4%) from the main part of these Abs with a serine protease‐like β‐casein‐hydrolyzing activity. The relative activity of this metalloprotease sIgA fraction containing intrinsically bound metal ions increases ～1.2–1.9‐fold after addition of external metal ions (Mg²⁺ > Fe²⁺ > Cu²⁺ ≥ Ca²⁺ ≥ Mn²⁺) but decreases by 85 ± 7% after the removal of the intrinsically bound metals. The metalloprotease sIgA fraction free of intrinsic metal ions demonstrates a high β‐casein‐hydrolyzing activity in the presence of individual external metal ions (Fe²⁺ > Ca²⁺ > Co²⁺ ≥ Ni²⁺) and especially several combinations of metals: Co²⁺ + Ca²⁺ < Mg²⁺ + Ca²⁺ < Ca²⁺ + Zn²⁺ < Fe²⁺ + Zn²⁺ < Fe²⁺ + Co²⁺ < Fe²⁺ + Ca²⁺. The patterns of hydrolysis of a 22‐mer oligopeptide corresponding to one of sIgA‐dependent specific cleavage sites in β‐casein depend significantly on the metal used. Metal‐dependent sIgAs demonstrate an extreme diversity in their affinity for casein‐Sepharose and chelating Sepharose, and interact with Sepharoses bearing immobilized monoclonal mouse IgGs against λ‐ and κ‐type light chains of human Abs. Possible ways of the production of metalloprotease abzymes (Abz) by human immune system are discussed. Copyright © 2010 John Wiley & Sons, Ltd.     

Anti-idiotypic antibody mimics proteolytic function of parent antigen

Functional imaging of subtilisin Carlsberg active center by the idiotypic network yielded a catalytic anti-idiotypic antibody with endopeptidase, amidase, and esterase activities. A monoclonal antibody inhibitory to subtilisin (Ab1 5-H4) was employed as the template for guiding the idiotypic network to produce the catalytic anti-idiotypic Ab2 6B8-E12. Proteolytic activity of 6B8-E12 was demonstrated by zymography using self-quenched fluorescein-BSA conjugate and in a coupled assay detecting Ab2-dependent RNase A inactivation. Cleavage of peptide substrates by 6B8-E12 revealed distinct patterns of hydrolysis with high preference for aromatic residues before or after the scissile bond. Catalytic activity of Ab2 was inhibited by phenylmethylsulfonyl fluoride, a mechanism-based inhibitor of serine hydrolases. 5-H4 and 6B8-E12 were cloned, produced in Escherichia coli as single-chain variable fragments (scFvs), and purified. Kinetic parameters for amidolytic and esterolytic activities were similar in Ab2 and its scFv derivative. Although the antigen-specific portion of 6B8-E12 possesses no primary structure similarity to subtilisin, it mimics proteolytic and amidolytic functions of the parental antigen, albeit with 4 orders of magnitude slower acceleration rates. The lack of detectable endopeptidase activity of 6B8-E12 scFv raises interesting issues concerning general evolution of catalytic activity. The in silico 3D models of Ab1 and Ab2 revealed strong structural similarity to known anti-protease antibodies and to abzymes, respectively. These results indicate that the idiotypic network is capable, to a significant extent, of reproducing catalytic apparatus of serine proteases and further validate the use of imaging of enzyme active centers by the immune system for induction of abzymes accelerating energy-demanding amide bond hydrolysis.     

IgG antibodies with peroxidase-like activity from the sera of healthy Wistar rats

Various catalytic antibodies or abzymes (Abzs) have been detected recently in the sera of patients and animals with many autoimmune diseases, where their presence is most probably associated with autoimmunization. Normal humans or animals usually do not contain Abzs. In contrast, polyclonal Abzs from healthy humans and animals have an intrinsic superoxide dismutase activity and catalyze formation of H(2)O(2) (Wentworth et al., 2000, Proc. Natl. Acad. Sci. USA; 2001, Science). Here, we present the first evidence showing that highly purified native IgGs from the sera of healthy Wistar rats interact with H(2)O(2) and possess peroxidase-like activity. Specific peroxidase activity of IgG preparations from the sera of 10 rats varied in the range 1.6-27% as compared with that for horseradish peroxidase (100%). Antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases are known to represent critical defence mechanisms for preventing oxidative modifications of DNA, proteins, and lipids. Antioxidant peroxidase activity of Abzs can also play an important role in the protection of organisms from oxidative stress as well as in oxidation of toxic compounds.     

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.     

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.     

Degradation of connective tissue proteins by serine proteases from Streptococcus pneumoniae.

The proteolytic activity of pneumococcal culture supernatants was investigated. Phenylmethylsulfonyl fluoride and diisopropylfluorophosphate inhibited the proteolytic activity by 94% indicating that the enzymes are serine proteases. Zymogram analysis with inhibitors utilizing a non-denaturing gelatin substrate gel revealed two classes of serine proteases; one sensitive to calcium chelators and one resistant. Enzymes from the culture supernatant cleaved fibronectin, fibrinogen, elastin, and laminin; whereas bovine albumin, and the human immunoglobulins, IgG, IgM, and IgA, were not cleaved. These results indicate that pneumococci produce previously unrecognized serine proteases that degrade several tissue and blood proteins.     

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.     

Catalytically active antibodies (abzymes) in human milk

The review is focused on the analysis of published data and the results obtained by the authors about the catalytic activity of antibodies (abzymes) of human colostrum and milk. Possible mechanisms of origination of these abzymes and their potential role in the regulation of biological activity of human milk compounds are considered. A hypothesis about the role of secretoty abzymes in non-specific humoral defense for the epithelial cells against viral infections is proposed.     

Hepatocyte growth factor activator inhibitor type 1 inhibits protease activity and proteolytic activation of human airway trypsin-like protease

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type transmembrane serine protease inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA), a serine protease that converts pro-HGF to the active form. HAI-1 also has inhibitory activity against serine proteases such as matriptase, hepsin and prostasin. In this study, we examined effects of HAI-1 on the protease activity and proteolytic activation of human airway trypsin-like protease (HAT), a transmembrane serine protease that is expressed mainly in bronchial epithelial cells. A soluble form of HAI-1 inhibited the protease activity of HAT in vitro. HAT was proteolytically activated in cultured mammalian cells transfected with its expression vector, and a soluble form of active HAT was released into the conditioned medium. The proteolytic activation of HAT required its own serine protease activity. Co-expression of the transmembrane full-length HAI-1 inhibited the proteolytic activation of HAT. In addition, full-length HAI-1 associated with the transmembrane full-length HAT in co-expressing cells. Like other target proteases of HAI-1, HAT converted pro-HGF to the active form in vitro. These results suggest that HAI-1 functions as a physiological regulator of HAT by inhibiting its protease activity and proteolytic activation in airway epithelium.     

Serine protease activity in developmental stages of Eimeria tenella

A number of complex processes are involved in Eimeria spp. survival, including control of sporulation, intracellular invasion, evasion of host immune responses, successful reproduction, and nutrition. Proteases have been implicated in many of these processes, but the occurrence and functions of serine proteases have not been characterized. Bioinformatic analysis suggests that the Eimeria tenella genome contains several serine proteases that lack homology to trypsin. Using RT-PCR, a gene encoding a subtilisin-like and a rhomboid protease-like serine protease was shown to be developmentally regulated, both being poorly expressed in sporozoites (SZ) and merozoites (MZ). Casein substrate gel electrophoresis of oocyst extracts during sporulation demonstrated bands of proteolytic activity with relative molecular weights (Mr) of 18, 25, and 45 kDa that were eliminated by coincubation with serine protease inhibitors. A protease with Mr of 25 kDa was purified from extracts of unsporulated oocysts by a combination of affinity and anion exchange chromatography. Extracts of SZ contained only a single band of inhibitor-sensitive proteolytic activity at 25 kDa, while the pattern of proteases from extracts of MZ was similar to that of oocysts except for the occurrence of a 90 kDa protease, resistant to protease inhibitors. Excretory-secretory products (ESP) from MZ contained AEBSF (4-[2-Aminoethyl] benzenesulphonyl fluoride)-sensitive protease activity with a specific activity about 10 times greater than that observed in MZ extracts. No protease activity was observed in the ESP from SZ. Pretreatment of SZ with AEBSF significantly reduced SZ invasion and the release of the microneme protein, MIC2. The current results suggest that serine proteases are present in all the developmental stages examined.     

Affinity and catalytic heterogeneity of polyclonal myelin basic protein‐hydrolyzing IgGs from sera of patients with multiple sclerosis

Human myelin basic protein (hMBP)‐hydrolyzing activity was recently shown to be an intrinsic property of antibodies (Abs) from multiple sclerosis (MS) patients. Here, we present the first evidence demonstrating a significant diversity of different fractions of polyclonal IgGs (pIgGs) from MS patients in their affinity for hMBP and in the ability of pIgGs to hydrolyze hBMP at different optimal pHs (3–10.5). IgGs containing λ‐ and κ‐types of light chains demonstrated comparable relative activities in the hydrolysis of hMBP. IgGs of IgG1–IgG4 sub‐classes were analyzed for catalytic activity. IgGs of all four sub‐classes were catalytically active, with their contribution to the total activity of Abzs in the hydrolysis of hMBP and its 19‐mer oligopeptide increasing in the order: IgG1 (1.5–2.1%) < IgG2 (4.9–12.8%) < IgG3 (14.7–25.0%) < IgG4 (71–78%). Our findings suggest that the immune systems of individual MS patients generate a variety of anti‐hMBP abzymes with different catalytic properties, which can attack hMBP of myelin‐proteolipid shell of axons, playing an important role in MS pathogenesis.     

Identification and characterisation of the excreted/secreted serine proteases of larvae of the old world screwworm fly, Chrysomya bezziana

Serine proteases are the major proteolytic activity excreted or secreted from Chrysomya bezziana larvae as demonstrated by gelatin gel analyses and the use of specific substrates, benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Serine proteases were identified through their inhibition by 4-(2-aminoethyl)-benzene sulphonyl fluoride and classified as trypsin- and chymotrypsin-like on the basis of inhibition by tosyl-L-lysine chloromethyl ketone and tosyl-L-phenylalanine chloromethyl ketone, respectively. Like most insect serine proteases, the C. bezziana enzymes were active over broad pH range from mildly acidic to alkaline. The excreted or secreted serine proteases were purified by affinity chromatography using soybean trypsin inhibitor. A different subset of the serine proteases was isolated by salt elution from washed larval peritrophic matrices. Amino-terminal sequencing identified both trypsin and chymotrypsin-like sequences in the excreted or secreted pool with the latter being the dominant protease, whereas trypsin was the dominant species in the peritrophic matrix eluant. These results suggest that trypsin was possibly preferably adsorbed by the peritrophic matrix and may act as a final proteolytic processing stage as partially digested and ingested polypeptides pass through the peritrophic matrix. Immunoblot analysis on dissected gut tissues indicated that the anterior and posterior midguts were the main source of the serine proteases, although a novel species of 32 kDa was predominantly associated with the peritrophic matrix. Proteases are a target for a partially protective immune response and understanding the complexity of the secreted and digestive proteases is a necessary part of understanding the mechanism of the host's immunological defence against the parasite.