Georgy A. Nevinsky

Human catalytic RNA- and DNA-hydrolyzing antibodies

In patients with autoimmune diseases, anti-idiotypic antibodies directed to nucleoprotein complexes, DNA, and enzymes that participate in nucleic acid metabolism may be induced spontaneously by primary antigens and can have characteristics of the primary antigen, including catalytic activity. The first natural catalytic antibody, now termed abzyme, which hydrolyzes intestinal vasoactive peptide, was discovered by Paul et al. [Science 244 (1989) 1158]. Subsequently, other abzymes able to hydrolyze proteins, DNA, RNA, or polysaccharides have been found in the sera of patients with autoimmune and also viral pathologies. Further, we have discovered in the milk of healthy human mothers antibodies that catalyze the hydrolysis of RNA, DNA, nucleotides, and the phosphorylation of lipids and proteins. The phenomenon of catalysis by autoantibodies is extremely interesting and can potentially be applied to many different objectives including new types of efficient catalysts, evaluation of the functional roles of abzymes in innate and adaptive immunity, and understanding of certain aspects of self-tolerance and of the destructive responses in autoimmune diseases. In this review, we collate methods for purifying and characterizing natural abzymes especially those catalyzing DNA and RNA hydrolysis. We also describe new methods that we have developed to provide rigorous criteria that catalytic activity is an intrinsic property of some antibodies. Some major current themes are discussed as well as potential applications of abzymes in scientific, medical, and biotechnological fields.

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.

Catalytic heterogeneity of polyclonal DNA-hydrolyzing antibodies 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 activity is associated with IgGs from the sera of patients with multiple sclerosis (MS) but not with those from the sera of normal humans. Here we present evidence showing that MS IgG, its F(ab) fragments, and separated L-chains catalyze DNA hydrolysis. The properties of the DNase activity of these polyclonal IgGs distinguish them from other known human DNases. In addition, their specific activities with different oligonucleotide substrates and the range of optimal pHs, apparent K(M) values and substrate specificities varied widely for different patients. The findings speak in favor of the generation by the immune systems of individual patients of a variety of polyclonal catalytic IgG pools, from relatively small to extremely large ones.

Kinetics of substrate recognition and cleavage by human 8-oxoguanine-DNA glycosylase

Human 8-oxoguanine-DNA glycosylase (hOgg1) excises 8-oxo-7,8-dihydroguanine (8-oxoG) from damaged DNA. We report a pre-steady-state kinetic analysis of hOgg1 mechanism using stopped-flow and enzyme fluorescence monitoring. The kinetic scheme for hOgg1 processing an 8-oxoG:C-containing substrate was found to include at least three fast equilibrium steps followed by two slow, irreversible steps and another equilibrium step. The second irreversible step was rate-limiting overall. By comparing data from Ogg1 intrinsic fluorescence traces and from accumulation of products of different types, the irreversible steps were attributed to two main chemical steps of the Ogg1-catalyzed reaction: cleavage of the N-glycosidic bond of the damaged nucleotide and β-elimination of its 3′-phosphate. The fast equilibrium steps were attributed to enzyme conformational changes during the recognition of 8-oxoG, and the final equilibrium, to binding of the reaction product by the enzyme. hOgg1 interacted with a substrate containing an aldehydic AP site very slowly, but the addition of 8-bromoguanine (8-BrG) greatly accelerated the reaction, which was best described by two initial equilibrium steps followed by one irreversible chemical step and a final product release equilibrium step. The irreversible step may correspond to β-elimination since it is the very step facilitated by 8-BrG.

Uracil-DNA glycosylase: Structural, thermodynamic and kinetic aspects of lesion search and recognition

Uracil appears in DNA as a result of cytosine deamination and by incorporation from the dUTP pool. As potentially mutagenic and deleterious for cell regulation, uracil must be removed from DNA. The major pathway of its repair is initiated by uracil-DNA glycosylases (UNG), ubiquitously found enzymes that hydrolyze the N-glycosidic bond of deoxyuridine in DNA. This review describes the current understanding of the mechanism of uracil search and recognition by UNG. The structure of UNG proteins from several species has been solved, revealing a specific uracil-binding pocket located in a DNA-binding groove. DNA in the complex with UNG is highly distorted to allow the extrahelical recognition of uracil. Thermodynamic studies suggest that UNG binds with appreciable affinity to any DNA, mainly due to the interactions with the charged backbone. The increase in the affinity for damaged DNA is insufficient to account for the exquisite specificity of UNG for uracil. This specificity is likely to result from multistep lesion recognition process, in which normal bases are rejected at one or several pre-excision stages of enzyme-substrate complex isomerization, and only uracil can proceed to enter the active site in a catalytically competent conformation. Search for the lesion by UNG involves random sliding along DNA alternating with dissociation-association events and partial eversion of undamaged bases for initial sampling.

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 mothers 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.

Secretory immunoglobulin A from healthy human mothers' milk catalyzes nucleic acid hydrolysis.

The human milk secretory immune system is known to be the first line of protection for the newborn infant against various pathogens. Secretory IgA (sIgA), the typical immunoglobulin found in secretions, can fight infections through many mechanisms. Using different methods, we have shown that sIgA from the milk of healthy women possesses DNAse and RNAse activities. The catalytic center is localized in the light chain of catalytic sIgA, while the DNA-binding center is predominantly formed by its heavy chain. The enzymic properties and substrate specificity of catalytic sIgA distinguish it from other known DNases and RNases. It is reasonable to assume, that the milk DNA- and RNA-hydrolyzing antibodies are capable not only of neutralizing viral and bacterial nucleic acids by binding these antigens as well as by hydrolyzing them. The DNA-hydrolyzing activity of Abs raises the possibility that these catalytic Abs may provide protective functions for the newborn through the hydrolysis of viral and bacterial nucleic acids.

Catalytic diversity of polyclonal RNA-hydrolyzing IgG antibodies from the sera of patients with systemic lupus erythematosus

Various catalytic antibodies or abzymes have been detected in the sera of patients with several autoimmune pathologies, and recently we have shown that RNase activity is associated with IgGs and IgMs from the sera of patients with systemic lupus erythematosus (SLE) but not with those from the sera of normal humans. Here we present for the first time convincing evidence showing that highly purified SLE IgG, its F(ab) fragments and separated L-chains catalyze RNA hydrolysis. These IgGs hydrolyze RNA about one to three orders of magnitude faster than DNA. The enzymatic properties of the RNase activity of these polyclonal IgGs distinguish them from other known human RNases. Their specific activity in hydrolysis of ribooligoadenylates is 2-100-fold higher than that of RNase A and human serum RNases, and they are markedly more thermolabile. In addition, their specific activities with different oligonucleotide substrates, optimal pHs, apparent K(m) values for substrates, and substrate specificities varied very much for different patients. These findings show that a pool of polyclonal RNA-hydrolyzing IgG, which may be relatively small or extremely large, is generated by the immune system of SLE patients.

Amylolytic activity of IgM and IgG antibodies from patients with multiple sclerosis

IgG and IgM antibodies from the sera of patients with multiple sclerosis (MS) were found to possess amylolytic activity hydrolyzing alpha-(1-->4)-glucosyl linkages of maltooligosaccharides, glycogen, and several artificial substrates. Individual IgM fractions isolated from 54 analyzed patients with the clinically definite diagnoses of MS had approximately three orders of magnitude higher specific amylolytic activity than that for healthy donors, whereas IgG from only a few patients had high amylolytic activity. Strict criteria were used to prove that the amylolytic activity of IgMs and IgGs is their intrinsic property and is not due to any enzyme contamination. Fab fragments produced from IgM and IgG fractions of the MS patients displayed the same amylolytic activity. IgMs from various patients demonstrated different modes of action in hydrolyzing maltooligosaccharides.

Catalytic DNA-and RNA-hydrolyzing antibodies from milk of healthy human mothers

Various catalytically active antibodies (Abs), or abzymes, have been detected recently in the sera of patients with autoimmune pathologies, in whom their presence is probably associated with autoimmunization. Normal humans are generally not considered to have abzymes, since no obvious immunizing factors are present. Here is shown by different methods that IgG from the milk of normal females possesses both DNase and RNase activities. The activities were also present in the IgG F(ab′)2 and Fab fragments.Affinity modification of IgG by the chemically reactive derivative of an oligonucleotide led to preferential modification of the L chain of IgG. After separation of the subunits by sodium dodecyl sulfate electrophoresis in a gel containing DNA, an in-gel assay showed DNase activity in the L chain. The L chain separated by affinity chromatography on DNA-cellulose was catalytically active. These findings speak in favor of the generation of cat alytic Abs by the immune system of healthy mothers. It is known that the treatment of adults with DNases and RNases offers protection from viral and bacterial diseases. Since breast milk protects the infants from infec tions until the immune system is developed, this raises the possibility that catalytic Abs like nucleases, may possess a protective role.