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Georgy A Nevinsky - One of the best experts on this subject based on the ideXlab platform.
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secretory immunoglobulin a from human milk hydrolyzes microrna
Journal of Dairy Science, 2020Co-Authors: Ivan Yu Kompaneets, Valentina N Uneva, Evgeny A Ermakov, Sergey E Sedykh, Georgy A NevinskyAbstract:ABSTRACT For breast-fed infants, human milk is a source of various nutrients (e.g., proteins, peptides, antibodies) and bioactive components that promote neonatal growth and protect infants from viral and bacterial infection. Moreover, in terms of infant nutrition and protection the functions of many human milk components are very different from those of blood and other biological fluids of healthy adults. For example, catalytic antibodies (“Abzymes”) with synthetic activities (protein, oligosaccharide, and lipid kinase activities) have been found in human breast milk that are absent in the blood of healthy people. Abzymes with hydrolyzing functions have been detected not only in milk, but also in the blood of patients with autoimmune diseases. Obviously, feeding newborns human milk has a very specific role and it is a unique aspect of mammalian nutrition. Ribonuclease and DNase autoantibodies or Abzymes are found in milk and blood of lactating women, but not in blood sera of healthy men and nonpregnant woman. Here, we present the first evidence that human milk secretory IgA molecules (sIgA) can effectively hydrolyze ribooligonucleotide containing 23 different bases [(pN)23 ribooligonucleotides] and 4 microRNAs: miR-9-5p, miR-219-2-3p, miR-137, and miR-219a-5p. Ribonuclease activity is an inherent property of sIgAs. We showed that 7 individual sIgAs hydrolyzed the ribooligonucleotides (pA)23, (pU)23, and (pC)23 nonspecifically and with comparable efficiency, whereas hydrolysis of the 4 microRNAs by sIgAs was site-specific. Sites of hydrolysis of 4 microRNAs by IgG from blood of patients with schizophrenia have been previously identified. The sites of hydrolysis of 4 microRNAs by sIgA-Abzymes were very different from the previously identified sites of hydrolysis by IgG in patients with schizophrenia. In addition, in contrast to IgG, milk sIgAs efficiently hydrolyzed microRNAs in their loop and duplex regions.
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affinity and catalytic heterogeneity and metal dependence of polyclonal myelin basic protein hydrolyzing iggs from sera of patients with systemic lupus erythematosus
Journal of Molecular Recognition, 2011Co-Authors: Anna M Bezuglova, Georgy A Nevinsky, Valentina N Buneva, Ludmila P Konenkova, Boris M DoroninAbstract: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: Ca2+ > Mg2+ ≥ Co2+ ≥ Fe2+ ≥ Ni2+ ≥ Zn2+ ≥ Cu2+ ≥ Mn2+. Combinations of Ca2+ + Mg2+ and Ca2+ + Co2 lead to a significant increase in the antibody proteolytic activity as compared with Ca2+, Co2+, or Mg2+ 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. Copyright © 2011 John Wiley & Sons, Ltd.
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natural catalytic antibodies in norm autoimmune viral and bacterial diseases
The Scientific World Journal, 2010Co-Authors: Georgy A Nevinsky, Valentina N BunevaAbstract:In human patients with autoimmune, viral, and bacterial diseases, the generation of antibodies (Abs) to foreign antigens and/or autoantibodies to self-antigens usually occurs. Some Abs with different catalytic activities (Abzymes, Abzs) may be induced spontaneously by primary antigens and can have characteristics of the primary antigen, including the catalytic activity of idiotypic and/or anti-idiotypic Abs. Healthy humans usually do not develop Abzs or their activities are low, often on the borderline of sensitivity of the detection methods. Detection of Abzs was shown to be the earliest indicator of development of different autoimmune diseases (ADs). At the early stages of ADs, the repertoire of Abzs is usually relatively narrow, but it greatly expands with the progress of the disease, leading to the generation of catalytically diverse Abzs with different activities and functions. Some Abzs are cytotoxic and can play an important negative role in the pathogenesis of ADs, while positive roles have been proposed for other Abzs. Abzs with some low activities can temporarily be present in the blood of patients in the course of viral and bacterial diseases, but their activity increases significantly if these infections stimulate development of ADs. A significant increase in the relative Abz activities associated with a specific reorganization of the immune system, including changes in the differentiation and proliferation of bone marrow hematopoietic stem cells and lymphocyte proliferation in different organs. Different mechanisms of Abz production can be proposed for healthy externally immunized and for autoimmune mammals during the development of pathology.
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antibodies against pancreatic ribonuclease a hydrolyze rna and dna
International Immunology, 2008Co-Authors: Michael A Krasnorutskii, Valentina N Buneva, Georgy A NevinskyAbstract:The sera of patients with autoimmune (AI) diseases contain antibodies with DNase and RNase activities. We have shown for the first time that immunization of healthy rabbits with RNase A conjugated with BSA produces a better immune response than immunization with pure RNase and induced IgGs with RNase and DNase activities, which were intrinsic properties of IgGs, while polyclonal IgGs (pIgGs) from non-immunized rabbits and animals immunized with BSA were catalytically inactive. It was shown that 74-85% of the total IgG DNase and RNase activities belongs to anti-idiotypic antibodies to RNase A (0.6-0.8% of total pIgGs), while 15-26% of the activities cannot interact with Sepharose-bearing antibodies against RNase A and may be antibodies to nucleic acids bound to RNase. Affinity chromatography on DNA-cellulose separated catalytic IgGs into several antibody subfractions demonstrating only DNase or RNase activity or hydrolyzing RNA faster than DNA. Our data suggest that a fraction of Abzymes (or catalytically active antibodies) from AI patients hydrolyzing both DNA and RNA may be antibodies against RNase or its complexes with other proteins.
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catalytic antibodies in healthy humans and patients with autoimmune and viral diseases
Journal of Cellular and Molecular Medicine, 2003Co-Authors: Georgy A Nevinsky, Valentina N BunevaAbstract: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.
Gui Min Luo - One of the best experts on this subject based on the ideXlab platform.
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a selenium containing single chain Abzyme with potent antioxidant activity
FEBS Journal, 2003Co-Authors: Delin You, Gui Min Luo, Tong Shu Yang, Xiaojun Ren, Yan Xue, Jia Cong ShenAbstract:Reactive oxygen species (ROS) are products of normal metabolic activities and are thought to be the cause of many diseases. A selenium-containing single-chain Abzyme 2F3 (Se-2F3-scFv) that imitates glutathione peroxidase has been produced which has the capacity to remove ROS. To evaluate the antioxidant ability of Se-2F3-scFv, we constructed a ferrous sulfate/ascorbate (Vc/Fe2+)-induced mitochondrial damage model system and investigated the capacity of Se-2F3-scFv to protect mitochondria from oxidative damage. Se-2F3-scFv markedly decreased mitochondrial swelling, inhibited lipid peroxidation, and maintained the activity of cytochrome c oxidase, in comparison with Ebselen, a well-studied glutathione peroxidase mimic, indicating that Se-2F3-scFv has potential for treating diseases mediated by ROS.
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preparation of a selenium containing single chain Abzyme with glutathione peroxidase activity by protein engineering
Chinese Journal of Biologicals, 2001Co-Authors: Shujuan Gao, Gui Min Luo, Yan Zhang, Xiaojun Ren, Delin You, Yuanming Luo, Ganglin Yan, Hualiang Huang, Zi LiuAbstract:Objective To prepare a selenium-containing single-chain Abzyme with glutathione peroxidase(GPX)activity.Methods The genes were amplified from the heavy(VH)and light(VL)chain variable regions of hybridoma cell strain 2F3 by RT-PCR and identified by DNA sequencing. The recombinant plasmid pTMF-scFv was constructed with the genes and transformed to E. coli ,and expressed product was purified by Co2+ -IMAC affinity chromatography. A selenium-containing single-chain Abzyme with glutathione peroxidase activity was obtained by the renaturation and chemical mutagenesis of the purified expressed product. Results SDS-PAGE showed that the expressed scFv existed in the form of inclusion body with a molecular weight of 30000.The scFv expressed in E. coli .1M109(DE3) ,B121(DE3)and BL21(coden plus)contained about 5% - 10% ,15% - 20% and 25% - 30% of total protein respectively.The GPX activity of the selenium-containing single-chain Abzyme( 3400U/μmol) was close to that of natural GPX enzyme. Conclusion The study laid a foundation of industrial production of selenium-containing single-chain Abzyme with glutathione peroxidase activity.
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protection of myocardial mitochondria against oxidative damage by selenium containing Abzyme m4g3
Applied Biochemistry and Biotechnology, 1999Co-Authors: Gui Min Luo, Li Zhou, Tong Shu YangAbstract:Selenium-containing Abzyme (m4G3) was prepared and its protection of myocardial mitochondria against oxidative damage was studied using the swelling of mitochondria, quantity of lipid peroxidation products, and change in cytochrome-c oxidase activity as a measure of mitochondrial damage. The results showed that m4G3 could inhibit mitochondrial damage caused by the hypoxanthine-xanthine oxidase system in vitro. Electronic spin resonance (ESR) studies demonstrated that m4G3 could decrease the amount of free radicals generated in the damage system.
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a selenium containing Abzyme the activity of which surpassed the level of native glutathione peroxidasea
Annals of the New York Academy of Sciences, 1998Co-Authors: Gui Min Luo, Lan Ding, Zhi Liu, Tong Shu YangAbstract:Using two different glutathione derivatives as hapten, we have prepared two Abzymes, which display glutathione peroxidase (GPX) activity. Their GPX activities are 0.2 and 1.6 times that of natural GPX from rabbit liver, respectively. Selenium content analysis indicates that the activity difference between the two Abzymes is possibly attributed to the conformation difference of the Abzymes.
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construction of scfv gene of the Abzyme with glutathione peroxidase catalytic activity
Chinese journal of veterinary science, 1998Co-Authors: Shuzhang Feng, Gui Min Luo, Zi Liu, Xuejun Guo, Guoli Zhang, Meijuan GaoAbstract:Following a chemical mutation, the McAb secreted by mouse hybridoma 5C_(9) showed a higher catalytic activity with its activity being 3.6 times as high as that of native glutathione peroxidase from rabbit liver. ScFv gene of the Abzyme was successfully constructed successively by total RNA and mRNA isolation, cDNA synthesis, V_(H) and V_(L) PCR amplification and assembly.
Valentina N Buneva - One of the best experts on this subject based on the ideXlab platform.
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changes in different parameters lymphocyte proliferation and hematopoietic progenitor colony formation in eae mice treated with myelin oligodendrocyte glycoprotein
Journal of Cellular and Molecular Medicine, 2016Co-Authors: Vasilii B Doronin, Valentina N Buneva, Taisiya A Parkhomenko, Alexey Korablev, L B Toporkova, Julia A Lopatnikova, Alina A Alshevskaja, Sergei V Sennikov, Thomas BuddeAbstract:Myelin oligodendrocyte glycoprotein (MOG) is an antigen of the myelin sheath, which may trigger immune cell responses and the production of auto-antibodies in multiple sclerosis (MS). In this study, we used MOG(35-55) -induced experimental autoimmune encephalomyelitis (EAE), a model of human MS, to assess the production of catalytically active immunoglobulin G (IgG) antibodies or Abzymes which have been shown to be present in sera of patients with several autoimmune diseases. Here, we show that IgGs from the sera of control C57BL/6 mice are catalytically inactive. During development of EAE, a specific reorganization of the immune system of mice occurred leading to a condition which was associated with the generation of catalytically active IgGs hydrolysing DNA, myelin basic protein (MBP) and MOG which was associated with increased proteinuria, changes in differentiation of mice bone marrow hematopoietic stem cells (HSCs) and an increase in proliferation of lymphocytes in bone marrow, spleen and thymus as well as a significant suppression of cell apoptosis in these organs. The strongest alterations were found in the early disease phase (18-24 days after immunization) and were less pronounced in later EAE stages (40 days after EAE induction). We conclude that a significant increase in DNase and proteolytic activities of antibodies may be considered the earliest statistically significant marker of MOG-induced EAE in mice. The possible differences in immune system reorganizations during preclinical phases of the disease, acute and late EAE, leading to production of different auto-antibodies and Abzymes as well other changes are discussed.
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affinity and catalytic heterogeneity and metal dependence of polyclonal myelin basic protein hydrolyzing iggs from sera of patients with systemic lupus erythematosus
Journal of Molecular Recognition, 2011Co-Authors: Anna M Bezuglova, Georgy A Nevinsky, Valentina N Buneva, Ludmila P Konenkova, Boris M DoroninAbstract: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: Ca2+ > Mg2+ ≥ Co2+ ≥ Fe2+ ≥ Ni2+ ≥ Zn2+ ≥ Cu2+ ≥ Mn2+. Combinations of Ca2+ + Mg2+ and Ca2+ + Co2 lead to a significant increase in the antibody proteolytic activity as compared with Ca2+, Co2+, or Mg2+ 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. Copyright © 2011 John Wiley & Sons, Ltd.
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natural catalytic antibodies in norm autoimmune viral and bacterial diseases
The Scientific World Journal, 2010Co-Authors: Georgy A Nevinsky, Valentina N BunevaAbstract:In human patients with autoimmune, viral, and bacterial diseases, the generation of antibodies (Abs) to foreign antigens and/or autoantibodies to self-antigens usually occurs. Some Abs with different catalytic activities (Abzymes, Abzs) may be induced spontaneously by primary antigens and can have characteristics of the primary antigen, including the catalytic activity of idiotypic and/or anti-idiotypic Abs. Healthy humans usually do not develop Abzs or their activities are low, often on the borderline of sensitivity of the detection methods. Detection of Abzs was shown to be the earliest indicator of development of different autoimmune diseases (ADs). At the early stages of ADs, the repertoire of Abzs is usually relatively narrow, but it greatly expands with the progress of the disease, leading to the generation of catalytically diverse Abzs with different activities and functions. Some Abzs are cytotoxic and can play an important negative role in the pathogenesis of ADs, while positive roles have been proposed for other Abzs. Abzs with some low activities can temporarily be present in the blood of patients in the course of viral and bacterial diseases, but their activity increases significantly if these infections stimulate development of ADs. A significant increase in the relative Abz activities associated with a specific reorganization of the immune system, including changes in the differentiation and proliferation of bone marrow hematopoietic stem cells and lymphocyte proliferation in different organs. Different mechanisms of Abz production can be proposed for healthy externally immunized and for autoimmune mammals during the development of pathology.
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antibodies against pancreatic ribonuclease a hydrolyze rna and dna
International Immunology, 2008Co-Authors: Michael A Krasnorutskii, Valentina N Buneva, Georgy A NevinskyAbstract:The sera of patients with autoimmune (AI) diseases contain antibodies with DNase and RNase activities. We have shown for the first time that immunization of healthy rabbits with RNase A conjugated with BSA produces a better immune response than immunization with pure RNase and induced IgGs with RNase and DNase activities, which were intrinsic properties of IgGs, while polyclonal IgGs (pIgGs) from non-immunized rabbits and animals immunized with BSA were catalytically inactive. It was shown that 74-85% of the total IgG DNase and RNase activities belongs to anti-idiotypic antibodies to RNase A (0.6-0.8% of total pIgGs), while 15-26% of the activities cannot interact with Sepharose-bearing antibodies against RNase A and may be antibodies to nucleic acids bound to RNase. Affinity chromatography on DNA-cellulose separated catalytic IgGs into several antibody subfractions demonstrating only DNase or RNase activity or hydrolyzing RNA faster than DNA. Our data suggest that a fraction of Abzymes (or catalytically active antibodies) from AI patients hydrolyzing both DNA and RNA may be antibodies against RNase or its complexes with other proteins.
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catalytic antibodies in healthy humans and patients with autoimmune and viral diseases
Journal of Cellular and Molecular Medicine, 2003Co-Authors: Georgy A Nevinsky, Valentina N BunevaAbstract: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.
Ikuo Fujii - One of the best experts on this subject based on the ideXlab platform.
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directed evolution governed by controlling the molecular recognition between an Abzyme and its haptenic transition state analog
Journal of Immunological Methods, 2004Co-Authors: Naoko Takahashiando, Hiroyuki Kakinuma, Ikuo Fujii, Yoshisuke NishiAbstract:Abstract The catalytic antibody, 6D9, was subjected to directed evolution in the phage-display system using two structurally related transition–state analogs (TSAs) for panning. One analog, TSA 3, was originally used for immunization, and the other, TSA 4, a derivative of TSA 3, was designed to optimize the differential affinity for the transition state relative to the ground state so as to provide variants with improved reaction rates. We previously reported that by panning with TSA 4, we could obtain variants with highly improved catalytic rate enhancement (kcat/kuncat), and Tyr (L27e) seemed to play a key role in stabilizing the transition–state structure [Nat. Biotechnol. 19 (2001) 563]. Here, we examined in detail a large number of the variants selected by these haptens, in order to elucidate the mechanism of the directed evolution driven by them. ELISA with 3- and 4-bovine serum albumin (BSA) showed that variants selected by these TSAs exhibited distinct binding patterns. All the variants whose rate enhancement was greater than five-fold of that of 6D9 had Tyr (L27e) and were obtained from the library panned with TSA 4, but not from the library panned with TSA 3. Kinetic studies showed that TSA 4 could efficiently select variants with increased differential binding affinity for the transition state relative to the ground state, and these variants exhibited improved rate enhancements. This study verified the difference of in vitro evolution driven by the two structurally related TSAs and stresses the importance of designing an appropriate hapten for panning.
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Selective chemotherapeutic strategies using catalytic antibodies: a common pro-moiety for antibody-directed Abzyme prodrug therapy.
Journal of immunological methods, 2002Co-Authors: Hiroyuki Kakinuma, Ikuo Fujii, Yoshisuke NishiAbstract:Prodrug activation by catalytic antibodies (Abzymes) conjugated with anti-tumor antibodies, called antibody-directed Abzyme prodrug therapy (ADAPT), has been proposed as a strategy for site-specific drug delivery systems for anti-tumor drugs. The delivery of Abzymes is achieved by making a bi-specific antibody with a monovalent catalytic antibody and a monovalent binding antibody. To achieve ADAPT, we focused on specific requirements for prodrugs and catalytic antibodies, the stability of the prodrugs against natural enzymes, and the applicability of Abzymes for a wide range of prodrugs. Attention was paid to the design of a pro-moiety rather than a parent drug. As a common pro-moiety, we chose vitamin B(6), because the bulky vitamin B(6) esters are relatively stable against hydrolytic enzymes in serum. We have generated catalytic antibodies by immunization of a vitamin B(6) phosphonate transition state analog. The elicited antibodies were found to hydrolyze several anti-cancer and anti-inflammatory prodrugs with the vitamin B(6) pro-moiety. Finally, we evaluated antibody-catalyzed prodrug activation by examining the growth inhibition of human cervical cancer (HeLa) cells with the vitamin B(6) ester of butyric acid. These results suggest that the pro-moiety of vitamin B(6) ester is stable enough to resist natural enzymes in serum and is removed by the tailored catalytic antibodies. The combination of catalytic antibodies and prodrugs masked with vitamin B(6) would allow hydrophobic and highly toxic drugs to be used.
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in vitro Abzyme evolution to optimize antibody recognition for catalysis
Nature Biotechnology, 2001Co-Authors: Naoko Takahashi, Hiroyuki Kakinuma, Yoshisuke Nishi, Lidong Liu, Ikuo FujiiAbstract:Enzymes have evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreasing the affinity for the ground state. To evolve Abzymes toward higher catalytic activity, we have reconstructed an enzyme-evolutionary process in vitro. Thus, a phage-displayed combinatorial library from a hydrolytic Abzyme, 6D9, generated by the conventional in vivo method with immunization of the transition-state analog (TSA), was screened against a newly devised TSA to optimize the differential affinity for the transition state relative to the ground state. The library format successfully afforded evolved variants with 6- to 20-fold increases in activity (kcat) as compared with 6D9. Structural analysis revealed an advantage of the in vitro evolution over the in vivo evolution: an induced catalytic residue in the evolved Abzyme arises from double mutations in one codon, which rarely occur in somatic hypermutation in the immune response.
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a structural basis for transition state stabilization in antibody catalyzed hydrolysis crystal structures of an Abzyme at 1 8 a resolution
Journal of Molecular Biology, 1998Co-Authors: Ole Kristensen, Fujie Tanaka, Dmitry G Vassylyev, Kosuke Morikawa, Ikuo FujiiAbstract:Abstract The three-dimensional structure of a catalytic antibody, 6D9, has been solved as a complex with a transition state analog. The structure was determined from two different crystal forms, and was refined at a resolution of 1.8 A. The antibody 6D9, which was induced by immunization with the phosphonate transition state analog 3 , hydrolyzes a prodrug of chloramphenicol monoester 1 to generate the parent drug 2 . The kinetic studies have shown that the antibody is catalytic by virtue of the theoretical relationship between the affinity for the transition state and the catalytic efficiency ( k cat / k uncat = K S / K TSA ). The crystal structure makes it possible to visualize the theoretical relationship. A side-chain (N ϵ ) of His L27D is placed in a key position to make a hydrogen bond to the phosphonate oxygen of the transition state analog with a distance of 2.72 A, suggesting a hydrogen bond to the oxyanion developing in the transition state of the hydrolysis. There are no catalytic residues, other than the histidine, around the phosphonate moiety. In addition, in the antibody-hapten complex, the hapten bears a folded conformation and the two stacked aromatic rings are buried deep in the antigen-combining site through aromatic-aromatic interaction with Trp H100I and Tyr H58 . The conformation of the bound hapten suggests that the antibody binds the substrate to change the conformation of the ester moiety to a thermodynamically unstable E -form, thereby making it easy for the substrate to reach the transition-state during catalysis. These observations reveal that the catalytic mechanism is explained purely on the basis of the stabilization of the transition state. The refined high resolution structures reported here are envisaged to have an impact on the understanding of other hydrolytic antibodies, since their haptens share some unique features with the hapten used in this study.
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pyridoxal mediated Abzyme system for aldol and retro aldol reactions
Tetrahedron Letters, 1998Co-Authors: Fujie Tanaka, Motoko Oda, Ikuo FujiiAbstract:Abstract A pyridoxal-mediated Abzyme system for aldol and retro-aldol reactions is demonstrated. Antibody 1OH2 catalyzes the aldol and retro-aldol reactions of 4-acetamidobenzaldehyde and glycine to β-hydroxy α-amino acid, using pyridoxal 5′-phosphate as a cofactor.
Jia Cong Shen - One of the best experts on this subject based on the ideXlab platform.
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a selenium containing single chain Abzyme with potent antioxidant activity
FEBS Journal, 2003Co-Authors: Delin You, Gui Min Luo, Tong Shu Yang, Xiaojun Ren, Yan Xue, Jia Cong ShenAbstract:Reactive oxygen species (ROS) are products of normal metabolic activities and are thought to be the cause of many diseases. A selenium-containing single-chain Abzyme 2F3 (Se-2F3-scFv) that imitates glutathione peroxidase has been produced which has the capacity to remove ROS. To evaluate the antioxidant ability of Se-2F3-scFv, we constructed a ferrous sulfate/ascorbate (Vc/Fe2+)-induced mitochondrial damage model system and investigated the capacity of Se-2F3-scFv to protect mitochondria from oxidative damage. Se-2F3-scFv markedly decreased mitochondrial swelling, inhibited lipid peroxidation, and maintained the activity of cytochrome c oxidase, in comparison with Ebselen, a well-studied glutathione peroxidase mimic, indicating that Se-2F3-scFv has potential for treating diseases mediated by ROS.
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some physicochemical and enzymatic properties of selenium containing Abzyme
Biochemical and Biophysical Research Communications, 1994Co-Authors: Zhen Qi Zhu, Lan Ding, Gui Min Luo, Zhi Liu, Qi An Sun, Tong Shu Yang, Jia Cong ShenAbstract:Abstract We successfully prepared the Se-containing Abzyme (Se-Abzyme) with glutathione peroxidase (GPX) activity and futher studied its physicochemical and enzymic properties and stabilities. Data showed that the isoelectric point of the Abzyme was 6.95-7.08, and its molecular weight was 158 KD. The ranges of optimum pH and temperature of the Se-Abzyme were wider than the native GPX. The store stability of the Abzyme was higher than the native GPX. The Se content in the Abzyme was found to be 5 mol Se/mol Abzyme by X-ray photoelectron spectrum, and binding constant 1.11×10 7 M −1 by using ELISA method. The Se-Abzyme was inhibited competitively by dithiobis (2-nitrobenzoic acid) (DTNB), and inhibition constant was determined to be 1.25×10 −3 M −1 .
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generation of selenium containing Abzyme by using chemical mutation
Biochemical and Biophysical Research Communications, 1994Co-Authors: Gui Min Luo, Zhen Qi Zhu, Lan Ding, Zhi Liu, Qi An Sun, Tong Shu Yang, Gui Gao, Jia Cong ShenAbstract:Abstract A new strategy for generating Abzyme was developed. Glutathione peroxidase (GPX, EC 1.11.1.9 ) is one of the important members of antioxidation enzyme system; it catalyzes the reductions of a variety of hydroperoxides in presence of glutathione (GSH). We have first prepared the monoclonal antibody (McAb) with GSH binding sites, then incorporated GPX catalytic group selenocystein (SeCys) into the antibody combining sites by using chemical mutation. Thus the mutated antibody displays high GPX activity, which approaches the magnitude level of native GPX, exhibits the kinetic behavior similar to native GPX, and has some advantages over native GPX.