The Experts below are selected from a list of 7248 Experts worldwide ranked by ideXlab platform
Taizo Uda - One of the best experts on this subject based on the ideXlab platform.
-
A unique method for Antibody to possess the Catalytic function (3rd report)
The FASEB Journal, 2018Co-Authors: Taizo Uda, Hiroaki Taguchi, Yuko Akiyoshi, Emi HifumiAbstract:Since a natural Catalytic Antibody found at 1989, many mouse- and human-type of Catalytic antibodies cleaving the targeting antigens such as peptides, nucleotides, viruses and bacterial proteins, m...
-
a novel method of preparing the monoform structure of Catalytic Antibody light chain
The FASEB Journal, 2016Co-Authors: Emi Hifumi, Mitsue Arakawa, Shingo Matsumoto, Hiroki Nakashima, Shogo Itonaga, Yoshiki Katayama, Ryuichi Kato, Taizo UdaAbstract:Along with the development of Antibody drugs and Catalytic antibodies, the structural diversity (heterogeneity) of antibodies has been given attention. For >20 yr, detailed studies on the subject have not been conducted, because the phenomenon presents many difficult and complex problems. Structural diversity provides some (or many) isoforms of an Antibody distinguished by different charges, different molecular sizes, and modifications of amino acid residues. For practical use, the Antibody and the subunits must have a defined structure. In recent work, we have found that the copper (Cu) ion plays a substantial role in solving the diversity problem. In the current study, we used several Catalytic Antibody light chains to examine the effect of the Cu ion. In all cases, the different electrical charges of the molecule converged to a single charge, giving 1 peak in cation-exchange chromatography, as well as a single spot in 2-dimensional gel electrophoresis. The Cu-binding site was investigated by using mutagenesis, ultraviolet-visible spectroscopy, atomic force microscope analysis, and molecular modeling, which suggested that histidine and cysteine residues close to the C-terminus are involved with the binding site. The constant region domain of the Antibody light chain played an important role in the heterogeneity of the light chain. Our findings may be a significant tool for preparing a single defined, not multiple, isoform structure.
-
biochemical features and antiviral activity of a monomeric Catalytic Antibody light chain 23d4 against influenza a virus
The FASEB Journal, 2015Co-Authors: Emi Hifumi, Mitsue Arakawa, Shingo Matsumoto, Yoshiki Katayama, Tatsuhiro Yamamoto, Taizo UdaAbstract:Catalytic antibodies have exhibited interesting functions against some infectious viruses such as HIV, rabies virus, and influenza virus in vitro as well as in vivo. In some cases, a Catalytic Antibody light chain takes on several structures from the standpoint of molecular size (monomer, dimer, etc.) and/or isoelectronic point. In this study, we prepared a monomeric 23D4 light chain by mutating the C-terminal Cys to Ala of the wild-type. The mutated 23D4 molecule took a simple monomeric form, which could hydrolyze synthetic 4-methyl-coumaryl-7-amide substrates and a plasmid DNA. Because the monomeric 23D4 light chain suppressed the infection of influenza virus A/Hiroshima/37/2001 in an in vitro assay, the corresponding experiments were conducted in vivo, after the virus strain (which was taken from a human patient) was successfully adapted into BALB/cN Sea mice. In the experiments, a mixture of the monomeric 23D4 and the virus was nasally administered 1) with preincubation and 2) without preincubation. A...
-
biochemical features of a Catalytic Antibody light chain 22f6 prepared from human lymphocytes
Journal of Biological Chemistry, 2013Co-Authors: Emi Hifumi, Naoko Fujimoto, Mitsue Arakawa, Shingo Matsumoto, Eri Saito, Nobuyuki Kobayashi, Taizo UdaAbstract:Human Antibody light chains belonging to subgroup II of germ line genes were amplified by a seminested PCR technique using B-lymphocytes taken from a human adult infected with influenza virus. Each gene of the human light chains was transferred into the Escherichia coli system. The recovered light chain was highly purified using a two-step purification system. Light chain 22F6 showed interesting Catalytic features. The light chain cleaved a peptide bond of synthetic peptidyl-4-methyl-coumaryl-7-amide (MCA) substrates, such as QAR-MCA and EAR-MCA, indicating amidase activity. It also hydrolyzed a phosphodiester bond of both DNA and RNA. From the analysis of amino acid sequences and molecular modeling, the 22F6 light chain possesses two kinds of active sites as amidase and nuclease in close distances. The 22F6 Catalytic light chain could suppress the infection of influenza virus type A (H1N1) of Madin-Darby canine kidney cells in an in vitro assay. In addition, the Catalytic light chain clearly inhibited the infection of the influenza virus of BALB/c mice via nasal administration in an in vivo assay. In the experiment, the titer in the serum of the mice coinfected with the 22F6 light chain and H1N1 virus became considerably lowered compared with that of 22F6-non-coinfected mice. Note that the Catalytic light chain was prepared from human peripheral lymphocyte and plays an important role in preventing infection by influenza virus. Considering the fact that the human light chain did not show any acute toxicity for mice, our procedure developed in this study must be unique and noteworthy for developing new drugs.
-
highly efficient method of preparing human Catalytic Antibody light chains and their biological characteristics
The FASEB Journal, 2012Co-Authors: Emi Hifumi, Akira Nishizono, Eijiro Honjo, Naoko Fujimoto, Mitsue Arakawa, Taizo UdaAbstract:The ultimate goal of Catalytic Antibody research is to develop new patient therapies that use the advantages offered by human Catalytic antibodies. The establishment of a high-throughput method for obtaining valuable candidate Catalytic antibodies must be accelerated to achieve this objective. In this study, based on our concept that we can find Antibody light chains with a high probability of success if they include a serine protease-like Catalytic triad composed of Ser, His, and Asp on a variable region of the Antibody structure, we amplified and cloned DNAs encoding human Antibody light chains from germline genes of subgroup II by seminested PCR using two primer sets designed for this purpose. Seven DNA fragments encoding light chains in 17 clones were derived from germline gene A18b, 6 DNA fragments from A3/A19, 2 DNA fragments from A17, and a clone DNA fragment from A5 and O11/O1. All light chains expressed in Escherichia coli and highly purified under nondenaturing conditions exhibited amidolytic ac...
Emi Hifumi - One of the best experts on this subject based on the ideXlab platform.
-
A unique method for Antibody to possess the Catalytic function (3rd report)
The FASEB Journal, 2018Co-Authors: Taizo Uda, Hiroaki Taguchi, Yuko Akiyoshi, Emi HifumiAbstract:Since a natural Catalytic Antibody found at 1989, many mouse- and human-type of Catalytic antibodies cleaving the targeting antigens such as peptides, nucleotides, viruses and bacterial proteins, m...
-
a novel method of preparing the monoform structure of Catalytic Antibody light chain
The FASEB Journal, 2016Co-Authors: Emi Hifumi, Mitsue Arakawa, Shingo Matsumoto, Hiroki Nakashima, Shogo Itonaga, Yoshiki Katayama, Ryuichi Kato, Taizo UdaAbstract:Along with the development of Antibody drugs and Catalytic antibodies, the structural diversity (heterogeneity) of antibodies has been given attention. For >20 yr, detailed studies on the subject have not been conducted, because the phenomenon presents many difficult and complex problems. Structural diversity provides some (or many) isoforms of an Antibody distinguished by different charges, different molecular sizes, and modifications of amino acid residues. For practical use, the Antibody and the subunits must have a defined structure. In recent work, we have found that the copper (Cu) ion plays a substantial role in solving the diversity problem. In the current study, we used several Catalytic Antibody light chains to examine the effect of the Cu ion. In all cases, the different electrical charges of the molecule converged to a single charge, giving 1 peak in cation-exchange chromatography, as well as a single spot in 2-dimensional gel electrophoresis. The Cu-binding site was investigated by using mutagenesis, ultraviolet-visible spectroscopy, atomic force microscope analysis, and molecular modeling, which suggested that histidine and cysteine residues close to the C-terminus are involved with the binding site. The constant region domain of the Antibody light chain played an important role in the heterogeneity of the light chain. Our findings may be a significant tool for preparing a single defined, not multiple, isoform structure.
-
biochemical features and antiviral activity of a monomeric Catalytic Antibody light chain 23d4 against influenza a virus
The FASEB Journal, 2015Co-Authors: Emi Hifumi, Mitsue Arakawa, Shingo Matsumoto, Yoshiki Katayama, Tatsuhiro Yamamoto, Taizo UdaAbstract:Catalytic antibodies have exhibited interesting functions against some infectious viruses such as HIV, rabies virus, and influenza virus in vitro as well as in vivo. In some cases, a Catalytic Antibody light chain takes on several structures from the standpoint of molecular size (monomer, dimer, etc.) and/or isoelectronic point. In this study, we prepared a monomeric 23D4 light chain by mutating the C-terminal Cys to Ala of the wild-type. The mutated 23D4 molecule took a simple monomeric form, which could hydrolyze synthetic 4-methyl-coumaryl-7-amide substrates and a plasmid DNA. Because the monomeric 23D4 light chain suppressed the infection of influenza virus A/Hiroshima/37/2001 in an in vitro assay, the corresponding experiments were conducted in vivo, after the virus strain (which was taken from a human patient) was successfully adapted into BALB/cN Sea mice. In the experiments, a mixture of the monomeric 23D4 and the virus was nasally administered 1) with preincubation and 2) without preincubation. A...
-
biochemical features of a Catalytic Antibody light chain 22f6 prepared from human lymphocytes
Journal of Biological Chemistry, 2013Co-Authors: Emi Hifumi, Naoko Fujimoto, Mitsue Arakawa, Shingo Matsumoto, Eri Saito, Nobuyuki Kobayashi, Taizo UdaAbstract:Human Antibody light chains belonging to subgroup II of germ line genes were amplified by a seminested PCR technique using B-lymphocytes taken from a human adult infected with influenza virus. Each gene of the human light chains was transferred into the Escherichia coli system. The recovered light chain was highly purified using a two-step purification system. Light chain 22F6 showed interesting Catalytic features. The light chain cleaved a peptide bond of synthetic peptidyl-4-methyl-coumaryl-7-amide (MCA) substrates, such as QAR-MCA and EAR-MCA, indicating amidase activity. It also hydrolyzed a phosphodiester bond of both DNA and RNA. From the analysis of amino acid sequences and molecular modeling, the 22F6 light chain possesses two kinds of active sites as amidase and nuclease in close distances. The 22F6 Catalytic light chain could suppress the infection of influenza virus type A (H1N1) of Madin-Darby canine kidney cells in an in vitro assay. In addition, the Catalytic light chain clearly inhibited the infection of the influenza virus of BALB/c mice via nasal administration in an in vivo assay. In the experiment, the titer in the serum of the mice coinfected with the 22F6 light chain and H1N1 virus became considerably lowered compared with that of 22F6-non-coinfected mice. Note that the Catalytic light chain was prepared from human peripheral lymphocyte and plays an important role in preventing infection by influenza virus. Considering the fact that the human light chain did not show any acute toxicity for mice, our procedure developed in this study must be unique and noteworthy for developing new drugs.
-
highly efficient method of preparing human Catalytic Antibody light chains and their biological characteristics
The FASEB Journal, 2012Co-Authors: Emi Hifumi, Akira Nishizono, Eijiro Honjo, Naoko Fujimoto, Mitsue Arakawa, Taizo UdaAbstract:The ultimate goal of Catalytic Antibody research is to develop new patient therapies that use the advantages offered by human Catalytic antibodies. The establishment of a high-throughput method for obtaining valuable candidate Catalytic antibodies must be accelerated to achieve this objective. In this study, based on our concept that we can find Antibody light chains with a high probability of success if they include a serine protease-like Catalytic triad composed of Ser, His, and Asp on a variable region of the Antibody structure, we amplified and cloned DNAs encoding human Antibody light chains from germline genes of subgroup II by seminested PCR using two primer sets designed for this purpose. Seven DNA fragments encoding light chains in 17 clones were derived from germline gene A18b, 6 DNA fragments from A3/A19, 2 DNA fragments from A17, and a clone DNA fragment from A5 and O11/O1. All light chains expressed in Escherichia coli and highly purified under nondenaturing conditions exhibited amidolytic ac...
Stephen J Benkovic - One of the best experts on this subject based on the ideXlab platform.
-
detection of a Catalytic Antibody species acylated at the active site by electrospray mass spectrometry
Biochemistry, 1995Co-Authors: Joseph F Krebs, Jon D Stewart, Gary Siuzdak, H J Dyson, Stephen J BenkovicAbstract:The chemical interactions between a Catalytic Antibody Fv fragment and ester substrates were examined using pneumatically assisted electrospray (ion spray) mass spectrometry. Upon addition of the p-nitrophenyl ester substrate to the Antibody fragment, an Antibody fragment species that represents approximately 8% of the total Fv concentration is clearly observed in the electrospray spectrum. The observed increase in molecular weight of the Fv fragment corresponds to the mass of the acyl group of the substrate. Formation of the acyl-Fv species is blocked by preincubation of the Antibody fragment with hapten inhibitor, suggesting that the acyl linkage involves a residue in the active site of the Antibody. The acyl-Fv species is not observed when the corresponding p-chlorophenyl ester substrate is used, indicating that the level of this species is dependent on the leaving group of the substrate. The acylated species is not observed for a site-directed mutant lacking Catalytic activity, His L91 Gln. The present results are consistent with modeling studies of the structure of the Fv fragment and provide strong confirmatory evidence for the multistep kinetic mechanism previously proposed for this Antibody.
-
site directed mutagenesis of a Catalytic Antibody an arginine and a histidine residue play key roles
Biochemistry, 1994Co-Authors: Jon D Stewart, Neil R. Thomas, Victoria A Roberts, Elizabeth D Getzoff, Stephen J BenkovicAbstract:Individual residues important for ligand binding and Catalytic activity were identified by computer modeling and investigated by site-directed mutagenesis for Catalytic Antibody 43C9, which accelerates amide hydrolysis by a factor of 10(6). On the basis of a computer model, Tyr L32, His L91, Arg L96, His H35, and Tyr H95 were chosen for replacement by site-directed mutagenesis. To facilitate these studies, an expression system was developed in which properly folded 43C9 single-chain Antibody was secreted from an engineered Escherichia coli host. Substitution of His L91 by Gln produced a mutant with no Catalytic activity, but whose affinities for ligands were nearly the same as those of the wild-type, identifying His L91 as the nucleophile that forms the acyl intermediate implicated by previous kinetic studies. Arg L96 is also critical for Catalytic activity and appears to function as a oxyanion hole for the tetrahedral transition states. Two substitutions for His H35 resulted in mutant proteins with no Catalytic activity as well as altered affinities for ligands, indicating an important structural role for this residue. Substitutions for Tyr L32 and Tyr H95 were made in an attempt to improve the Catalytic efficiency of 43C9. The results of these mutations allow us to propose a mechanism for 43C9-catalyzed hydrolysis: Substrate binding to 43C9 orients the scissile carbonyl group adjacent to both the His L91 and Arg L96 side chains. The imidazole of His L91 acts as a nucleophile, forming an acyl-Antibody intermediate that breaks down by hydroxide attack to afford the products and regenerate the catalyst.
-
Catalytic Antibody model and mutagenesis implicate arginine in transition state stabilization
Journal of Molecular Biology, 1994Co-Authors: Victoria A Roberts, Jon D Stewart, Stephen J Benkovic, Elizabeth D GetzoffAbstract:To probe the mechanism of the Catalytic Antibody NPN43C9, we have constructed a three-dimensional model of the NPN43C9 variable region using our Antibody structural database (ASD), which takes maximal advantage of immunoglobulin sequence and structural information. The ASD contains separately superimposed variable light and variable heavy chains, which reveal not only conserved backbone structure, but also structurally conserved side-chain conformations. The NPN43C9 model revealed that the guanidinium group of light chain Arg L96 was positioned at the bottom of the antigen-binding site and formed a salt bridge with the antigen's phosphonamidate group, which mimics the negatively charged, tetrahedral transition states in the hydrolysis reaction. Thus, the model predicts both binding and Catalytic functions for Arg L96, which previously had not been implicated in either. First, Arg L96 should enhance antigen binding by electrostatically complementing the negative charge of the antigen, which is buried upon complex formation. Second, Arg L96 should promote catalysis by electrostatically stabilizing the negatively charged transition states formed during catalysis. These hypotheses were tested experimentally by design and characterization of the R-L96-Q mutant, in which Arg L96 was replaced with Gln by site-directed mutagenesis. As predicted, antigen binding in the R-L96-Q mutant was decreased relative to that in the parent NPN43C9 Antibody, but binding of antigen fragments lacking the phosphonamidate group was retained. In addition, the R-L96-Q mutant had no detectable esterase activity. Thus, the computational model and experimental results together suggest a mechanism by which the Catalytic Antibody NPN43C9 stabilizes high-energy transition states during catalysis.
-
Catalytic antibodies: Perusing combinatorial libraries
Trends in biochemical sciences, 1994Co-Authors: Bruce A. Posner, Jeffrey A. Smiley, Irene Lee, Stephen J BenkovicAbstract:Combinatorial libraries are a promising alternative for isolating Catalytic antibodies produced by the immune system in response to the transition-state analog of a given reaction. Large, diverse panels of antibodies with high affinity for the transition-state analog can be isolated using screening or selection approaches. Furthermore, we have estimated that nucleotide sequences that bear close similarity to the sequence for a known Catalytic Antibody occur in combination at frequencies sufficient for their detection in such libraries.
-
an unexpectedly efficient Catalytic Antibody operating by ping pong and induced fit mechanisms
Science, 1991Co-Authors: Peter Wirsching, Kim D. Janda, Stephen J Benkovic, Jon A Ashley, Richard A. LernerAbstract:A transition state analogue was used to produce a mouse Antibody that catalyzes transesterification in water. The Antibody behaves as a highly efficient catalyst with a covalent intermediate and the characteristic of induced fit. While some features of the Catalytic pathway were programmed when the hapten was designed and reflect favorable substrate-Antibody interactions, other features are a manifestation of the chemical potential of Antibody diversity. The fact that antibodies recapitulate mechanisms and pathways previously thought to be a characteristic of highly evolved enzymes suggests that once an appropriate binding cavity is achieved, reaction pathways commensurate with the intrinsic chemical potential of proteins ensue.
Naoki Yamamoto - One of the best experts on this subject based on the ideXlab platform.
-
Mechanistic analysis of the phosphonate transition-state analogue-derived Catalytic and non-Catalytic Antibody.
Journal of biochemistry, 2007Co-Authors: Yoshisuke Nishi, Naoki Yamamoto, Kazuko Shimazaki, Naoko Takahashi-ando, Hiroyuki Kakinuma, Sun Jialin, Sergey N. Ruzheinikov, Tatyana A. Muranova, David W. Rice, Yasuhiro KajiharaAbstract:The esterolytic Catalytic Antibody (catAb) has the positive charged region interacting with the carbonyl group of the ester substrate. To examine how such a region interacts with the substrate, we compared the catAb with the non-Catalytic Antibody (non-catAb) for interaction with the non-cleavable amide substrate (a mimic of the ester substrate) and the two end products. Surface plasmon resonance (SPR) analysis revealed that the amide substrate gave the equivalent K d values for the two antibodies, whereas both the on-rate and off-rate of the catAb were five-times lower than those of the non-catAb. In agreement with SPR analysis, saturation transfer difference (STD) NMR spectroscopy detected the STD signals only between the catAb and one of the product, suggesting the slower exchange rates of the amide substrate in the catAb as compared with the mixing times, whereas it was not the case with the non-catAb. Transferred nuclear Overhauser effect NMR spectroscopy showed the negative signals for only between the non-catAb and the amide substrate or the product, again suggesting the lower off-rates of the catAb as compared with the mixing times. The decreased interaction rates should be the primary consequence of the positively charged region in the combining site in the catAb.
-
Catalytic Antibody light chain capable of cleaving a chemokine receptor ccr 5 peptide with a high reaction rate constant
Biotechnology and Bioengineering, 2004Co-Authors: Yukie Mitsuda, Kumi Tsuruhata, Hiroko Fujinami, E. Hifumi, Naoki YamamotoAbstract:A monoclonal Antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the Antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two Catalytic triad(s), composed of Asp1, Ser27a (or Ser27e), and His93 (or His27d), in the light chain of ECL2B-2. The three amino acid residues, Asp1, Ser27a, and His93, are identical to those of Catalytic Antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high Catalytic reaction rate constant (kcat) of 2.23 min−1, which is greater by factors of tens to hundreds than those of natural Catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no Catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide. © 2004 Wiley Periodicals, Inc.
-
Catalytic Antibody light chain capable of cleaving a chemokine receptor CCR‐5 peptide with a high reaction rate constant
Biotechnology and Bioengineering, 2004Co-Authors: Yukie Mitsuda, Kumi Tsuruhata, Hiroko Fujinami, E. Hifumi, Naoki YamamotoAbstract:A monoclonal Antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the Antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two Catalytic triad(s), composed of Asp1, Ser27a (or Ser27e), and His93 (or His27d), in the light chain of ECL2B-2. The three amino acid residues, Asp1, Ser27a, and His93, are identical to those of Catalytic Antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high Catalytic reaction rate constant (kcat) of 2.23 min−1, which is greater by factors of tens to hundreds than those of natural Catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no Catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide. © 2004 Wiley Periodicals, Inc.
-
Catalytic Antibody light chain capable of cleaving a chemokine receptor ccr 5 peptide with a high reaction rate constant
Biotechnology and Bioengineering, 2004Co-Authors: Yukie Mitsuda, Kumi Tsuruhata, Hiroko Fujinami, E. Hifumi, Naoki Yamamoto, Taizo UdaAbstract:A monoclonal Antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the Antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two Catalytic triad(s), composed of Asp(1), Ser(27a) (or Ser(27e)), and His(93) (or His(27d)), in the light chain of ECL2B-2. The three amino acid residues, Asp(1), Ser(27a), and His(93), are identical to those of Catalytic Antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high Catalytic reaction rate constant (k(cat)) of 2.23 min(-1), which is greater by factors of tens to hundreds than those of natural Catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no Catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide.
Yukie Mitsuda - One of the best experts on this subject based on the ideXlab platform.
-
specific amyloid β clearance by a Catalytic Antibody construct
Journal of Biological Chemistry, 2015Co-Authors: Stephanie Planque, Yukie Mitsuda, Yasuhiro Nishiyama, Hiroaki Taguchi, Mariko Hara, Sari Sonoda, Yan Lin, Steven J Kolodziej, Veronica Gonzalez, Hameetha B R SaitAbstract:Classical immunization methods do not generate Catalytic antibodies (catabodies), but recent findings suggest that the innate Antibody repertoire is a rich catabody source. We describe the specificity and amyloid β (Aβ)-clearing effect of a catabody construct engineered from innate immunity principles. The catabody recognized the Aβ C terminus noncovalently and hydrolyzed Aβ rapidly, with no reactivity to the Aβ precursor protein, transthyretin amyloid aggregates, or irrelevant proteins containing the catabody-sensitive Aβ dipeptide unit. The catabody dissolved preformed Aβ aggregates and inhibited Aβ aggregation more potently than an Aβ-binding IgG. Intravenous catabody treatment reduced brain Aβ deposits in a mouse Alzheimer disease model without inducing microgliosis or microhemorrhages. Specific Aβ hydrolysis appears to be an innate immune function that could be applied for therapeutic Aβ removal.
-
metal dependent amyloid β degrading Catalytic Antibody construct
Journal of Biotechnology, 2014Co-Authors: Yasuhiro Nishiyama, Yukie Mitsuda, Stephanie Planque, Hiroaki Taguchi, Mariko Hara, Sudhir PaulAbstract:Catalytic antibodies (catabodies) that degrade target antigens rapidly are rare. We describe the metal-dependence of catabody construct 2E6, an engineered heterodimer of immunoglobulin light chain variable domains that hydrolyzes amyloid β peptides (Aβ) specifically. In addition to the electrophilic phosphonate inhibitor of serine proteases, the metal chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline completely inhibited the hydrolysis of Aβ by catabody 2E6. Formation of catabody-electrophilic phosphonate inhibitor adducts was unaffected by EDTA, suggesting that the metal exerts a favorable effect on a Catalytic step after the initial catabody nucleophilic attack on Aβ. The EDTA inactivated catabody failed to disaggregate fibrillar Aβ, indicating the functional importance of the Aβ hydrolytic activity. Treating the EDTA-inactivated catabody with Zn2+ or Co2+ restored the Aβ hydrolytic activity, and Zn2+-induced catabody conformational transitions were evident by fluorescence emission spectroscopy. The studies reveal the absolute catabody dependence on a metal cofactor.
-
Catalytic Antibody light chain capable of cleaving a chemokine receptor ccr 5 peptide with a high reaction rate constant
Biotechnology and Bioengineering, 2004Co-Authors: Yukie Mitsuda, Kumi Tsuruhata, Hiroko Fujinami, E. Hifumi, Naoki YamamotoAbstract:A monoclonal Antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the Antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two Catalytic triad(s), composed of Asp1, Ser27a (or Ser27e), and His93 (or His27d), in the light chain of ECL2B-2. The three amino acid residues, Asp1, Ser27a, and His93, are identical to those of Catalytic Antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high Catalytic reaction rate constant (kcat) of 2.23 min−1, which is greater by factors of tens to hundreds than those of natural Catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no Catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide. © 2004 Wiley Periodicals, Inc.
-
Catalytic Antibody light chain capable of cleaving a chemokine receptor CCR‐5 peptide with a high reaction rate constant
Biotechnology and Bioengineering, 2004Co-Authors: Yukie Mitsuda, Kumi Tsuruhata, Hiroko Fujinami, E. Hifumi, Naoki YamamotoAbstract:A monoclonal Antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the Antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two Catalytic triad(s), composed of Asp1, Ser27a (or Ser27e), and His93 (or His27d), in the light chain of ECL2B-2. The three amino acid residues, Asp1, Ser27a, and His93, are identical to those of Catalytic Antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high Catalytic reaction rate constant (kcat) of 2.23 min−1, which is greater by factors of tens to hundreds than those of natural Catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no Catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide. © 2004 Wiley Periodicals, Inc.
-
Catalytic Antibody light chain capable of cleaving a chemokine receptor ccr 5 peptide with a high reaction rate constant
Biotechnology and Bioengineering, 2004Co-Authors: Yukie Mitsuda, Kumi Tsuruhata, Hiroko Fujinami, E. Hifumi, Naoki Yamamoto, Taizo UdaAbstract:A monoclonal Antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the Antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two Catalytic triad(s), composed of Asp(1), Ser(27a) (or Ser(27e)), and His(93) (or His(27d)), in the light chain of ECL2B-2. The three amino acid residues, Asp(1), Ser(27a), and His(93), are identical to those of Catalytic Antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high Catalytic reaction rate constant (k(cat)) of 2.23 min(-1), which is greater by factors of tens to hundreds than those of natural Catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no Catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide.