Emi Hifumi

Catalytic digestion of human tumor necrosis factor-α by antibody heavy chain.

It has long been an important task to prepare a catalytic antibody capable of digesting a targeting crucial protein that controls specific life functions. Tumor necrosis factor‐α (TNF‐α) is a cytokine and an important molecule concerned with autoimmune diseases such as rheumatoid arthritis, chronic obstructive pulmonary disease, and Crohn’s disease. A mAb (ETNF‐6 mAb) raised against human TNF‐α was prepared, and the steric conformation was created by using molecular modeling after the cDNA was sequenced. The heavy chain (ETNF‐6‐H) of the mAb was considered to possess a catalytic triad‐like structure in the complementarity determining regions (CDRs). As a result, ETNF‐6‐H exhibited a peptidase and a protease activity. In fact, ETNF‐6‐H predominantly cleaved the Ser5‐Arg6 bond of TNF‐α at the first step, resulting in the generation of a fragment of ∼u200317u2003kDa. This fragment was digested to a smaller molecule of 15u2003kDa by scission of the Gln21‐Ala22 bond. The intermediate product was further converted into a fragment of 13.3u2003kDa by successive cleavage of the Leu36‐Leu37 and Asn39‐Gly40 bonds. The heavy chain possessed a protease activity against TNF‐α with a multicleavage site.

Biochemical Features of a Catalytic Antibody Light Chain, 22F6, Prepared from Human Lymphocytes * □

Background: We explored catalytic antibodies applicable to patients. Results: A human light chain (22F6) possessing both amidase and nuclease activities and preventing infection of influenza virus was obtained. Conclusion: The 22F6 light chain holds a huge potential as a new drug for patient therapies in the future. Significance: The procedure developed in this study is highly noteworthy for developing new drugs. 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.

Therapeutic effects of molecularly designed antigen UREB138 for mice infected with Helicobacter pylori

Helicobacter pylori (H. pylori) is a bacteria that is well known as the principal cause of chronic gastritis and peptic ulcer disease in humans. Because no effective vaccine has yet been established, we designed a new biomolecule as a vaccination antigen capable of preventing the infection of H. pylori. The designed biomolecule involves a 138 stretch (aa 201–aa 338 of β‐subunit of H. pylori urease), which is the functionally important region for urease activity. The region was expressed as a recombinant protein, called UREB138. The therapeutic vaccination was performed using UREB138 in mice persistently infected with H. pylori. The subcutaneous administration of UREB138 remarkably reduced the number of bacteria in the mice stomach compared with the control. Immunization with UREB138 enhanced the urease‐specific IgA and IgG1 in the serum. Immunohistochemical staining for IgA in gastric mucosa showed that the number of mice positively stained with IgA was significantly higher in UREB138‐immunized mice than in control mice. Furthermore, the expression of interferon‐gamma mRNA in the gastric tissues with eradicated bacteria was higher than in the non‐eradicated group. The combination of Th1‐ and Th2‐mediated immunity plays a role in reducing the colonization of bacterial numbers of H. pylori. Biotechnol. Bioeng. 2008;100: 634–643.

A novel method of preparing the monoform structure of catalytic antibody light chain

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.—Hifumi, E., Matsumoto, S., Nakashima, H., Itonaga, S., Arakawa, M., Katayama, Y., Kato, R., Uda T. A novel method of preparing the monoform structure of catalytic antibody light chain. FASEB J. 30, 895–908 (2016). www.fasebj.org

Detection of influenza virus by a biosensor based on the method combining electrochemiluminescence on binary SAMs modified Au electrode with an immunoliposome encapsulating Ru (II) complex

Recently, point of care testing (POCT) used for diagnosis of influenza infection has a problem showing false negative diagnosis because of the low sensitivity. We would like to report detection of influenza virus A (H1N1) by an immunosensor based on electrochemiluminescence (ECL) that uses an immunoliposome encapsulating tris(2,2′-bipyridyl)ruthenium(II) complex. By using the sensor, we could detect the virus that competed with hemagglutinin (HA) peptide immobilized on self-assembled monolayers (SAMs) in immunoreaction of the antibody bound on the surface of liposome. The HA peptide was 19 mer (TGLRNGITNKVNSVIEKAA). We demonstrated great improvement of sensitivity and accuracy by introducing binary SAMs instead of mono SAMs. The binary SAMs was prepared from 3,3′-dithiodipropionic acid and 1-hexanethiol. Use of the binary SAMs enabled to increase the SAMs coverage on Au electrode; the fact was confirmed by observation of the cathodic desorption currents. By using suchxa0an electrode, firstxa0the detection method of BSA was optimized to lower ECL background signal. Then we applied the method to the detection of influenza virus. We could successfully detect the virus with higher sensitivity compared with that by POCT and ELISA. The detection range was from a concentration of 2.7u2009×u2009102 to 2.7u2009×u2009103 PFU/mL.

Role of the constant region domain in the structural diversity of human antibody light chains

Issues regarding the structural diversity (heterogeneity) of an antibody molecule have been the subject of discussion along with the development of antibody drugs. Research on heterogeneity has been extensive in recent years, but no clear solution has been reached. Heterogeneity is also observed in catalytic antibody k light chains (CLs). In this study, we investigated how the constant region domain of CLs concerns structural diversity because it is a simple and good example for elucidating heterogeneity. By means of cation‐exchange chromatography, SDS‐PAGE, and 2‐dimensional electrophoresis for the CL, multimolecular forms consisting of different electrical charges and molecular sizes coexisted in the solution, resulting in the similar heterogeneity of the full length of CLs. The addition of copper ion could cause the multimolecular forms to change to monomolecular forms. Copper ion contributed greatly to the enrichment of the dimer form of CL and the homogenization of the differently charged CLs. Two molecules of the CL protein bound one copper ion. The binding affinity of the ion was 48.0 μM21. Several divalent metal ions were examined, but only zinc showed a similar effect. —Hifumi, E., Taguchi, H., Kato, R., Uda, T. Role of the constant region domain in the structural diversity of human antibody light chains. FASEB J. 31, 1668–1677 (2017) www.fasebj.org

Biochemical features and antiviral activity of a monomeric catalytic antibody light-chain 23D4 against influenza A virus

Catalytic antibodies have exhibited interesting functions against some infections 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. As a result, the monomeric 23D4 clearly exhibited the ability to suppress the influenza virus infection in both cases, indicating a potential drug for preventing infection of the influenza A virus.— Hifumi, E., Arakawa, M., Matsumoto, S., Yamamoto, T., Katayama, Y., Uda, T. Biochemical features and antiviral activity of a monomeric catalytic antibody light‐chain 23D4 against influenzaAvirus. FASEB J. 29, 2347‐2358 (2015). www.fasebj.org

Emzyme and Antibody Are Brothers

標記のタイトルを見て驚き,あるいは,興味をもつ読者があろうかと思う。これまで酵素は「酵素学」で,抗体は「免疫学」でおもに学習してきた。しかしながら近年,通常の免疫によっても,あるいは,ある病気(おもに自己免疫疾患)の患者血清中にも酵素活性をもつ抗体が次々に見いだされてきている。初期には微量の酵素のコンタミが原因ではないかと指摘(指弾?)されたが,現在では天然に抗体酵素が存在することが明白になってきた。なぜ抗体が生まれながらにして酵素活性をもつのか?抗体は酵素と何らかの関係があるのかなど,最近の知見に基づいて筆者らの考えを概説したい。