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.

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 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. 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.

Biochemical features of a catalytic antibody light chain, 22F6, prepared from human lymphocytes.

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 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 activity against synthetic peptides. Some of the light chains exhibited unique features that suppressed the infectious activity of the rabies virus. Furthermore, the survival rate of mice in which a lethal level of the rabies virus was coinoculated directly into the brain with light chain 18 was significantly improved. In the case of humans, these results demonstrate that high-throughput selection of light chains possessing catalytic functions and specificity for a target molecule can be attained from a light-chain DNA library amplified from germline genes belonging to subgroup II.

Morphological varieties of the Purkinje fiber network in mammalian hearts, as revealed by light and electron microscopy.

Purkinje fibers in mammalian hearts are known to comprise the following three groups depending on their structure: group I found commonly in ungulates, group II in humans, monkeys and dogs, and group III in rodents. The aim of the present study was to document precisely the cytoarchitecture of a network of Purkinje fibers in different species by light and electron microscopy. Light microscopy of silver impregnated tissues revealed the reticular fibers ensheathing individual Purkinje strands consisting of 2-8 cells in both the ungulates (i.e., sheep and goats) and cetaceans (whales and dolphins) while they encircled each Purkinje cell in the primates (humans and monkeys), carnivores (dogs and seals), and rodents (rats). Scanning electron microscopy of NaOH digested tissues showed the ungrates (group I) to have a Purkinje fiber network composed of Purkinje strands; the cells in the strands were oval and made side-to-side and/or end-to-end connections. The Purkinje fiber network in the primates and carnivores (group II) was delicate and complicated; the Purkinje cells were usually cylindrical and connected end-to-end, the exception being their polygonal or stellate shapes at the bifurcations. Purkinje cells in the rodents (group III) resembled ventricular cardiac myocytes in cytoarchitecture. Morphologically, whales and seals respectively belonged to Purkinje cells of group I and group II. These findings indicate that the structural variety of the Purkinje fiber network may reflect the conducting function and be related to the phylogeny of the mammalian species.

Construction of human Fab (gamma1/kappa) library and identification of human monoclonal Fab possessing neutralizing potency against Japanese encephalitis virus.

A combinatorial human Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from Japanese encephalitis virus hyper-immune volunteers on pComb3H phagemid vector. The size of the constructed Fab library was 3.3x10(8) Escherichia coli transformants. The library was panned 3 times on the purified Japanese encephalitis virus (JEV) virion, and phage clones displaying JEV antigen-specific Fab were enriched. The enriched phage pool was then screened for clones producing Fab molecule with JEV neutralizing activity by the focus reduction-neutralizing test. Among 188 randomly selected clones, 9 Fab preparations revealed neutralizing activities against JEV strain Nakayama. An E. coli transformed with TJE12B02 clone, which produced human monoclonal Fab with the highest neutralizing activity was cultured in a large scale, and the Fab molecule was purified using affinity chromatography. The purified FabTJE12B02 showed the 50% focus reduction endpoint at the concentration of 50.2 microg/ml (ca. 1,000 nM) when JEV strain Nakayama was used. The FabTJE12B02 recognized E protein of JEV strain Nakayama, and the dissociation equilibrium constant (Kd) of the FabTJE12B02 against purified JEV antigen was calculated as 1.21x10(-8) M. Sequence analysis demonstrated that TJE12B02 used a VH sequence homologous to the VH3 family showing 88.8% homology to germline VH3-23, and used a Vkappa sequence homologous to the VkappaII subgroup showing 92.8% homology to germline A17.