Toyokazu Yokota

Regulation of human Fc epsilon RI alpha-chain gene expression by multiple transcription factors.

Transcriptional regulation of the gene-encoding human FcεRI α-chain was analyzed in detail. EMSA revealed that either YY1 or PU.1 bound to the region close to that recognized by Elf-1. The α-chain promoter activity was up-regulated ∼2-fold by exogenously expressed YY1 or PU.1 and ∼7-fold by GATA-1, respectively, in KU812 cells. In contrast, coexpression of GATA-1 with either of PU.1 or YY1 dramatically activated the promoter ∼41- or ∼27-fold, respectively. Especially synergic activation by GATA-1 and PU.1 was surprising, because these transcription factors are known to inhibit the respective transactivating activities of each other. These up-regulating effects of PU.1 and YY1 with GATA-1 were inhibited by overexpression of Elf-1, indicating that Elf-1 serves as a repressor for the α-chain gene expression. Transcriptional regulation of the α-chain gene through four transcriptional factors is discussed.

Production of humanized Fab fragment against human high affinity IgE receptor in Pichia pastoris.

Binding of allergen-IgE complexes to the high affinity IgE receptor (FcεRI) on mast cells and basophils leads to the release of various mediaters such as histamine. Fab fragments prepared by the papain digestion of humanized antibody against human FcεRI inhibited the release of histamine from human basophils. Here we established an expression system to directly produce Fab fragments of the humanized anti-human FcεRI antibody in methylotropic yeast, P. pastoris. Fab fragments were efficiently secreted into the medium at a concentration of 10-40 mg/L using a signal sequence from the P. pastoris phosphatase gene. They were consisted of disulfide-linked light and heavy chains correctly starting from the first amino acid residues by proper cleavage of the signal peptides. The obtained Fab fragments inhibited the binding between IgE and FcεRI as efficiently as the counterpart prepared by papain digestion of the whole antibody.

Hyposensitization to allergic reaction in rDer f 2-sensitized mice by the intranasal administration of a mutant of rDer f 2, C8/119S.

C8/119S is a mutant of recombinant Der f 2 (rDer f 2), and lacks a disulphide bond possessed by wild‐type rDer f 2. In humans and mice, C8/119S has a very weak IgE‐binding capacity compared with the wild‐type, but possesses a T cell reactivity comparable to that of the wild‐type. C8/119S may thus be a safe immunotherapeutic agent for house dust mite allergy. The aim of the present study was to evaluate whether the intranasal administration of C8/119S could suppress an immediate allergic reaction in mice sensitized with wild‐type rDer f 2, possessing an allergic activity comparable to native counterparts purified from mite extract. Seven‐week‐old male A/J mice were immunized with wild‐type rDer f 2 four times, and then intranasally administered 0.2–2 μg of wild‐type, 0.2–20 μg of C8/119S, or PBS alone, three times a week for 4 weeks. Seven days after the last administration, the mice were examined for an immediate allergic reaction. The animals administered 2 μg of C8/119S (C2.0 group) showed significantly reduced immediate bronchoconstriction provoked by the i.v. injection of 1 and 10 μg of wild‐type rDer f 2, compared with the PBS‐treated mice. Similar results were obtained when we examined mice 10 weeks after the last administration. The reactions in the other groups given wild‐type or C8/119S also tended to decrease in severity in comparison with the animals of the PBS group. The allergic phenotypes of the T cells, B cells, and basophils in the C2.0 group were shifted to that of naive mice without immunization. We conclude that C8/119S has hyposensitizing activities in mice sensitized with wild‐type rDer f 2. C8/119S may be useful for immunotherapy of house dust mite allergy.

A complex composed of USF1 and USF2 activates the human FcεRI α chain expression via a CAGCTG element in the first intron

The high‐affinity IgE receptor, FcϵRI, is a key regulatory molecule in the allergic reaction. During the course of studies to find cis‐acting elements for FcϵRI α chain gene expression, a CAGCTG sequence located in the first intron was revealed to serve as a crucial enhancer element. Electromobility shift assays using antibodies and in vitro translation products showed that the CAGCTG element was recognized by the USF1 / USF2 complex. As was the case for other intronic cis‐elements, the CAGCTG element regulated the promoter in an orientation‐ and position‐dependent manner. Overexpression of USF2 antisense repressed the FcϵRI α chain gene promoter and decreased the amount of α chain mRNA in mast cell lines. All these results indicated that the USF1 / USF2 complex activates the human FcϵRI α chain gene expression via the CAGCTG element in the first intron.

Production of enzymatically and immunologically active Der f 1 in Escherichia coli

Der f 1 is a major house dust mite allergen belonging to the cysteine protease family. Because of the great demand for clinical and research use of this allergen, much effort to establish an efficient method of preparing purified Der f 1 has been made. We constructed an isopropyl-β-D-thiogalactopyranoside-inducible expression plasmid to produce the pro-form of Der f 1 in Escherichia coli. The recombinant product was accumulated as insoluble inclusion bodies in cells. The solubilized inclusion bodies were found to be successfully renatured by two-step gel filtration chromatography. About 70 mg of pro Der f 1 were properly refolded by this method from 1 liter of culture. Acid treatment of the renatured pro Der f 1 resulted in the autocatalytic removal of the pro-sequence. The obtained mature form of Der f 1 bound IgE in patient sera and induced the release of histamine from peripheral blood leukocytes equally to native Der f 1. Furthermore, mature Der f 1 obtained by this method had identical protease activity with native Der f 1. We also discussed the contribution of the pro-sequence and the sugar chain of Der f 1 to its antigenic and enzymatic activity. This is the first report to produce an active mature form of recombinant Der f 1 in E. coli.

Unlocking the allergenic structure of the major house dust mite allergen Der f 2 by elimination of key intramolecular interactions

We report on the structural background of the remarkable reduction of allergenicity in engineering of the major house dust mite allergen Der f 2. Disruption of intramolecular disulfide bonds in Der f 2 caused extensive conformational change that was monitored by circular dichroism and gel‐filtration analysis. The degree of conformational change correlated well with the degree of reductions in the capacity to bind IgE and to induce histamine release from basophils in mite‐allergic patients. Loosening the rigid tertiary structure by elimination of key intramolecular interactions is an effective strategy to reduce the number of high affinity IgE epitopes of allergen vaccine.

Experimental Monkey Model Sensitized with Mite Antigen

Background: Monkeys are considered to have an immune system very similar to that of humans, as compared with mice, rats, and guinea pigs. Although primate allergic models to several pollen allergens have been developed, no model of house dust mite allergy has been reported. In this study, we attempted to induce type I allergy to mite allergens in rhesus monkeys. Methods: Six rhesus monkeys were immunized subcutaneously with crude mite extract adsorbed on aluminum hydroxide for 4 months. Then 5 monkeys positive for IgE production to mite extract were further immunized subcutaneously and conjunctivally with recombinant Der f 2 (rDer f 2). The status of sensitization to mite extract and rDer f 2 in monkeys was examined before and after the immunization. Plasma antigen-specific IgE and IgG levels, cutaneous reaction, and histamine release from peripheral blood leukocytes were measured. After conjunctival immunization, immediate conjunctivitis and leukocyte influx into conjunctiva after rDer f 2 challenge were examined. Results: After immunization with crude mite extract, 5 of 6 sensitized monkeys showed IgE response to the mite, and 4 out of 5 rDer f 2-sensitized monkeys exhibited IgE production to rDer f 2. Three monkeys sensitized with rDer f 2 showed immediate conjunctivitis and conjunctival eosinophilia after applying rDer f 2 to their eyes. Sensitized animals also showed IgG response to mite antigens. Conclusion: Four rhesus monkeys were positive for IgE production and allergic reactions to both mite extract and rDer f 2. These monkeys could represent a useful model for studying the development and regulation of house dust mite allergy.

Effects of site-directed mutagenesis in the cysteine residues and the N-glycosylation motif in recombinant Der f 1 on secretion and protease activity.

Background: The group 1 allergens from mite feces, which belong to the papain-like cysteine protease family, are the most significant in-door allergens. In this study, we analyzed the contribution of the cysteine residues and N-glycosylation in Der f 1, the group 1 allergen from Dermatophagoides farinae, to secretion and maturation by using systems for expression of recombinant Der f 1 (rDer f 1). Methods: The rDer f 1 and its mutants were expressed in yeast Pichia pastoris and insect SF9 cells. Secretion of their proforms was checked by SDS-PAGE or immunoblotting. Protease activities of the secreted proform of a mutant and the mature form were compared with that of native Der f 1. Results: The proform of a mutant Der f 1, pro-N53Q, whose consensus motif for N-glycosylation was disrupted, was not secreted in insect SF9 cells although secreted in P. pastoris. Indirect evidence was obtained to support the disulfide bond formation between Cys4 and Cys118, which were not conserved in papain. A mutant for Cys35 in the catalytic site of the cysteine protease, pro-C35S/N53Q, was secreted, but the other mutants for cysteines concerning intramolecular disulfide bonds were not secreted in P. pastoris. The prosequence of pro-C35S/N53Q was removed by an in vitro activation process. The mature C35S/N53Q showed low protease activity. Conclusion: N-glycosylation is essential for secretion in insect SF9 cells but not in P. pastoris. Disulfide bonds are essential for secretion in P. pastoris. A mutation in the catalytic site, C35S, is not completely critical to removal of the prosequence and protease activity. The findings are useful for future design of recombinant products for application in immunotherapy.

Production of Humanized Antibody against Human High-affinity IgE Receptor in a Serum-free Culture of CHO Cells, and Purification of the Fab Fragments

We describe the preparation of Fab fragments of a humanized anti-human high-affinity IgE receptor (FcεRIα) antibody potentially useful for treatment of IgE-mediated allergic diseases. IgE-binding capacities of sixteen combinations of light and heavy chains of four recombinant anti-FcεRIα antibodies, chimeric CRA2, humanized CRA2, chimeric CRA4, and humanized CRA4, were compared. A combination in which both chains were of humanized CRA2 had the highest activity. Stable transfectant clones of four kinds of host cells expressing recombinant antibodies were established. CHO-K1 cells were the most productive. Serum-free media suitable for culture of the stable CHO-transfectant clones were screened. The concentration of the humanized CRA2, which the most productive clone secreted into the chosen serum-free medium, was approximately 100 μg/ml. A procedure for the purification of the antibody, papain-digestion, and purification of Fab fragments was established. The highly purified humanized Fab fragments are suitable for use to examine their in vivo activity and immunogenicity in primates.

Molecular cloning of rat transcription factor YY1.

YY1 is a ubiquitously expressed multifunctional transcription factor that is involved in both positive and negative regulation of gene expression as well as initiation of transcription. Here, we isolated cDNA encoding a full-length open reading frame (ORF) of rat YY1. Rat YY1 is composed of 411 amino acid residues and its amino acid sequence is 97.6% identical to that of mouse YY1 and 97.8% identical to that of human YY1. The transactivating abilities of wild-type rat YY1 and four truncated mutant forms of YY1 were examined by transient reporter assays. When residues 114-193, which sequence includes a portion of the activation region and most of the Gly/Lys-rich region, were lacking, transactivation activity decreased somewhat, but the further deletion in the activation region (of residues 56-113) did not cause further decrease of the activity. On the other hand, N-terminus of the activation region (1-78/100-106) did not have transactivation activity by itself as well as synergistic activity with an erythroid specific transcription factor GATA-1.