Der f 34, a Novel Major House Dust Mite Allergen Belonging to a Highly Conserved Rid/YjgF/YER057c/UK114 Family of Imine Deaminases.

The high prevalence of house dust mite (HDM) allergy is a growing health problem worldwide, and the characterization of clinically important HDM allergens is a prerequisite for the development of diagnostic and therapeutic strategies. Here, we report a novel HDM allergen that belongs structurally to the highly conserved Rid/YjgF/YER057c/UK114 family (Rid family) with imine deaminase activity. Isolated HDM cDNA, named der f 34, encodes 128 amino acids homologous to Rid-like proteins. This new protein belongs to the Rid family and has seven conserved residues involved in enamine/imine deaminase activity. Indeed, we demonstrated that purified Der f 34 had imine deaminase activity that preferentially acted on leucine and methionine. Native Der f 34 showed a high IgE binding frequency as revealed by two-dimensional immunoblotting (62.5%) or ELISA (68%), which was comparable with those of a major HDM allergen Der f 2 (77.5 and 79%, respectively). We also found that Der f 34 showed cross-reactivity with another prominent indoor allergen source, Aspergillus fumigatus This is the first report showing that the Rid family imine deaminase represents an additional important pan-allergen that is conserved across organisms.

A flavanone derivative from the Asian medicinal herb (Perilla frutescens) potently suppresses IgE-mediated immediate hypersensitivity reactions.

Perilla frutescens is a dietary leafy herb consumed as a traditional Japanese condiment as well as used for Chinese medicine with anti-inflammatory activity. Here we report a hitherto-unrecognized P. frutescens phytochemical that potently suppresses IgE-mediated type I hypersensitivity reactions. Structural analysis reveals that the purified anti-allergic compound (Perilla-derived methoxyflavanone, PDMF) is identified as 8-hydroxy-5,7-dimethoxyflavanone. PDMF significantly inhibits IgE-mediated histamine release from RBL-2H3 rat basophilic leukemia cells as compared with those seen in known P. frutescens-derived anti-inflammatory polyphenols. We also show that oral administration of PDMF not only suppresses passive cutaneous anaphylaxis, but also prevents allergic rhinitis-like nasal symptoms in a murine model of Japanese cedar pollinosis. Mechanistically, PDMF negatively regulates Akt phosphorylation and intracellular Ca2+ influx, both of which are essential for mast cell secretory granule translocation and its exocytosis upon high-affinity IgE receptor (FcεRI) cross-linking. These results represent PDMF as a new potent anti-allergic phytochemical useful for prevention of IgE-driven hypersensitivity reactions.

Plasma Cluster Ions Reduce the IgE-Binding Capacity of House Dust Mite Allergens under a Simulated Indoor Environmental Condition.

BACKGROUND: A high level of house dust mite (HDM) allergens in a living environment is a risk factor for both sensitization to these allergens and asthmatic attacks. We previously showed that plasma cluster ions (PCIs) impaired the IgE-binding capacity of atomized crude allergens prepared from Japanese cedar pollen and fungus under experimental conditions. OBJECTIVE: We evaluated the capacity of PCIs to impair the IgE-binding capacity of airborne HDM allergens under a simulated indoor environmental condition. METHODS: For the determination of the effects of PCIs on HDM allergens under an experimental condition, HDM extract was atomized as aqueous mist into a cylindrical experimental apparatus filled with PCIs. For the evaluation of the effects of PCIs under a simulated natural indoor environmental condition, dried HDM allergens were floated as airborne particles in an acryl cubic apparatus in the presence of PCIs. The IgE-binding capacities of the PCI- and sham-treated HDM allergens were analyzed by an ELISA. RESULTS: The IgE-binding capacity of the HDM allergens was significantly impaired after PCI treatment compared to that after sham treatment under both experimental and simulated environmental conditions. The ELISA results demonstrated that the IgE-binding capacities of HDM allergens after PCI treatment showed 68 and 74% reductions compared to those after sham treatment under the experimental and simulated environmental conditions, respectively. CONCLUSIONS: PCIs have the capacity to impair the IgE-binding capacity of airborne HDM allergens in a simulated environmental condition.

Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases.

Membrane-bound desaturases are physiologically and industrially important enzymes that are involved in the production of diverse fatty acids such as polyunsaturated fatty acids and their derivatives. Here, we identified amino acid residues that determine the substrate specificity of rat Δ6 desaturase (D6d) acting on linoleoyl-CoA by comparing its amino acid sequence with that of Δ5 desaturase (D5d), which converts dihomo-γ-linolenoyl-CoA. The N-terminal cytochrome b5-like domain was excluded as a determinant by domain swapping analysis. Substitution of eight amino acid residues (Ser209, Asn211, Arg216, Ser235, Leu236, Trp244, Gln245, and Val344) of D6d with the corresponding residues of D5d by site-directed mutagenesis switched the substrate specificity from linoleoyl-CoA to dihomo-γ-linolenoyl-CoA. In addition, replacement of Leu323 of D6d with Phe323 on the basis of the amino acid sequence of zebra fish Δ5/6 bifunctional desaturase was found to render D6d bifunctional. Homology modeling of D6d using recent crystal structure data of human stearoyl-CoA (Δ9) desaturase revealed that Arg216, Trp244, Gln245, and Leu323 are located near the substrate-binding pocket. To our knowledge, this is the first report on the structural basis of the substrate specificity of a mammalian front-end fatty acid desaturase, which will aid in efficient production of value-added fatty acids.

Unexpected T cell regulatory activity of anti-histone H1 autoantibody: its mode of action in regulatory T cell-dependent and -independent manners.

Induction of anti-nuclear antibodies against DNA or histones is a hallmark of autoimmune disorders, but their actual contribution to disease predisposition remains to be clarified. We have previously reported that autoantibodies against histone H1 work as a critical graft survival factor in a rat model of tolerogeneic liver transplantation. Here we show that an immunosuppressive anti-histone H1 monoclonal antibody (anti-H1 mAb) acts directly on T cells to inhibit their activation in response to T cell receptor (TCR) ligation. Intriguingly, the T cell activation inhibitory activity of anti-H1 mAb under suboptimal dosages required regulatory T (Treg) cells, while high dose stimulation with anti-H1 mAb triggered a Treg cell-independent, direct negative regulation of T cell activation upon TCR cross-linking. In the Treg cell-dependent mode of immunosuppressive action, anti-H1 mAb did not induce the expansion of CD4(+-)Foxp3(+) Treg cells, but rather potentiated their regulatory capacity. These results reveal a previously unappreciated T cell regulatory role of anti-H1 autoantibody, whose overproduction is generally thought to be pathogenic in the autoimmune settings.

A novel peptide mimotope identified as a potential immunosuppressive vaccine for organ transplantation.

We reported that anti-histone H1 autoantibody is one of the main immunosuppressive factors in serum that is induced after orthotopic liver transplantation in a rat tolerogenic model. We generated a novel anti-histone H1 IgM mAb produced by hybridoma 16G9 (16G9 mAb) that shows MLR-inhibitory activity. Identification of a functional epitope responsible for the immunosuppressive activity of 16G9 mAb may lead to the establishment of a novel therapeutic strategy. We used a combinatorial phage display peptide library to screen for peptides that bind to 16G9 mAb. Consequently, two peptides that bind to 16G9 mAb, SSV and LPQ, were selected from the library. The binding of 16G9 mAb to histone H1 was inhibited by SSV. SSV was recognized by rat tolerogenic post-orthotopic liver transplantation serum and the binding to SSV was inhibited by histone H1. Mice were immunized with keyhole limpet hemocyanin-conjugated SSV and LPQ. Abs induced by SSV immunization inhibited Con A-stimulated splenocyte proliferation, and the inhibition was neutralized by preincubation with SSV. Splenocytes stimulated by anti-CD3 Ab were inhibited by SSV-induced Abs using CFSE labeling. SSV immunization in rats before heterotopic heart transplantation resulted in significant prolonged allograft survival. These findings suggested that SSV is a functional histone H1-binding epitope for 16G9 mAb. SSV is capable of determining serum immunoreactivity against histone H1 as an index marker for tolerance. The inhibitory activity of SSV-induced Abs on blast cell proliferation and the prolonged graft survival that results from SSV immunization imply a potential for the development of an immunosuppressive vaccine.

Sake lees fermented with lactic acid bacteria prevents allergic rhinitis-like symptoms and IgE-mediated basophil degranulation.

We tested the effect of oral administration of fermented sake lees with lactic acid bacteria (FESLAB) on a murine model of allergic rhinitis upon immunization and nasal sensitization with ovalbumin (OVA). We used Lactobacillus paracasei NPSRIk-4 (isolated from sake lees), and L. brevis NPSRIv-8 (from fermented milk) as starter strains to produce the FESLAB. Oral FESLAB administration resulted in the development of significantly fewer sneezing symptoms than those seen in sham control animals given sterile water. We also found that FESLAB suppressed the allergen-induced degranulation of RBL2H3 rat basophilic leukemia cells.

Immunological aspects and therapeutic significance of an autoantibody against histone H1 in a rat model of concanavalin A-induced hepatitis.

We previously demonstrated the immunosuppressive activity of anti-histone H1 autoantibody induced in experimental and clinical liver allograft tolerance. This study aimed to explore the immunological aspects of anti-histone H1 autoantibody in liver injury induced by concanavalin A (Con A). To establish a Con A-hepatitis model, 20 mg/kg Con A was intravenously injected into rats, after which liver function and histopathological analyses were performed. In this model, anti-histone H1 autoantibody was transiently induced in the sera during the natural recovery stage, 3-7 days after Con A injection. To evaluate the therapeutic significance of anti-histone H1 autoantibody, a polyclonal antibody against histone H1 was intraperitoneally injected immediately after Con A injection. We found that injection of anti-histone H1 antibody could reduce Con A-induced liver damage. Further mechanical analyses revealed that anti-histone H1 antibody altered the intracellular activation of mitogen-activated protein kinase, nuclear factor-kappaB and calcineurin via T-cell receptor signalling, suggesting that anti-histone H1 antibody may protect the liver from Con A-induced injury by inhibiting activation of effector T cells. These findings suggest that anti-histone H1 autoantibody may be a natural immune regulatory factor that protects inflamed livers suffering from autoimmune hepatitis and may lead to T-cell unresponsiveness through the selective regulation of mitogen-activated protein kinase/nuclear factor-kappaB and calcineurin signalling.

Hydrolyzed Konjac glucomannan suppresses IgE production in mice B cells.

BACKGROUND: Oral administration of pulverized Konjac glucomannan (KGM) reduces increased plasma IgE and the amount of epsilon-germline transcript (epsilonGT) in the spleen, as well as preventing the development of dermatitis in mice. To elucidate the mechanism of action of pulverized KGM, we solubilized KGM and studied its effect on IgE in vitro and in vivo. METHODS: Solubilized KGM was prepared by acid hydrolysis, and we analyzed the effective molecular size for the suppression of IgE production and epsilonGT in vitro and the level of plasma IgE induced by immunization with ovalbumin in BALB/c mice. RESULTS: The production of IgE and epsilonGT in splenic cells, but not purified B cells, was inhibited by hydrolyzed KGM (KGM hydrolyzed with 0.25 N HCl; H-KGM) at the optimal size of between 10 and 500 kDa. However, no effect was observed when H-KGM was substituted with unhydrolyzed KGM in vitro. IgE production from purified B cells cocultured with purified monocytes, but not with purified T cells, was inhibited by H-KGM. The release of IFNgamma in cultures of monocytes but in purified B cells with or without T cells was enhanced in the presence of H-KGM. Injection of mice with H-KGM also suppressed the production of plasma IgE and IgG1 but not IgG2a in vivo. CONCLUSION: KGM at an optimal size prevents germline class-switching and IgE production both in vitro and in vivo. H-KGM may be useful as a tool to study the mechanism of action of KGM and as a dietary supplement to prevent atopic diseases.

Molecular cloning and immunochemical characterization of a novel major Japanese cedar pollen allergen belonging to the aspartic protease family.

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis in Japan. Protease activity in the pollen grains may trigger pro-allergic responses but no such proteases have yet been identified as pollen allergens. OBJECTIVES: We report the molecular cloning and immunochemical characterization of a novel C. japonica pollen allergen belonging to the aspartic protease family. METHODS: We focused on the C. japonica pollen allergen spot No. 63 (CPA63, 47.5% IgE binding frequency) on our 2-dimensional IgE immunoblot map. The internal amino acid sequences were determined using time-of-flight mass spectrometry. Full-length cpa63 cDNA was cloned by rapid amplification of cDNA ends (RACE)-PCR. Recombinant CPA63 (r-CPA63) was expressed using the baculovirus-insect cell culture system and its IgE binding capacity was analyzed by enzyme-linked immunosorbent assay (ELISA). The proteolytic activity of r-CPA63 was also assessed using a putative mature enzyme produced upon autolysis. RESULTS: cpa63 cDNA encoded a 472 amino acid polypeptide showing about 40% sequence identity to members of the plant atypical aspartic protease family. ELISA showed that r-CPA63 was recognized by IgE antibodies in the serum of 58% (18/31) of Japanese cedar pollinosis patients. We also demonstrated an aspartic protease-like enzyme activity of the putative mature r-CPA63. CONCLUSIONS: We have identified the first plant aspartic protease allergen from Japanese cedar pollen. The availability of the CPA63 sequence and its recombinant allergen production system are useful not only for pharmaceutical applications but also for further examination of the role of protease activity in the pathogenesis of cedar pollinosis.

Effects of single and combined administration of fermented barley extract and gamma-aminobutyric acid on the development of atopic dermatitis in NC/Nga mice.

We examined the effects single and combined administration of fermented barley extract P (FBEP), prepared from barley-shochu distillery by-products, and gamma-aminobutyric acid (GABA) on the development of atopic dermatitis (AD)-like skin lesions in NC/Nga mice. Single administration of FBEP and GABA dose-dependently reduced the development of AD-like skin lesions in mice. GABA reduced the development of AD-like skin lesions by suppressing serum immunoglobulin E (IgE) and splenocyte interleukin (IL)-4 production, while FBEP reduced skin lesions without affecting the IgE or cytokine production. However, in mice with induced AD-like skin lesions, combined administration of FBEP and GABA decreased serum IgE levels and splenic cell IL-4 production, and increased splenic cell interferon-gamma production. These results suggest that combined administration of FBEP and GABA alleviated AD-like skin lesions in the NC/Nga mice by adjusting the Th1/Th2 balance to a Th1-predominant immune response.

Effects of a fermented barley extract on subjects with slightly high serum uric acid or mild hyperuricemia.

The uric acid-lowering effect and safety of a fermented barley extract P (FBEP) prepared from barley-shochu distillery by-products were investigated in a randomized, placebo-controlled, parallel-group, double-blinded study. A total of 111 subjects with serum uric acid levels of 6.0-7.9 mg/dl were provided with either a drink containing 2 g/d of FBEP (test group) or a placebo drink. After 12 weeks, the serum uric acid levels changed by -0.21+/-0.56 mg/dl in the test group, showing a significant decrease in comparison to those of the placebo group (+0.02+/-0.54 mg/dl). Additionally, the uric acid clearance in the test group showed a tendency to increase after 12 weeks more than in the placebo group (p=0.054). No abnormalities in the physical and clinical tests were observed, and no adverse diagnostic findings were attributed to the intake of the test meal. These results demonstrated the benefits and safety of the FBEP treatment to subjects with slightly high serum uric acid or mild hyperuricemia.

A new lipid transfer protein homolog identified as an IgE-binding antigen from Japanese cedar pollen.

Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal rhinitis and conjunctivitis in Japan, and an understanding of its full allergen repertoire is prerequisite for the development of future molecular diagnostics and immunotherapeutic strategies. Here we report the identification of a new C. japonica pollen IgE-binding antigen (CJP-8) homologous to lipid transfer proteins (LTPs), a class of plant cross-reactive allergens found in foods, latex, and pollen grains. The cjp-8 cDNA encodes a 165-amino acid polypeptide possessing the conserved eight cysteines characteristic of plant LTP family members. Escherichia coli-expressed recombinant CJP-8 (r-CJP-8) reacted with IgE antibody from Japanese cedar pollinosis patients at a 37.5% frequency (6/16).

Improvement of Stearidonic acid production in Oleaginous Saccharomyces cerevisiae.

Rat Delta6 desaturase was introduced in "oleaginous" Saccharomyces cerevisiae genetically modified by the snf2 disruption, DGA1 overexpression, and LEU2 expression to improve stearidonic acid (18:4n-3 (Delta6, 9, 12, 15)) production. When this transformed yeast was cultured in the presence of 0.7 g/l alpha-linolenic acid for 7 d, 0.4 g of alpha-linolenic acid was accumulated and 44 mg of stearidonic acid was produced, mainly as a triacylglycerol in the 1 liter broth.

Codon optimization increases steady-state mRNA levels in Aspergillus oryzae heterologous gene expression.

We investigated the effect of codon optimization on the expression levels of heterologous proteins in Aspergillus oryzae, using the mite allergen Der f 7 as a model protein. A codon-optimized Der f 7 gene was synthesized according to the frequency of codon usage in A. oryzae by recursive PCR. Both native and optimized Der f 7 genes were expressed under the control of a high-level-expression promoter with their own signal peptides or in a fusion construct with A. oryzae glucoamylase (GlaA). Codon optimization markedly increased protein and mRNA production levels in both nonfused and GlaA-fused Der f 7 constructs. For constructs with native codons, analysis by 3' rapid amplification of cDNA ends revealed that poly(A) tracts tended to be added within the coding region, producing aberrant mRNAs that lack a termination codon. Insertion of a termination codon between the carrier GlaA and native Der f 7 proteins in the GlaA fusion construct resulted in increases in mRNA and secreted-carrier-GlaA levels. These results suggested that mRNAs without a termination codon as a result of premature polyadenylation are degraded, possibly through the nonstop mRNA decay pathway. We suggest that codon optimization in A. oryzae results in elimination of cryptic polyadenylation signals in native Der f 7, thereby circumventing the production of truncated transcripts and resulting in an increase in steady-state mRNA levels.

Involvement of autoimmunity against nuclear histone H1 in liver transplantation tolerance.

BACKGROUND: Our recent studies suggested that anti-histone H1 autoantibody (auto-Ab) plays an important role in experimental and clinical liver allograft tolerance as a natural immunosuppressive factor. The present study aimed to explore how the autoimmune response against histone H1 is involved in tolerance induction. METHODS: The measurement of anti-histone H1 auto-Ab and immunohistochemical analysis were performed in serum and liver allografts after orthotopic liver transplantation (OLT). To compare the auto-Ab response against histone H1 between the recipients of rejector (DA-LEW) and tolerogenic (DA-PVG) OLT models, naïve recipients were immunized with calf thymus histone H1. The immunosuppressive state of histone H1-immunized rats was assayed by mixed lymphocyte reaction (MLR). RESULTS: Anti-histone H1 Ab titer was transiently increased during the rejection phase after OLT (days 7-21) in the DA-PVG combination, while no such response was confirmed in the DA-LEW acute rejection model. Nuclear histone H1 antigens were found in the cytoplasm and the extracellular environment in liver allografts at the rejection phase in the tolerogenic model but not in the rejector model, resulting from the transient induction of anti-histone H1 auto-Ab in recipient PVG rats after OLT. Low dose and short-term immunization with histone H1 upregulated the anti-histone H1 Ab titer in naïve PVG rats, which exhibited a low allogeneic immune response, while no such response was found in naïve LEW rats. CONCLUSIONS: These results suggest that the sensitivity to nuclear antigens such as histone H1 may be a key factor determining the acceptance or rejection of donor liver grafts, at least in DA-PVG and DA-LEW combinations.

A methoxyflavanone derivative from the Asian medicinal herb (Perilla frutescens) induces p53-mediated G2/M cell cycle arrest and apoptosis in A549 human lung adenocarcinoma.

Perilla frutescens is an Asian dietary herb consumed as an essential seasoning in Japanese cuisine as well as used for a Chinese medicine. Here, we report that a newly found methoxyflavanone derivative from P. frutescens (Perilla-derived methoxyflavanone, PDMF; 8-hydroxy-5,7-dimethoxyflavanone) shows carcinostatic activity on human lung adenocarcinoma, A549. We found that treatment with PDMF significantly inhibited cell proliferation and decreased viability through induction of G2/M cell cycle arrest and apoptosis. The PDMF stimulation induces phosphorylation of tumor suppressor p53 on Ser15, and increases its protein amount in conjunction with up-regulation of downstream cyclin-dependent kinase inhibitor p21Cip1/Waf1 and proapoptotic caspases, caspase-9 and caspase-3. We also found that small interfering RNA knockdown of p53 completely abolished the PDMF-induced G2/M cell cycle arrest, and substantially abrogated its proapoptotic potency. These results suggest that PDMF represents a useful tumor-preventive phytochemical that triggers p53-driven G2/M cell cycle arrest and apoptosis.

A novel moonlight function of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for immunomodulation.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an energy metabolism-related enzyme, which generates NADH in glycolysis. Our previous study revealed a novel role of exogenous GAPDH in the amelioration of lipopolysaccharide (LPS)-induced sepsis-related, severe acute lung injury (ALI) in mice. Here, we show the effect of extracellular GAPDH on the physiological functions of macrophages, which play an important role in the onset of sepsis and ALI. GAPDH has no effect on cell viability, while it strongly suppressed cell adhesion, spreading, and phagocytic function of LPS-stimulated macrophages. GAPDH treatment significantly reduced tumor necrosis factor (TNF)-α, while it induced interleukin (IL)-10 production from LPS-stimulated macrophages in a dose-dependent manner. It is noteworthy that heat inactivation of GAPDH lost its immunomodulatory activity. Correspondingly, NADH significantly inhibited TNF-α and enhanced IL-10 production with elevation of both M1/M2 macrophage markers. These data suggest that extracellular GAPDH induces intermediate M1/M2 macrophages for termination of inflammation, partly through its enzyme activity for generation of NADH. © 2018 BioFactors, 44(6):597-608, 2018.

Synergistic tumor suppression by a Perilla frutescens-derived methoxyflavanone and anti-cancer tyrosine kinase inhibitors in A549 human lung adenocarcinoma.

Anti-cancer tyrosine kinase inhibitors (TKIs) are effective in many types of cancers including non-small cell lung cancer, while appearance of TKI-resistant tumors suggests a need for the development of their potentiation strategies. We have previously shown that a methoxyflavanone derivative from the Asian medicinal herb Perilla frutescens (Perilla-derived methoxyflavanone; PDMF) shows a prominent anti-tumor activity against A549 human lung adenocarcinoma. Here we show that PDMF and anti-cancer TKIs (nilotinib, bosutinib, dasatinib, and ponatinib) synergistically suppress proliferation of A549 cells. Flow cytometric analysis indicated that co-stimulation with nilotinib (4 µM) and PDMF induced G2/M cell cycle arrest in low PDMF doses (10-50 µM), whereas this combination triggered de novo G1 arrest in higher PDMF dosages (50-125 µM). We also found that co-administration with nilotinib and PDMF significantly suppressed in vivo tumorigenicity of A549 cells in athymic nude mice.

Prominent IgE-binding and cytokine-inducing capacities of a newly cloned N-terminal region of Der f 14, an apolipophorin-like house dust mite allergen.

We previously characterized a 177-kDa allergen, M-177, from Dermatophagoides farinae. Thereafter, a counterpart to M-177 for Euroglyphus maynei was cloned as Eur m 14, and its sequence revealed that two environmental allergens, Mag 1 and Mag 3, are digested fragments of M-177. The aims of this study were to clone the cDNA of Der f 14 corresponding to M-177 and to elucidate the allergenic capacities of the N-terminal fragment of Der f 14 (Der f 14-N). Recombinant allergens were produced as trigger-factor-fused proteins in Escherichia coli. Der f 14-N showed the highest IgE-binding frequency among Der f 14-derived fragments in patients allergic to house dust mite by enzyme-linked immunosorbent assay. Der f 14-N showed the highest capacity to induce cell proliferation in murine lymphocyte and human peripheral mononuclear cells among Der f 14-derived fragments. Der f 14-N induced IL-13, IFN-γ and IL-17 production more than Der f 1 and Der f 2 in mouse, and induced IL-5 and IFN-γ production at levels comparable to those of Der f 1 and Der f 2 in some patients. The high prevalence of IgE binding to the Der f 14-N indicates that it could be an important mite allergen.