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.

Molecular cloning and immunochemical characterization of a new Japanese cedar pollen allergen homologous to plant subtilisin-like serine protease.

Protease activities in allergen sources are thought to be involved in triggering allergic inflammation through the disruption of epithelial barrier or the induction of proinflammatory cytokines. Protease allergens may also work as type 2 helper T cell (TH2) adjuvants through the cleavage of cell surface receptors. Here, we report molecular cloning and immunochemical characterization of a new Japanese cedar (Cryptomeria japonica) pollen allergen (CPA9) homologous to serine protease, which is initially found as a high IgE-binding spot on our two-dimensional (2-D) IgE immunoblotting map. The cpa9 cDNA encoded a 757 amino acid polypeptide showing a significant sequence identity with plant subtilisin-like serine protease family members including melon major allergen Cuc m 1. We found that native CPA9 purified from C. japonica pollen showed a high IgE-binding frequency and IgE cross-reactivity with melon extract.

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.

Peritoneal injection of fucoidan suppresses the increase of plasma IgE induced by OVA-sensitization.

We previously reported that fucoidan, a dietary fiber purified from seaweed, inhibited IgE production by B cells in vitro. In this study, we examined the effect of fucoidan on IgE production in vivo. The OVA-induced increase of plasma IgE was significantly suppressed when fucoidan was intraperitoneally, but not orally, administered prior to the first immunization with OVA. The production of IL-4 and IFN-gamma in response to OVA in spleen cells isolated from OVA-sensitized mice treated with fucoidan in vivo was lower than that from mice treated without fucoidan. Moreover, the flow cytometric analysis and ELISpot assay revealed that the administration of fucoidan suppressed a number of IgE-expressing and IgE-secreting B cells, respectively. These results indicate that fucoidan inhibits the increase of plasma IgE through the suppression of IgE-producing B cell population, and the effect of fucoidan in vivo is crucially dependent on the route and timing of its administration.

Development of autoantibody responses in NC/Nga mice: its prevention by pulverized konjac glucomannan feeding.

Dietary pulverized konjac glucomannan (PKGM) suppresses the development of eczema in NC/Nga mice, a model of atopic dermatitis (AD). Although NC/Nga mice were originally recognized as an autoimmune disease model, recent studies on their autoimmunity are still poorly performed. Here, we show that cervical lymphadenopathy, splenomegaly, and increases in plasma levels of anti-dsDNA, rheumatoid factor IgG autoantibodies, and B cell-activating factor of the TNF family (BAFF) were co-elicited in eczematous NC/Nga mice; however, these symptoms were all prevented in PKGM-fed mice. Our results imply the possible involvement of autoimmunity on the pathogenesis of dermatitis and hyper-IgE syndrome in NC/Nga mice. PKGM might be effective in preventing autoimmune responses in AD.

Dietary pulverized konjac glucomannan prevents the development of allergic rhinitis-like symptoms and IgE response in mice.

Konjac is a traditional Japanese food with a peculiar texture, and it has been suggested that its main ingredient, konjac glucomannan (KGM), ameliorates metabolic disorders such as diabetes and hypercholesteremia. We have found that feeding with pulverized KGM (PKGM) prevents skin inflammation in a murine model of atopic dermatitis. Here, we show that dietary PKGM suppresses allergic rhinitis-like symptoms in mice upon immunization and nasal sensitization with ovalbumin (OVA). The PKGM-fed mice showed a much lower frequency of sneezing than in control animals. We also found that PKGM supplementation exclusively suppressed OVA-specific IgE response without affecting IgG1/IgG2a responses as well as systemic Th1/Th2 cytokine production. These results suggest that PKGM can be a beneficial foodstuff in preventing nasal allergy such as seasonal pollinosis.

Dietary pulverized konjac glucomannan suppresses scratching behavior and skin inflammatory immune responses in NC/Nga mice.

BACKGROUND: Feeding with pulverized konjac glucomannan (PKGM) suppresses the development of eczema and hyper-IgE production in NC/Nga mice, a model of atopic dermatitis. This study aimed to examine the effects of PKGM on scratching behavior and skin inflammatory immune responses in NC/Nga mice. METHODS: Four-week-old NC/Nga mice were maintained for 8 or 9 weeks on diet containing PKGM. Scratching behavior and clinical symptoms were evaluated every 2 weeks. Effects of PKGM on cutaneous inflammation were evaluated by histopathological analysis. Local expression levels of substance P and proinflammatory cytokines were measured by ELISA. RESULTS: An increase in scratching behavior was evident from 6 weeks of age in control mice, but this symptom was dose-dependently inhibited in PKGM-fed mice. Continuous PKGM feeding then significantly inhibited eczematous skin lesions including hyperkeratosis, dermal mastocytosis and eosinophilia. Concomitantly, cutaneous overproductions of substance P, IL-10, IL-4, and TNF-alpha were all suppressed in PKGM-fed mice. CONCLUSIONS: PKGM feeding markedly suppressed development of scratching behavior, substance P expression with mastocytosis, and skin inflammatory immune responses in NC/Nga mice.

A new immunomodulatory function of low-viscous konjac glucomannan with a small particle size: its oral intake suppresses spontaneously occurring dermatitis in NC/Nga mice.

BACKGROUND: Konjac glucomannan (GM) is a well-known dietary fiber with various beneficial functions: the higher viscosity displayed the stronger potency. However, the high-viscous GM powders, ordinary konjac powder and highly purified GM were mostly unsuitable for the application to various food industries. Our aims are to develop new physiological functions of low-viscous GM powder, pulverized GM or re-granulated fine GM, using a murine model of atopic dermatitis. METHODS: Male 4-week-old NC/Nga mice were fed for 8 weeks on diets containing 5% of two high-viscous and two low-viscous GM powders, respectively. RESULTS: Striking suppression against the aggravation of dermatitis, the increase in scratching behaviors, and the rise in IgE levels was recognized only in mice fed on the pulverized GM diet, but not in mice fed on the other GM diets or a control diet. Eczema prevention in the fine GM-fed mice was accompanied by a significant decrease in their plasma IFN-gamma levels, a positive regulatory cytokine for atopic skin inflammation. CONCLUSION: Only the pulverized GM possessed the ability to suppress the development of dermatitis in NC/Nga mice. This is the new immunomodulatory function of low-viscous GM with a small particle size.

The ability of konjac-glucomannan to suppress spontaneously occurring dermatitis in NC/Nga mice depends upon the particle size.

Konjac glucomannan (GM) is a well-known dietary fiber with various beneficial functions, which the higher viscous GM is proposed to have the stronger potencies. Application of highly viscous GM to various food industries is limited for the sake of long elapsed time required to solubilize the GM. Thus, the exploration into any physiological function of low-viscous GM remained undone for a long time. Male 4-week-old NC/Nga mice, a model of atopic dermatitis (AD), were fed for 8 weeks on diets containing 5% each of a high-viscous GM powder, ordinary konjac or highly purified GM, and a low-viscous GM powder, pulverized GM or re-granulated fine GM. Striking suppression to the aggravation of dermatitis and the increases in scratching behaviors and IgE levels was significantly observed only in mice fed on the pulverized GM diet, but not in mice fed on the other GM diets or a control diet. We concluded that the low-viscous GM possesses a novel immunoregulatory function and the intensity of the function depends upon the particle size rather than the viscosity.