Effects of heat treatment on the antigenicity, antigen epitopes, and structural properties of ß-conglycinin.

In this study, the effects of heat treatment on antigenicity, antigen epitopes, and structural changes in ß-conglycinin were investigated. Results showed that the IgG (Immunoglobulin G) binding capacity of heated protein was inhibited with increased temperature, although IgE (Immunoglobulin E) binding capacity increased. Linear antigen epitopes generally remained intact during heat treatment. After heat treatment, ß-conglycinin was more easily hydrolyzed by digestive enzymes, and a large number of linear epitopes was destroyed. In addition, heat denaturation of ß-conglycinin led to the formation of protein aggregates and reduction of disulfide bonds. The contents of random coils and ß-sheet of heated ß-conglycinin decreased, but the contents of ß-turn and α-helix increased. Moreover, the protein structure of heated ß-conglycinin unfolded, more hydrophobic regions were exposed, and the tertiary structure of ß-conglycinin was destroyed. Heat treatment affected the antigenicity and potential sensitization of ß-conglycinin by changing its structure.

Cloning, expression and immunological characterisation of Coc n 1, the first major allergen from Coconut pollen.

Coconut pollen has been documented to be a major contributor to the aeroallergen load in India, causing respiratory allergy in a large cohort of susceptible individuals. Here, we report the identification of the first major allergen from Coconut pollen, Coc n 1. The full-length sequence of the allergen was determined from previously identified peptides and overexpressed in E. coli. Recombinant Coc n 1 folded into a trimer and was found to possess allergenicity equivalent to its natural counterpart. Proteolytic processing of Coc n 1 led to the formation of an immunodominant ∼20 kDa C-terminal subunit and the site of cleavage was determined by amino acid microsequencing. Five linear IgE binding epitopes were predicted and mapped on the homology modelled structure of Coc n 1. Amongst three immunodominant epitopes, two were present towards the C-terminal end. Coc n 1 was found to belong to the highly diverse cupin superfamily and mimics its structure with known 7S globulin or vicilin allergens but lacks sequence similarity. Using sequence similarity networks, Coc n 1 clustered as a separate group containing unannotated cupin domain proteins and did not include known vicilin allergens except Gly m Bd 28 kDa, a Soybean major allergen. 7S globulins are major storage proteins and food allergens, but presence of such protein in pollen grains is reported for the first time. Further study on Coc n 1 may provide insights into its function in pollen grains and also in the development of immunotherapy to Coconut pollen allergy.

MHC II-PI3K/Akt/mTOR Signaling Pathway Regulates Intestinal Immune Response Induced by Soy Glycinin in Hybrid Grouper: Protective Effects of Sodium Butyrate.

Soy glycinin (11S) is involved in immune regulation. As an additive, sodium butyrate (SB) can relieve inflammation caused by 11S. To further delve into the mechanisms. A diet containing 50% fishmeal was the control group (FM group), and the experimental groups consisted of the FM group baseline plus 2% glycinin (GL group), 8% glycinin (GH group), and 8% glycinin + 0.13% sodium butyrate (GH-SB group). The specific growth ratio (SGR), feed utilization, and density of distal intestinal (DI) type II mucous cells were increased in the GL group. In the serum, IFN-γ was significantly upregulated in the GL group, and IgG and IL-1ß were upregulated in the GH group. IgG, IL-1ß, and TNF-α in the GH-SB group were significantly downregulated compared to those in the GH group. The mRNA levels of mTOR C1, mTOR C2, and Deptor were upregulated in the GL, GH, and GH-SB groups in the DI compared with those in the FM group, while the mRNA levels of mTOR C1 and Deptor in the GH group were higher than those in the GL and GH-SB groups. 4E-BP1, RICTOR, PRR5, MHC II, and CD4 were upregulated in the GH group. TSC1, mLST8, and NFY mRNA levels in the GL and GH-SB groups were upregulated compared with those in the FM and GH groups. Western blotting showed P-PI3KSer294/T-PI3K, P-AktSer473/T-Akt, and P-mTORSer2448/T-mTOR were upregulated in the GH group. Collectively, our results demonstrate that low-dose 11S could improve serum immune by secreting IFN-γ. The overexpression of IgG and IL-1ß is the reason that high-dose 11S reduces serum immune function, and supplementing SB can suppress this overexpression. Low-dose 11S can block the relationship between PI3K and mTOR C2. It can also inhibit the expression of 4E-BP1 through mTOR C1. High-dose 11S upregulates 4E-BP2 through mTOR C1, aggravating intestinal inflammation. SB could relieve inflammation by blocking PI3K/mTOR C2 and inhibiting 4E-BP2. Generally speaking, the hybrid grouper obtained different serum and DI immune responses under different doses of 11S, and these responses were ultimately manifested in growth performance. SB can effectively enhance serum immunity and relieve intestinal inflammation caused by high dose 11S.

Mining Novel Allergens from Coconut Pollen Employing Manual De Novo Sequencing and Homology-Driven Proteomics.

Coconut pollen, one of the major palm pollen grains is an important constituent among vectors of inhalant allergens in India and a major sensitizer for respiratory allergy in susceptible patients. To gain insight into its allergenic components, pollen proteins were analyzed by two-dimensional electrophoresis, immunoblotted with coconut pollen sensitive patient sera, followed by mass spectrometry of IgE reactive proteins. Coconut being largely unsequenced, a proteomic workflow has been devised that combines the conventional database-dependent analysis of tandem mass spectral data and manual de novo sequencing followed by a homology-based search for identifying the allergenic proteins. N-terminal acetylation helped to distinguish "b" ions from others, facilitating reliable sequencing. This led to the identification of 12 allergenic proteins. Cluster analysis with individual patient sera recognized vicilin-like protein as a major allergen, which was purified to assess its in vitro allergenicity and then partially sequenced. Other IgE-sensitive spots showed significant homology with well-known allergenic proteins such as 11S globulin, enolase, and isoflavone reductase along with a few which are reported as novel allergens. The allergens identified can be used as potential candidates to develop hypoallergenic vaccines, to design specific immunotherapy trials, and to enrich the repertoire of existing IgE reactive proteins.

Diversification of 13S globulins, allergenic seed storage proteins, of common buckwheat.

The α polypeptide of the 13S globulin subunit of common buckwheat is the counterpart of the major allergenic ß polypeptide. Trypsin digestibility varies between variants of the α polypeptide with and without a tandem repeat insert. To evaluate the intra-species diversity of 13S globulin, the comprehensive screening of a genomic DNA library was performed, resulting in the isolation of 14 and 3 genes for Met-poor and Met-rich subunits, respectively. Although most tandem repeat units were 45 bp in length, the two-repeat gene Glb2B and all one-repeat genes contained an additional 3 bp. Secondary structure predictions and polyacrylamide gel electrophoresis demonstrated that the sense strand of Glb2B-CCG, the additional 3 bp-deletion clone of Glb2B, formed a more rigid secondary structure than that of the wild-type. Thus, the large intra-species variation of 13S globulin revealed in this study and its diversification might be attributable to the unique nature of the tandem repeat sequences.

Role of soybean ß-conglycinin subunits as potential dietary allergens in piglets.

ß-Conglycinin, a major seed-storage protein in soybeans, is one of the primary antigenic proteins responsible for soybean-meal hypersensitivity in weaned piglets. The protein is a heterotrimer composed of subunits α, α' and ß. It is currently unknown which of the ß-conglycinin subunits are allergenic for piglets. The aim of this study was to identify potential allergenic subunits of ß-conglycinin for soybean sensitive piglets and to characterise these subunits by immunoglobulin (Ig) G and E immunoblotting, ELISA, 'skin prick' and whole blood histamine-release testing. The IgG and IgE binding capabilities of the purified α, α' and ß subunits of ß-conglycinin were determined by immunoblot analysis and ELISA with sera from ß-conglycinin sensitised piglets. Skin prick testing and whole blood histamine release testing were also performed to detect the activated effector cell response to specific allergens. Specific IgG and E antibodies were identified that recognised all three subunits of ß-conglycinin in the sera of ß-conglycinin sensitised piglets. All three subunits of ß-conglycinin elicited positive skin test and specific histamine release responses from the whole blood of ß-conglycinin sensitised piglets. These results suggest that all three ß-conglycinin subunits are potential allergens for piglets.

Saikosaponin-d inhibits ß-conglycinin induced activation of rat basophilic leukemia-2H3 cells.

ß-Conglycinin is one of the major storage proteins in soybean and has been identified as a potential diagnostic marker for severe allergic reactions to soybean. Unfortunately, there is a lack of information on the signal transduction pathways of ß-conglycinin induced mast cell activation and how to alleviate these allergic reactions. Bupleurum falcatum, a traditional oriental medicine, has been widely utilized in the treatment of influenza, fever, malaria and menstrual disorders. Furthermore, it has been reported that saikosaponins, the important principle of B. falcatum, possesses anti-allergic activities. Therefore, the present study investigated whether or not saikosaponin-d, an extract of B. falcatum, was effective in the treatment of allergic reactions cased by ß-conglycinin, using a rat basophilic leukemia-2H3 cell line. There were multiple signaling pathways contributing to the development of ß-conglycinin-mediated rat basophilic leukemia-2H3 cell activation. The intracellular calcium mobilization and tyrosine phosphorylation were early events, which in turn elicited reactive oxygen species production, gene activation of Cdc42 and c-Fos, and ultimately led to ß-hexosaminidase release. Saikosaponin-d inhibited rat basophilic leukemia-2H3 cell degranulation by suppressing these critical incidents in the signal transduction pathway. These results suggest that saikosaponin-d exhibited anti-allergic activity and could become an effective herbal therapy for alleviating soybean allergy.

Anaphylaxis to buckwheat in an atopic child: a risk factor for severe allergy to nuts and seeds?

Common buckwheat (Fagopyrum esculentum) is known to cause severe anaphylactic reactions in adult individuals. However, type I allergy to buckwheat is rarely seen in children. We report on a 7-year-old boy who developed a grade III anaphylactic reaction after consumption of a cake containing buckwheat flour. Prior to this incident, the boy had developed severe allergic reactions to hazelnuts and suffered from an oral allergy syndrome to poppy seed. Analysis of the patient's IgE reactivity by immunoblotting experiments revealed that he was sensitized to members of the 2S albumin and 11S globulin protein families in buckwheat. Additionally, cross-reactivity was found between the 11S globulins in buckwheat, poppy and hazelnut. IgE inhibition experiments indicated that the 11S globulin in buckwheat was the initial sensitizing protein. We conclude that 11S globulins in buckwheat have the potential to induce IgE antibodies cross-reactive with 11S globulins in other, botanically unrelated foods and may induce anaphylactic reactions.

The effects of lipoic acid on soybean beta-conglycinin-induced anaphylactic reactions in a rat model.

The purpose of this study was to evaluate the effects of feeding a low dose of lipoic acid on attenuating soybean beta-conglycinin-induced hypersensitivity using a rat model, with ovalbumin as the positive allergic control. Forty-eight recently weaned male Sprague-Dawley rats were assigned to four treatments and fed a cornstarch-casein-based diet either unsupplemented (Groups I, II and III) or supplemented with 25 mg/kg lipoic acid (Group IV). On days 1, 10, 17, and 24, Groups III and IV were sensitised with 20 mg beta-conglycinin by means of intragastric gavage, while Group II was sensitised with 20 mg ovalbumin and Group I (control) with casein. On day 31, rats received a double dose of beta-conglycinin, ovalbumin or casein, respectively. Ovalbumin-sensitised rats (Group II) and beta-conglycinin-sensitised rats (Group III) demonstrated an increase in serum IgE and histamine release, but reduced growth performance compared to the control (Group 1) (p < 0.05). A low dose of lipoic acid had no effect on average weight gain, but increased villus height in the jejunum (p < 0.05), while reducing serum beta-conglycinin-specific IgE and histamine content in the jejunum. Moreover, lipoic acid supplementation did not significantly affect interferon-gamma or interleukin-4. Taken together, our results suggest that a low dose of lipoic acid could potentially be used as an immunomodulator to attenuate soybean beta-conglycinin induced allergies.

Influence of early nutritional components on the development of murine autoimmune diabetes.

BACKGROUND/AIMS: Infant diet is suggested to modify autoimmune diabetes risk. The aim of this study was to determine whether infant food components affect diabetes development in the nonobese autoimmune diabetes (NOD) mouse. METHODS: A basal low-diabetogenic diet was identified by feeding litter-matched female NOD mice standardized diets with and without casein and wheat proteins after weaning. In subsequent trials, basal diet with supplements of wheat (5, 10 and 30%), gluten, wheat globulin/albumin, corn (5%), potato (5%), apple (5%) or carrot (5%) was fed to litter-matched female NOD mice after weaning. Mice were followed for diabetes development and insulin autoantibodies. RESULTS: A casein- and wheat-free diet was associated with the lowest rate of diabetes development (37% by age 25 weeks). Increased diabetes rates were observed when the basal diet was supplemented with 5% wheat (71% by age 25 weeks; p = 0.023) and 5% corn (57% by age 25 weeks; p = 0.05). Increasing wheat concentrations returned diabetes development to that in basal diet-fed mice. Other food supplements had no or minimal effects on diabetes development. CONCLUSIONS: Early supplementation of a basal low-diabetogenic diet with low concentrations of the cereals wheat or corn is associated with a moderate increase in the rate of diabetes. Removal of cereals, however, does not abrogate diabetes development in NOD mice.

IgE-binding epitopic peptide mapping on a three-dimensional model built for the 13S globulin allergen of buckwheat (Fagopyrum esculentum).

The three-dimensional model built for the 13S globulin allergen of buckwheat (Fagopyrum esculentum) consists of three protomers exhibiting the cupin motif, arranged in a homotrimer around a three-fold symmetry axis. Using the SPOT technique, 11 continuous IgE-binding epitopic peptides were characterized on the molecular surface of the 13S globulin allergen of buckwheat. Except for one of them, they all correspond to well exposed regions containing electropositiveley and/or electronegatively charged residues, which cover up to 40% of the molecular surface of the allergen. Some of these epitopes come in close contact to probably create more extended discontinuous epitopes, especially those located on the edge of the 13S globulin homotrimer. Half of the identified epitope peptides remain unaltered in a core structure protected against hydrolysis by digestive proteases and are thus assumed to promote the allergenicity of the 13S globulin. In addition, a few of these epitopes coincide with sequential IgE-binding epitopes previously characterized in soybean 11S globulins, that could account for the IgE-binding cross-reactions observed between soybean and buckwheat in Western blot experiments.

Differences in level and avidity of secretory IgA antibodies in breast milk of Swedish, Indian and Japanese mothers to soybean protein.

Colostrum was collected from Swedish, Indian and Japanese mothers. The samples were as a mean, collected 4.00-4.25 days after delivery of term infants. The level of specific IgA antibody to 2S, 7S and crude soybean antigen were measured by the enzyme-linked immunosorbent assay (ELISA). The avidity of the IgA antibodies to 7S soybean antigen was also measured with an ELISA system using different molarities of potassium thiocyanate for elution of the specific IgA antibody from solid phase-bound antigen. The level of specific IgA antibody to 7S and crude soybean antigen in the milk of the Indian mothers was significantly higher than in the milk of the Japanese mothers (p less than or equal to 0.01). In contrast, the avidity expressed as the molarity of KSCN for 50% elution of IgA antibody to 7S soybean antigen in the milk of the Japanese mothers was significantly higher than in the milk of the Indian mothers (p less than 0.01).

IgA subclasses of human colostral antibodies specific for microbial and food antigens.

The distribution of total and antigen-specific IgA1 and IgA2 antibodies in human colostrum was determined by ELISA using subclass-specific monoclonal reagents. In 18 samples of colostrum the mean ratio of total IgA1 to IgA2 was found to be 53:47, respectively, but significant individual variations were observed. In two samples we found unusually low levels of IgA1, while IgA2 was in the normal range. IgA1 and IgA2 antibody activities were determined against the following antigens: bovine gamma-globulin and beta-lactoglobulin, tetanus toxoid, protein antigen I/II of Streptococcus mutans, influenza virus vaccine, polysaccharides of pneumococcal, meningococcal and Haemophilus influenzae type b origin, and lipopolysaccharide (LPS) from Escherichia coli K235. The IgA antibody activity directed against the polysaccharides was almost equally distributed between the two subclasses. However, antibody activity specific for protein antigens was found predominantly in the IgA1 subclass while anti-LPS activity was mostly of the IgA2 subclass.