Subunits Of Β-conglycinin Research Articles

Preliminary X-ray analysis of the binding domain of the soybean vacuolar sorting receptor complexed with a sorting determinant of a seed storage protein.

β-Conglycinin is a major seed storage protein in soybeans, which are an important source of protein. The major subunits (α, α' and β) of β-conglycinin are sorted to protein-storage vacuoles in seed cells. Vacuolar sorting receptor (VSR) is an integral membrane protein that recognizes the sorting determinant of vacuolar proteins, including β-conglycinin, and regulates their sorting process. Vacuolar sorting determinants of the α' and β subunits of β-conglycinin exist in their C-terminal peptides. Here, the preliminary X-ray diffraction analysis of the binding domain of soybean VSR crystallized with the peptide responsible for the sorting determinant in β-conglycinin is reported. X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to space group P3121, with unit-cell parameters a = b = 116.4, c = 86.1 Å.

The Investigation of Virginiamycin-Added Fungal Fermentation on the Size and Immunoreactivity of Heat-Sensitive Soy Protein

The usage of soy protein for young monogastric animals is restricted due to potential allergens and high molecular weight. The investigation of fungi fermentation effect on soy protein has been interrupted by substrate sterilization. Virginiamycin at 0.05% was added together withAspergillus oryzaefor solid state fermentation (SSF) in unsterilized soy meal (SM). When compared toA. oryzaeSSF alone, virginiamycin did not cause the interference of fungal fermentation but elucidated the protein degradation. SDS-PAGE results showed that bothαandα′ subunits ofβ-conglycinin were degraded significantly. In addition, western blot results showed that the immunoreactive signals of soy protein were considerably reduced in virginiamycin-added fermentation with unsterilized SM. Furthermore, fungal fermentation increased total protein and essential amino acid contents, suggesting the value enhancement of SM products. Taken together, this study demonstrated for the first time that virginiamycin could help investigate fermentation effect on heat-sensitive soy protein. Fermented SM has several potential applications in feed industry.

Proteolytic activity of Lactobacillus strains on soybean proteins

The capacity of Lactobacillus strains for degrading soy proteins was evaluated. The hydrolytic action was monitored by SDS–PAGE and reverse phase-HPLC analyses. Peptidase activities were also determined using synthetic chromogenic substrates. All assayed strains were able to hydrolyze α'-subunit of β-conglycinin while for the other fractions the degree of degradation was strain-dependent. The pattern of peptides produced from soy proteins hydrolysis differed amongst the lactobacilli studied, suggesting different proteolytic enzyme specificities.

Development of a novel transgenic rice with hypocholesterolemic activity via high-level accumulation of the α′ subunit of soybean β-conglycinin

Soybean 7S globulin, known as β-conglycinin, has been shown to regulate human plasma cholesterol and triglyceride levels. Furthermore, the α' subunit of β-conglycinin has specifically been shown to possess low-density lipoprotein (LDL)-cholesterol-lowering activity. Therefore, accumulation of the α' subunit of β-conglycinin in rice seeds could lead to the production of new functional rice that could promote human health. Herein, we used the low-glutelin rice mutant 'Koshihikari' (var. a123) and suppressed its glutelins and prolamins, the major seed storage proteins of rice, by RNA interference. The accumulation levels of the α' subunit in the lines with suppressed glutelin and prolamin levels were >20mg in 1g of rice seeds, which is considerably higher than those in previous studies. Oral administration of the transgenic rice containing the α' subunit exhibited a hypocholesterolemic activity in rats; the serum total cholesterol and LDL cholesterol levels were significantly reduced when compared to those of the control rice (var. a123). The cholesterol-lowering action by transgenic rice accumulating the α' subunit induces a significant increase in fecal bile acid excretion and a tendency to increase in fecal cholesterol excretion. This is the first report that transgenic rice exhibits a hypocholesterolemic activity in rats in vivo by using the β-conglycinin α' subunit.

Heat-induced aggregation and sulphydryl/disulphide reaction products of soy protein with different sulphydryl contents

In this study, soy proteins were reduced with 0.1–10mM dithiothreitol (DTT) to obtain an increasing number of sulphydryl groups (SH) with a similar particle size. Aggregation was promoted by increasing the degree of reduction when heated (100°C, 30min), resulting in larger sized aggregates (from 40 to 70nm) and a higher viscosity of the aggregate dispersion. The disulphide bond (SS) content decreased and the less SS linked polymer, which was composed of acidic (A) polypeptide of glycinin, basic (B) polypeptides of glycinin, and a small amount of α′ and α subunits of β-conglycinin, was formed with increasing reduction degree, suggesting that SH/SS polymerisation was not the driving force for aggregation. The larger aggregates with increasing degrees of reduction were composed of more B of glycinin and β of β-conglycinin, suggesting that the A and the small amount of α′ and α in the SS linked polymer have an inhibiting effect on protein aggregates formation.

Protein particle and soluble protein structure in prepared soymilk

Systematically characterizing soymilk protein is important for understanding the formation of soymilk-related products and may be beneficial for enhancing the qualities of these products. Field emission scanning electron microscopy showed that soymilk protein could be divided into three categories: large (40–258 nm) and small (20–40 nm) stable protein particles and <20 nm soluble proteins. Regardless of soybean variety, the large protein particles contained lipoxygenase, γ-conglycinin, lectin, and Kunitz trypsin inhibitor as well as glycinin and β-conglycinin. These large protein particles were comprised of 40-nm protein aggregates (around 40 nm) formed by heat-dissociated soybean protein subunits. In addition, the small stable protein particles, which accounted for about 10% of soymilk protein, increased with β subunits of β-conglycinin while the soluble proteins (<20 nm) existed as monomers and oligomers of α′, α and acidic polypeptides.

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

β-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 β. It is currently unknown which of the β-conglycinin subunits are allergenic for piglets. The aim of this study was to identify potential allergenic subunits of β-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 β subunits of β-conglycinin were determined by immunoblot analysis and ELISA with sera from β-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 β-conglycinin in the sera of β-conglycinin sensitised piglets. All three subunits of β-conglycinin elicited positive skin test and specific histamine release responses from the whole blood of β-conglycinin sensitised piglets. These results suggest that all three β-conglycinin subunits are potential allergens for piglets.

The α′ subunit of β-conglycinin and the A1–5 subunits of glycinin are not essential for many hypolipidemic actions of dietary soy proteins in rats

This study examined the effects of dietary soy protein (SP) lacking different storage protein subunits and isoflavones (ISF) on the abdominal fat, blood lipids, thyroid hormones, and enzymatic activities in rats. Weanling Sprague-Dawley rats (8 males and 8 females/group) were fed diets containing either 20 % casein without or with supplemental isoflavones or alcohol-washed SP isolate or SP concentrates (SPC) prepared from 6 different soy bean lines for 8 weeks. Feeding of diets containing SPC regardless of their subunit compositions significantly lowered relative liver weights, blood total, free, and LDL cholesterol in both genders (P < 0.05) and also reduced serum free fatty acids (FFA) and abdominal fat in females (P < 0.05) compared to the casein or casein + ISF diets. Dietary SPC significantly elevated the plasma free triiodothyronine (T3) in both genders and total T3 in females compared to the casein diet (P < 0.05). The SPC lacking β-conglycinin α' and either the glycinin A1-3 or A1-5 subunits increased total T3 in males and reduced plasma enzymatic activities of creatine kinase and lactate dehydrogenase compared to casein or casein + ISF diet (P < 0.05). Soy isoflavones were mainly responsible for the hypocholesterolemic effects and increased plasma free T3, whereas reduction in FFA, abdominal fat, liver weight and increased plasma total T3 were the effects of the soy proteins. Neither the α' subunit of β-conglycinin nor the A1-5 subunits of glycinin are essential for the hypolipidemic properties of soy proteins.

Effect of pH and Pepsin Limited Hydrolysis on the Structure and Functional Properties of Soybean Protein Hydrolysates

Effects of limited enzymatic hydrolysis with pepsin on the functional properties and structure characteristics of soybean proteins were investigated. Hydrolysates with different incubation time (10 to 900 min) were prepared. Results showed that SPI hydrolyzed for 60 min exhibited the best emulsibility and the ability of resisting freezing/thawing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis proved that pepsin can degrade glycinin but had little effect on the α' subunit of β-conglycinin. The structure unfolding reached the largest extent after incubation for 60 min and the soluble and flexible aggregates were formed. After 120 min, glycinin was degraded totally and β-conglycinin formed insoluble aggregates. Moreover, 2 methods were applied for the deactivation of pepsin to obtain final hydrolysates at pH 2.0 and 7.0, respectively. The structure analysis revealed that the unfolding extent and structure characteristic were different in these 2 conditions. When adjusting the pH value from 2.0 to 7.0, the unfolding protein molecular would reaggregate again at pH 7.0 due to the charge neutralization, and the hydrodynamic diameter and λmax absorbance decreased compared to pH 2.0. Moreover, some of the insoluble aggregates formed at pH 2.0 became soluble at pH 7.0, because of the salt-in phenomenon.

Effects of Malondialdehyde Modification on the in Vitro Digestibility of Soy Protein Isolate

Soy protein isolate (SPI) was modified by lipid peroxidation product malondialdehyde (MDA), and the in vitro digestibility of modified SPI was investigated. Results indicated that incubation with increasing MDA concentration resulted in significant carbonyl group generation and loss of free amino groups of SPI. Fluorescence loss of natural tryptophan and formation of Schiff base were observed. Noncovalent interaction between molecules was enhanced and became the main force that led to the solubility reduction of MDA-modified SPI. Differential scanning calorimetry (DSC) indicated that SPI had higher thermal stability and lower total calorimetric enthalpy after MDA pretreatment. Electrophoresis showed that β-conglycinin was more sensitive to MDA modification. In vitro digestion indicated that MDA could induce non-disulfide covalent polymer of SPI, which could not be digested by pepsin and pancreatin. β subunits of β-conglycinin became more resistant to digestion with increasing MDA concentration. Evaluation of the free amino acid profile in the digests indicated that MDA-modified SPI had deteriorating nutritive quality.

Structural characterization of the N-glycosylation of individual soybean β-conglycinin subunits

Soybean (Glycine max) 7S β-conglycinin is a seed storage protein consisting of homo- and hetero-trimers of three subunits, namely α (∼67kDa), α′ (∼71kDa), and β (∼50kDa), non-covalently associated. The N-glycans released from the whole β-conglycinin have been already characterized by 1H NMR some decades ago. Nevertheless, the actual glycosylation of the potential sites and the glycoforms of the individual subunits have not been specifically investigated so far. In this study, up-to-date chromatographic, electrophoretic and mass spectrometric strategies have been combined to achieve the structural characterization of the glycoforms of the three individual β-conglycinin subunits. Glycosylation sites were assigned by analyzing the tryptic glycopeptides of the isolated subunits. Underivatized N-glycans were purified with a two-step clean-up, consisting in sequential reversed-phase and activated porous graphitized carbon micro-chromatography, and profiled by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS).

Reduction of the allergenic protein in soybean meal by enzymatic hydrolysis

The effects of Alcalase and Trypsin hydrolysis on the allergenic protein and its solubility of soybean meal (SBM) were investigated by SDS-PAGE and ELISA. After 10 min of hydrolysis with Alcalase, the solubility of the protein was 87.19%, the α′, α and β-subunits of β-conglycinin disappeared and the acidic and basic subunits of glycinin decreased obviously by SDS-PAGE pattern. However, with Trypsin for 10 min, the solubility of the protein was 72.74%. The contents of α′, α subunits of β-conglycinin decreased slightly in SDS-PAGE, and the acidic and basic subunits of glycinin were almost not affected. For the insoluble hydrolysates prepared by Alcalase, the α′, α subunits of β-conglycinin and acidic subunit of glycinin decreased significantly, that by trypsin did not. The content of immune-reactive β-conglycinin obtained by ELISA was consistent with them. These results confirmed that the allergenic proteins of SBM were more sensitive to Alcalase than to Trypsin.

LC-ESI-TOF MS Method for the Evaluation of the Immunostimulating Activity of Soybeans via the Determination of the Functional Peptide Soymetide

Bioactive peptides content in foodstuffs can seriously vary with many factors such as crop variety, food processing, animal breeding, etc. Because of this variability, quantitative methodologies are required to evaluate the content of bioactive peptides in foodstuffs. Progress in liquid chromatography and mass spectrometry technologies offer a great opportunity for the quantitation of bioactive peptides. This study undertook the development of a liquid chromatography-electrospray ionization-time-of-flight mass spectrometry method using a fused-core technology column for the sensitive and unambiguous determination of the immunostimulating peptide soymetide in soybean varieties. Soymetide precursor protein (α' subunit of β-conglycinin) was extracted with a Tris-HCl buffer (pH 8.0) containing urea and digested with trypsin. Soymetide separation conditions by reversed phase liquid chromatography (ion-pairing reagent, organic modifier, separation column, and elution gradient) and detection by MS were optimized, and a study of soymetide stability was also conducted. The method selectivity having been demonstrated, the linearity, accuracy, precision, and limits of detection and quantitation were evaluated. The developed method enabled the detection and quantitation of soymetide concentrations in the ppb range (7.5 ng/mL and 25 ng/mL, respectively), and about 30 times lower than those detected and determined in a previous work by capillary liquid chromatography with UV detection. These values could allow the quantitation of only 17 μg of soymetide per gram of soybean. The developed methodology was applied to the quantitation of soymetide in different soybean varieties from Europe, Japan, and USA observing great differences in soymetide content that ranged from 40 to 600 μg per gram of soybean depending on the soybean variety.

Prediction and characterization of the linear IgE epitopes for the major soybean allergen β-conglycinin using immunoinformatics tools

The α subunit of β-conglycinin is a major allergen in soybean. The objective of this study was to predict and identify the linear immunoglobulin (Ig)E epitopes of the soybean α subunit of β-conglycinin. Three immunoinformatics tools were used to predict the potential epitopes and were confirmed by dot-blot inhibition using sera from soybean allergic subjects. As a result, 15 peptides were predicted and assembled by solid-phase synthesis. Eleven epitopes were identified by the dot-blot inhibition test. Moreover, peptide 3 had IgE binding capability with all sera(5/5) tested, while peptide 1, 4, 6, 8 and12 could bind to 4/5 of the sera samples. Secondary structure prediction of peptide 3 and circular dichroism test validated that the structure of peptide 3 was a random coil.

Purification and IgE-binding properties of soybean β-conglycinin subunits

The purpose of this study was to purify soybean β-conglycinin subunits and analyze their IgE-binding properties with IgE immunoblotting and their response in a basophil histamine release test. The method presented in this paper offers an effective procedure to purify β-conglycinin subunits with higher yield and purity compared with previous methods by using a combination of ion exchange and metal chelate affinity chromatography. Beginning with a column load of 600mg β-conglycinin, the new method yielded 100.4mg of protein containing 96.7% α subunit, 97.0mg of protein containing 91.6% α′ subunit and 89.4mg of protein containing 96.1% β subunit. All of the purified subunits of β-conglycinin reacted with IgE from sera of soybean-allergic patients and induced dose-dependent histamine release from basophils from these patients, suggesting that purified α, α′, and β subunits of β-conglycinin retained allergenic activity and could be used for in vitro and in vivo diagnosis of soybean-induced food allergy.

Effect of concentration and incubation temperature on the acid induced aggregation of soymilk

The molecular details of the aggregation of soy protein particles in soymilk during acid-induced gelation using glucono-delta-lactone (GDL) were investigated. Soymilk samples were prepared from different water-to-bean ratios and contained approximately 4% and 7% protein. The effects of protein concentration and incubation temperature (30 or 7 °C) on soymilk gelation were observed using rheology and diffusing wave spectroscopy at two different GDL concentrations. During acidification, there was a decrease in electrostatic repulsion between particles which well correlated with the pH of aggregation determined by dynamic light scattering. Gelation of soymilk occurred at about pH 5.8, and neither the rate of acidification nor the protein concentration affected the gelation pH. Gel stiffness was affected by protein concentration. A detailed study of the soluble fraction during the preceding stages of aggregation clearly demonstrated that glycinin components were the first to destabilize during acidification, followed by the β-conglycinin subunits. Decreasing the incubation temperature from 30 °C to 7 °C increased the pH of gelation and the gel modulus (G′) measured by rheology. It was concluded that short range interactions play a major role in the formation of the protein network in soymilk curd.

The β-conglycinin deficiency in wild soybean is associated with the tail-to-tail inverted repeat of the α-subunit genes

β-conglycinin, a major seed protein in soybean, is composed of α, α', and β subunits sharing a high homology among them. Despite its many health benefits, β-conglycinin has a lower amino acid score and lower functional gelling properties compared to glycinin, another major soybean seed protein. In addition, the α, α', and β subunits also contain major allergens. A wild soybean (Glycine soja Sieb et Zucc.) line, 'QT2', lacks all of the β-conglycinin subunits, and the deficiency is controlled by a single dominant gene, Scg-1 (Suppressor of β-conglycinin). This gene was characterized using a soybean cultivar 'Fukuyutaka', 'QY7-25', (its near-isogenic line carrying the Scg-1 gene), and the F₂ population derived from them. The physical map of the Scg-1 region covered by lambda phage genomic clones revealed that the two α-subunit genes, a β-subunit gene, and a pseudo α-subunit gene were closely organized. The two α-subunit genes were arranged in a tail-to-tail orientation, and the genes were separated by 197 bp in Scg-1 compared to 3.3 kb in the normal allele (scg-1). In addition, small RNA was detected in immature seeds of the mutants by northern blot analysis using an RNA probe of the α subunit. These results strongly suggest that β-conglycinin deficiency in QT2 is controlled by post-transcriptional gene silencing through the inverted repeat of the α subunits.

Development of a competitive ELISA for the detection of soybean α subunit of β-conglycinin

The alpha subunit of β-conglycinin is one of the main allergens found in soybeans. For the preparation of a specific monoclonal antibody (mAb) against the α subunit, potential epitopes were predicted using Protean and evaluated by Wu's Antigenic Index. The specific epitope 85EQDERQFPFPR 95 was synthesized, and then conjugated to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) for use as an immunogen and the plate-coating antigen, respectively. The resulting mAb, termed mAb-60K, was characterized as belonging to the IgG1 isotype and containing the κ light chain; it exhibited high specificity for the α subunit and did not cross react with the α′ and β subunits of β-conglycinin or other proteins found within soybeans. A competitive enzyme-linked immunosorbent assay (cELISA) with high accuracy and reproducibility was developed based on mAb-60K and the plate-bound peptide. Co-incubation with the full-length α subunit showed a 50% mAb binding inhibition concentration (IC 50) value of 4.42 ng/mL, with a linear inhibition curve observed α subunit concentrations between 0.65 and 29.84 ng/mL. The newly developed mAb-60K and the companion cELISA could provide a valuable tool for determining sensitivity towards the α subunit of soybean β-conglycinin and for future studies on food allergies resulting from this protein.

Properties and microstructure of transglutaminase-set soy protein-stabilized emulsion gels

The development of cold-set protein-stabilized emulsion gels has attracted increasing interests, due to their potential to be applied as a kind of release-controlled carriers, especially for labile lipid-soluble bioactive compounds. This work aimed to elucidate the importance of changing oil volume fraction (ø=0.2–0.6) for the formation and properties of cold-set soy protein isolate (SPI)-stabilized emulsion gels, induced by microbial transglutaminase (MTGase). The gelation process, mechanical properties, water-holding capacity (WHC) and microstructure of the correspondingly formed gels at various ø values were evaluated. The results indicated that increasing ø progressively increased the storage modulus (gel strength) and WHC, from about 200 to 8000Pa and 52 to 88%, respectively. The high ø dependence of the gel strength and WHC was closely related to the microstructure of the formed gels. The network of the emulsion gels at high ø values (e.g. 0.6) was particulate in nature with coarse strands mainly composed of compact ‘aggregated’ oil droplets, while that at low ø values was filamentous or fine-stranded with oil droplets incorporated. The enzymatic treatment resulted in a progressive increase in amount of the proteins entrapped within the network, with highest extent observed at ø=0.5. The entrapped proteins involved the biopolymers of β-conglycinin subunits or glycinin acidic polypeptides, covalently cross-linked by the enzyme, together with glycinin basic polypeptides. The gelling mechanism at high ø values could be largely related to aggregation or coagulation of protein-coated oil droplets, while that at low ø values, the gels was more like enzyme-set protein gels. These results would be of great help for the understanding of the gelling mechanism and the development of enzyme-set protein-stabilized emulsion gels.

Growth Kinetics of Amyloid-like Fibrils Derived from Individual Subunits of Soy β-Conglycinin

The amyloid-like fibrillation of soy β-conglycinin subunits (α, α', and β) upon heating (0-20 h) at 85 °C and pH 2.0 was characterized using dynamic light scattering, circular dichroism (CD), binding to amyloid dyes (Thioflavin T and Congo red), and atomic force microscopy. The fibrillation of all three subunits was accompanied by progressive polypeptide hydrolysis. The hydrolysis behaviors, fibrillation kinetics, and morphologies of amyloid-like fibrils considerably varied among α, α', and β subunits. Faster hydrolysis rates and special fragments were observed for the α and α' subunits compared to the β subunit. However, the order of the fibrillation rate and capacity to form β-sheets was α' > β > α, as evidenced by CD and Thioflavin T data. Moreover, sequential growth of twisted screw-structure fibrils, leading to macroscopic fibrils with distinct morphological characteristics, was observed for β-conglycinin and individual subunits. The different fibrillation kinetics and morphologies of α, α', and β subunits appear to be associated with the differences in the amino acid composition and typical sequence of peptides. Besides, the disruption of ordered structure of fibrils occurred upon further heating (6-20 h) due to extensive hydrolysis. These results would suggest that all subunits are involved in the fibrillation of β-conglycinin, following multiple steps including polypeptide hydrolysis, assembly to amyloid structure, and growth into macroscopic fibrils with a fibril shaving process.