Alpha Subunit Of Beta-conglycinin Research Articles

The Mechanisms for Yuba Formation and Its Stable Lipid

Yuba is a protein-lipid film formed on soymilk surface by heating. It is characteristic of textured structure and long shelf life (3-6 months) in the dry state at room temperature. It was known that soymilk contained oil bodies, protein particles and soluble protein as main components. In this study their roles on Yuba formation were examined. Film was formed by heating an oil body suspension, but it was dispersed again by mixing; Tosan 205 soluble protein (mainly alpha' and alpha subunits of beta-conglycinin) formed film after a long time heating; Yumeminori soluble protein (mainly acidic peptides of glycinin) did not form a film even after all water was evaporated; Tosan 205 and Yumeminori non-lipid soymilks (containing protein particles and soluble protein) formed films by heating. Thus, it was concluded that protein particles were the most important for Yuba formation. In addition, Yuba was treated by liquid nitrogen, vacuum freeze-drying and observed by field emission scanning electron microscope (FESEM). The FESEM pictures showed that oil bodies, protein particles and soluble protein themselves were the "blocks" for Yuba network formation; the Yuba network was formed from protein, but oil bodies were incorporated in the network.

Structural Characterization of Heat-Induced Protein Particles in Soy Milk

This study analyzed the aggregation mode of polypeptides in protein particles of soy milk by using ultracentrifugation, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that the protein particles in soy milk were mainly formed by various complex protein aggregates. These protein aggregates were mainly composed of the basic and acidic polypeptides of glycinin (11S), which interact with each other via disulfide bonds, and a very small amount of the disulfide-linked alpha' and alpha subunits of beta-conglycinin (7S). Moreover, the protein aggregates and a part of monomeric subunits of 7S and 11S form protein particles through non-covalent interactions, especially hydrophobic interactions and hydrogen bonds. It is suggested that the polymerized basic polypeptides should be located inside the protein particles, whereas the acidic polypeptides of 11S, alpha' and alpha subunits are located outside them.

All Three Subunits of Soybean β-Conglycinin Are Potential Food Allergens

Soybeans are recognized as one of the "big 8" food allergens. IgE antibodies from soybean-sensitive patients recognize more than 15 soybean proteins. Among these proteins only the alpha-subunit of beta-conglycinin, but not the highly homologous alpha'- and beta-subunits, has been shown to be a major allergenic protein. The objective of this study was to examine if the alpha'- and beta-subunits of beta-conglycinin can also serve as potential allergens. Immunoblot analysis using sera collected from soybean-allergic patients revealed the presence of IgE antibodies that recognized several soy proteins including 72, 70, 52, 34, and 21 kDa proteins. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) analysis of trypsin-digested 72, 70, and 52 kDa proteins indicated that these proteins were the alpha'-, alpha-, and beta-subunits of beta-conglycinin, respectively. Additionally, purified alpha'-, alpha-, and beta-subunits of beta-conglycinin were recognized by IgE antibodies present in the soybean-allergic patients. The IgE reactivity to the beta-subunit of beta-conglycinin was not abolished when this glycoprotein was either deglycosylated using glycosidases or expressed as a recombinant protein in Escherichia coli . The results suggest that in addition to the previously recognized alpha-subunit of beta-conglycinin, the alpha'- and beta-subunits of beta-conglycinin also are potential food allergens.

Spatial and temporal control of transcription of the soybean .BETA.-conglycinin .ALPHA. subunit gene is conferred by its proximal promoter region and accounts for the unequal distribution of the protein during embryogenesis

Differentiation into specific embryo cell types correlates with the processes that lead to the accumulation of seed storage proteins in plants. The alpha subunit of beta-conglycinin, a major component of seed storage proteins in soybean, accumulates at a higher level in cotyledons than in the embryonic axis in developing embryos. To understand the mechanisms underlying this phenomenon, we characterized the upstream region of the alpha subunit gene in terms of transcriptional control using transgenic Arabidopsis thaliana plants carrying reporter gene constructs comprising the 1357-bp upstream sequence of the alpha subunit gene and the beta-glucuronidase (GUS) gene. Analysis of the time-course-dependent pattern of GUS expression revealed that the expression was first confined to the cotyledons and occurred later in the entire embryo during embryogenesis. The level of GUS expression was higher in cotyledons than in the embryonic axis throughout the period of its expression, coincident with the distribution of the alpha subunit protein in soybean embryos. By testing progressively shorter promoter fragments, the cis-acting elements responsible for transcriptional activation in the cotyledons and the embryonic axis were both localized to the region spanning -245 to -161 relative to the transcription start site. It is also concluded that the upstream region up to -245 is sufficient to control the spatial and temporal pattern of transcription, while further upstream regions influence transcription rate without affecting the transcriptional pattern. Overall, these results indicate that the unequal distribution of alpha subunit protein within the embryos is established primarily as a consequence of differential transcriptional activation controlled by a short proximal promoter region of the gene in different embryonic tissues.

Cosuppression of the alpha subunits of beta-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies.

The expression of the alpha and alpha' subunits of beta-conglycinin was suppressed by sequence-mediated gene silencing in transgenic soybean seed. The resulting seeds had similar total oil and protein content and ratio compared with the parent line. The decrease in beta-conglycinin protein was apparently compensated by an increased accumulation of glycinin. In addition, proglycinin, the precursor of glycinin, was detected as a prominent polypeptide band in the protein profile of the transgenic seed extract. Electron microscopic analysis and immunocytochemistry of maturing transgenic soybean seeds indicated that the process of storage protein accumulation was altered in the transgenic line. In normal soybeans, the storage proteins are deposited in pre-existing vacuoles by Golgi-derived vesicles. In contrast, in transgenic seed with reduced beta-conglycinin levels, endoplasmic reticulum (ER)-derived vesicles were observed that resembled precursor accumulating-vesicles of pumpkin seeds and the protein bodies accumulated by cereal seeds. Their ER-derived membrane of the novel vesicles did not contain the protein storage vacuole tonoplast-specific protein alpha-TIP, and the sequestered polypeptides did not contain complex glycans, indicating a preGolgi and nonvacuolar nature. Glycinin was identified as a major component of these novel protein bodies and its diversion from normal storage protein trafficking appears to be related to the proglycinin buildup in the transgenic seed. The stable accumulation of proteins in a protein body compartment instead of vacuolar accumulation of proteins may provide an alternative intracellular site to sequester proteins when soybeans are used as protein factories.

Soybean allergens and hypoallergenic soybean products.

About 15 soybean proteins were shown to be recognized by sera of soybean-sensitive patients with atopic dermatitis. Three of them were identified as major allergens and designated as Gly m Bd 60K, Gly m Bd 30K, and Gly m Bd 28K, respectively. Gly m Bd 60K is an alpha subunit of beta-conglycinin well known as a major soybean storage protein. Gly m Bd 30K is also known as a soybean oil-body-associated glycoprotein with a molecular weight of 34,000, which is homologous to Der p (or f) 1, a major allergen of house dust mite, classified under the papain super family. Gly m Bd 28K is a vicilin-like glycoprotein with a molecular weight of 26,000, a minor component fractionated into 7S globulin fraction. The reduction of allergenicity of soybean and soybean products has been developed with respect to the above-mentioned major three allergens as the targets by the use of the combined techniques of a chemical breeding, a physico-chemical treatment, and an enzymatic digestion. Among the three major allergens, the alpha subunit of beta-conglycinin and Gly m Bd 28K were eliminated from soybean seeds by the development of a mutant line, Tohoku 124, introduced by a chemical breeding technique. The strongest allergen, Gly m Bd 30K, was almost completely removed from defatted soymilk prepared from Tohoku 124 by a salting-out technique and a centrifugation under the limited pH and ionic strength and alternatively by an enzymatic digestion. By the application of these procedures, several hypoallergenic soybean products, such as cooked soybean grains, soybean curd (Tofu), and fermented soybean paste (Miso), soymilk, and a jelly-like soybean cake have been made to evaluate their usefulness by a challenge test for soybean-sensitive patients. It has been demonstrated by a preliminary trial that about 80% of the soybean-sensitive patients could ingest these hypoallegenic products without any adverse reactions.

Partial purification and characterization of a novel soybean protease which is inhibited by Kunitz and Bowman-Birk trypsin inhibitors.

A novel serine protease has been partially purified from dry seeds of the soybean (Glycine max) cultivar Keburi by cryoprecipitation at pH 6.4, fractional precipitation with ammonium sulfate, and a series of column chromatographic procedures on DEAE-Sepharose, SP-Sepharose, and Arginine-Sepharose 4B. Some properties of the purified enzyme were studied. The protease hydrolyzed the native storage globulins of soybean seeds, such as the alpha subunit of beta-conglycinin, at a pair of arginine residues, Arg126-Arg127. The proteolysis of the alpha subunit in the purified alpha 2 beta molecule of beta-conglycinin apparently followed first order kinetics. The enzyme was inhibited by both soybean Kunitz trypsin inhibitor and Bowman-Birk proteinase inhibitor in a competitive manner. Moreover, the enzyme could catalyze the specific proteolysis of the A3 polypeptide of the purified G5 glycinin at the Arg99-Gly100 linkage, or the carboxyl side of the Arg98-Arg99 paired basic residues.

Use of sodium dodecyl sulfate polyacrylamide gel electrophoresis to measure degradation of soluble soybean proteins by Prevotella ruminicola GA33 or mixed ruminal microbes in vitro.

Degradation of soluble soybean meal proteins by Prevotella ruminicola GA33 and mixed ruminal bacteria was studied using SDS-PAGE. This technique facilitated the quantification of peptides (molecular weight > or = kDa) released during the partial hydrolysis of soybean proteins. In P. ruminicola incubations, conglycinin alpha and alpha' subunits were degraded completely within 4 h, whereas the beta subunit of beta-conglycinin as well as the basic and acidic polypeptide components of glycinin were more resistant to degradation. Several peptides derived from digested soybean proteins appeared during the incubation. The N-terminal amino acid sequences of these peptides were similar to amino acid sequences in soybean proteins. The N-terminus of a 23-kDa peptide was identical to the N-terminus of the beta subunit of beta-conglycinin; the N-terminus of a 59-kDa peptide differed from amino acids 101 to 115 of the alpha subunit of beta-conglycinin by two of 15 amino acids; the N-terminus of a 64-kDa peptide differed from amino acids 67 to 76 of the alpha' subunit of beta-conglycinin by three of 10 amino acids. The transient accumulation of these peptides when soluble soybean proteins were incubated with mixed ruminal microorganisms suggests a role for P. ruminicola in the early stages of protein digestion in the rumen. Approximately 30% of the soluble soybean protein incubated with mixed ruminal microorganisms was not degraded after 9 h, and SDS-PAGE indicated that the basic subunit of glycinin and several peptides (molecular weights less than 23 kDa) make an appreciable contribution to metabolizable protein when soybean protein is fed to ruminants.

Alpha-subunit of beta-conglycinin, an allergenic protein recognized by IgE antibodies of soybean-sensitive patients with atopic dermatitis.

One of the major allergenic proteins in the soybean 7S-globulin fraction, which was recognized by sera of about 25% soybean-sensitive patients with atopic dermatitis, was identified as alpha-subunit of beta-conglycinin. The IgE antibodies recognizing the alpha-subunit did not show a cross-reactivity against both alpha'- and beta-subunits known to be highly homologous to alpha-subunit, and also against other allergenic components identified in soybeans [Ogawa et al., J. Nutr. Sci. Vitaminol., 35, 555-565 (1993)]. The IgE-binding site(s) was estimated to be located on the peptide 232-283 of alpha-subunit.

Characterization of a soybean beta-conglycinin-degrading protease cleavage site.

Protease C1, an enzyme from soybean (Glycine max [L.] Merrill cv Amsoy 71) seedling cotyledons, was previously determined to be the enzyme responsible for the initial degradation of the alpha' and alpha subunits, but not the beta subunit, of beta-conglycinin storage protein. The sizes of the proteolytic products generated by the action of protease C1 suggest that the cleavage sites on the alpha' and alpha subunits of beta-conglycinin may be located in their N-terminal domain, which is not found in the beta subunit of beta-conglycinin. To check this hypothesis, storage proteins from other plant species that are homologous to either the alpha'/alpha or the beta subunit of beta-conglycinin were tested as substrates. As expected, the convicilin from pea (Pisum sativum), a protein homologous to the alpha' and alpha subunits of beta-conglycinin, was digested by protease C1. The vicilins from pea as well as vicilins from adzuki bean (Vigna angularis), garden bean (Phaseolus vulgaris), black-eyed pea (Vigna unguiculata), and mung bean (Vigna radiata), storage proteins that are homologous to the beta subunit of soybean beta-conglycinin, were not degraded by protease C1. Degradation of soybean beta-conglycinin involves a sequential attack of the alpha subunit at multiple sites, culminating in the formation of a stable intermediate of 53.5 kD and a final product of 48.0 kD. The cleavage sites resulting in this formation of the intermediates and final product were determined by N-terminal analysis. These were compared to the known amino acid sequences of the three beta-conglycinin subunits. Results showed these two polypeptides to be generated by proteolysis of the alpha subunit at regions bearing long strings of acidic amino acid residues.

A Serine Protease in Soybean Seeds That Acts Specifically on the Native α Subunit of β-Conglycinin

A proteolytic activity directed against the alpha subunit of beta-conglycinin was detected in resting mature seeds of the soybean [Glycine max (L.) Merrill] cultivar Keburi. The relationship between pH and activity and the effect of protease inhibitors revealed that the enzyme was a neutral/alkaline serine protease. The proteolysis of the alpha subunit of beta-conglycinin yielded a specific product with a molecular weight of about 47,000, as determined by SDS-PAGE, but the enzyme had no activity against the beta subunit. The amino acid composition, the molecular weight and the amino-terminal amino acid sequence of the proteolytic product revealed that the action of the enzyme on the alpha subunit was specific, with cleavage occurring only at the R126-R127 peptide bond of the alpha subunit. These characteristics of the protease indicate that the enzyme is a novel protease that has not previously been recognized in soybean seeds.

Characterization of the Major Protease Involved in the Soybean β-Conglycinin Storage Protein Mobilization

Protease C1, the protease responsible for the initial degradation of the alpha' and alpha subunits of the soybean beta-conglycinin storage protein (Glycine max [L.] Merrill), has been purified. The enzyme was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular weight of 70,000 and a pH optimum of 3.5 to 4.5. Susceptibility to protease inhibitors indicates that protease C1 is a serine protease. Study of the proteolytic intermediates generated suggests that the cleavage of the alpha' and alpha subunits of beta-conglycinin by protease C1 results in intermediates that are 1 or 2 kilodaltons smaller than the native alpha' and alpha subunits. Following that, a succession of intermediates exhibiting molecular masses of 70.0 and 58.0 kilodaltons, then 63.0, 61.0, 55.0, and 53.5 kilodaltons, are observed. A 50.0- and a 48.0- kilodalton intermediate are the final products of protease C1 action. Comparison of these intermediates with the prominent anti-beta-conglycinin cross-reacting bands that increase during the first few days of germination and early growth show that protease C1 plays an important physiological role, but not an exclusive one, in the living plant.

Conserved amino acid sequences among plant proteins sorted to protein bodies and plant vacuoles

Amino acid sequence comparisons were made between the soybean alpha subunit of beta-conglycinin and 34 members of different plant protein families targeted to seed protein bodies or vacuoles. A number of short conserved amino acid sequences were identified in seed storage proteins, plant protease inhibitors and lectins, and the probable functions of these sequences are discussed. For proteins of known tertiary structure, these sequences map to the surface of the respective molecules. It is postulated that these regions produce a common secondary structure which could interact with other molecules involved in the sorting process. One of these regions, region A, is similar to the yeast carboxypeptidase Y (CPY) vacuolar targeting signal, and is present in both storage proteins and lectins. Computer modeling based upon the tertiary structure of concanavalin A (ConA) was used to generate models representing the structure of two highly related lectins from Dolichos biflorus, one of which is targeted to protein bodies and the other secreted. A different glycosylation pattern together with amino acid sequences upstream of the identified conserved amino acid sequences are predicted to modulate the presentation of the sorting domains in the lectins and be the determinant in the sorting of these lectins.

Regulation by ABA of β-Conglycinin Expression in Cultured Developing Soybean Cotyledons

The regulation of cotyledon-specific gene expression by exogenously applied abscisic acid (ABA) was studied in developing cultured cotyledons of soybean (Glycine max L. Merr. cv Provar). When immature cotyledons were cultured in modified Thompson's medium, the addition of ABA resulted in an increased concentration of the beta-subunit of beta-conglycinin, one of the major storage proteins of soybean seeds. The amount of the alpha'-and alpha-subunits of beta-conglycinin was relatively unaffected by the ABA treatment. When fluridone, an inhibitor of carotenoid biosynthesis that has been shown to decrease ABA levels in plant tissues, was added to the medium the level of ABA and the beta-subunit decreased in the cotyledons. Increasing the concentration of sucrose in the culture medium caused an increase in the concentration of ABA and beta-subunit in the cotyledons. When in vitro translation products from RNA isolated from cotyledons cultured with ABA were immunoprecipitated with antiserum against beta-conglycinin, there was an increased amount of pre-beta-subunit polypetide compared to the translation products from RNA isolated from control cotyledons. The pre-beta-subunit polypeptide was not detected in translation products from RNA isolated from fluridone-treated cotyledons. Nucleic acid hybridization reactions showed that the level of beta-subunit mRNA was higher in ABA-treated cotyledons compared to the control, and was lower in the fluridone-treated cotyledons. We have shown that exogenous ABA is able to modulate the accumulation of the beta-subunit of beta-conglycinin in developing cultured soybean cotyledons.