Accumulation of β-conglycinin in soybean cotyledon through the formation of disulfide bonds between α'- and α-subunits

β-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean β-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the α'- and α-subunits of β-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the β-conglycinin complexes containing the disulfide-linked α'/α-subunits were complexes of more than 720 kD. The α'- and α-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between α'/α-subunits residing in different β-conglycinin hexamers, but the binding of P34 to α'- and α-subunits reduces the linkage between β-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when β-conglycinin was expressed under nonreducing conditions.     

Identification of Gy4 nulls and development of multiplex PCR-based co-dominant marker for Gy4 and α’ subunit of β-conglycinin in soybean

Alpha prime (α’) subunit of β-conglycinin and Gy4 subunit of glycinin are two important subunits of soybean storage protein which have negative effects on food processing, total amino acid content, and hypersensitivity reactions. It has been possible to reduce or remove some of these problems from soybean by screening or developing mutant lines. The objective of this study was to establish a simple, cheap DNA marker for Gy4 and α’ subunit for use in non-seed destructive, marker-assisted selection (MAS) that can identify these two mutants at the same time in a unique PCR reaction. To achieve this objective, we identified eight of Gy4 mutants from diverse soybean accessions from the USDA Soybean Germplasm Collection and described a multiplex PCR based co-dominant DNA marker for Gy4 subunit of glycinin. Then we crossed one of these Gy4 mutants with Keburi (α’ mutant) for development of double mutant variety and established a multiplex PCR based, co-dominant DNA marker for screening Gy4 and α’ mutants. Thus, using this newly developed marker to identify Gy4 and α’ mutants in breeding programs we could save our time, labor, and resources.     

Synthesis and assembly of soybean β-conglycinin in vitro

The construction of SP6-derived expression plasmids that encode normal and modified -conglycinin subunits is described. With the exception of an additional methionine at their NH₂-terminal ends and the lack of glycans, the normal subunits synthesized at the direction of these plasmids coresponded to mature and subunits isolated from soybean seeds. The subunits assembled into trimers in vitro that were equivalent in size to those formed in vivo. This result shows that the glycans are not required either for protein folding or oligomer assembly. Subunits produced from other plasmids, which had modifications in a highly conserved hydrophobic region in the COOH-terminal end of the subunits, either did not assemble or assembled at an extremely low rate compared to unmodified subunits. Structural changes at the more hydrophilic NH₂-terminal end had mixed effects. Several subunits modified in this region assembled into trimers at rates that were either equal or greater than those for normal subunits. Others assembled less completely than the normal subunits. Our results indicate that the in vitro synthesis and assembly assay will be useful in evaluating structure-function relationships in modified -conglycinin subunits. The results also show that structural changes at the NH₂-terminal end of the subunits are tolerated to a greater extent than modifications in the hydrophobic conserved region in the COOH-terminal half of the subunits, and this information will be useful in efforts to improve soybean quality.     

Identification of the 7S globulin with β-conglycinin in soybean seeds

The 7S globulin, a major ultracentrifugal component with the 11S globulin, was identical with β-conglycinin one of four antigenic components in the reserve proteins of soybean seeds (Glycine max). Double gel immunodiffusion and immunoelectrophoresis in agar gel were used for their identification. In addition, some characteristic properties on ultracentrifugation and in carbohydrate content agreed well between the proteins. Their MWs were ca 180000.     

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 ⁸⁵EQDERQFPFPR⁹⁵ 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 (IC₅₀) 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.     

The C-terminal region of α′ subunit of soybean β-conglycinin contains two types of vacuolar sorting determinants

In maturing seed cells, proteins that accumulate in the protein storage vacuoles (PSVs) are synthesized on the endoplasmic reticulum (ER) and transported by vesicles to the PSVs. Vacuolar sorting determinants (VSDs) which are usually amino acid sequences of short or moderate length direct the proteins to this pathway. VSDs identified so far are classified into two types: sequence specific VSDs (ssVSDs) and C-terminal VSDs (ctVSDs). We previously demonstrated that VSDs of α′ and β subunits of β-conglycinin, one of major storage proteins of soybean (Glycine max), reside in the C-terminal ten amino acids. Here we show that both types of VSDs coexist within this region of the α′ subunit. Although ctVSDs can function only at the very C-termini of proteins, the C-terminal ten amino acids of α′ subunit directed green fluorescent protein (GFP) to the PSVs even when they were placed at the N-terminus of GFP, indicating that an ssVSD resides in the sequence. By mutation analysis, it was found that the core sequence of the ssVSD is Ser-Ile-Leu (fifth to seventh residues counted from the C-terminus) which is conserved in the α and β subunits and some vicilin-like proteins. On the other hand, the sequence composed of the C-terminal three amino acids (AFY) directed GFP to the PSVs when it was placed at the C-terminus of GFP, though the function as a VSD was disrupted at the N-terminus of GFP, indicating that the AFY sequence is a ctVSD.     

Isolation and characterization of cDNA clones for α- and β-subunits of ovine luteinizing hormone

A cDNA library of ovine pituitary DNA in plasmid pBR322 has been constructed by conventional methods with certain modifications. The library was screened using partial cDNAs for ratα-subunit and LHβ. We have isolated cDNA clones for ovineα-subunit and LHβ. The identification of these clones was confirmed by partial sequencing. The clones bear about 80% sequence homology with the respective rat cDNAs in the sequenced regions and hybridize with the rat clones in 5 X SSC at 55°C. The ovine LHβ clone has an insert of about 650 bp and selects an RNA of about 750 bases in a northern blot. The α-subunit cDNA clone has an insert of about 550 bp; it has two internalPst I sites and thus shows restriction-based differences from ratα-subunit cDNA, which does not have anyPst I site.     

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 >20 mg in 1 g 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.     

Co-expression of α′ and β subunits of β-conglycinin in rice seeds and its effect on the accumulation behavior of the expressed proteins

A transgenic rice that produces both the α′ and β subunits of β-conglycinin has been developed through the crossing of two types of transgenic rice. Although the accumulation level of the α′ subunit in the α′β-transgenic rice was slightly lower than that in the transgenic rice producing only the α′ subunit, the accumulation level of the β subunit in the α′β-transgenic rice was about 60% higher than that in the transgenic rice producing only the β subunit. Results from sequential extraction and gel-filtration experiments indicated that part of the β subunit formed heterotrimers with the α′ subunit in a similar manner as in soybean seeds and that the heterotrimers interacted with glutelin via cysteine residues. These results imply that the accumulation level of the β subunit in the α′β-transgenic rice increases by an indirect interaction with glutelin. Immunoelectron microscopy revealed that the α′ and β subunits are localized in a low electron-dense region of protein body-II (PB-II) and that α′ homotrimers in the α′β-transgenic rice seeds seem to accumulate outside of this low electron-dense region.     

Ultrastructural localization of glycinin and β-conglycinin in Glycine max (soybean) cv. Maple Arrow by the immunogold method

Summary -Conglycinin (7S globulin) and glycinin (11S globulin) are the major reserve proteins of soybean. They were localized by the protein A immunogold method in thin sections of glycine max (soybean) cv. Maple Arrow. In cotyledons, both globulins were simultaneously present in all protein bodies. Statistical analysis of marking intensities indicated no correlation between globulin concentration and size of protein bodies. The immunogold method failed to detect either globulin in the embryonic axis and in cotyledons of four-day-old seedlings. Similar observations were made with cotyledons of two soy varieties lacking either the lectin or the Kunitz trypsin inhibitor. In another variety (T-102) lacking the lectin, the 7S globulin could not be detected.     

A method for the separation of peptides and α-amino acids

1. Peptides and alpha-amino acids, occurring in mixtures from various sources, can be separated into one fraction containing the amino acids and several peptide fractions. This is achieved by chelation of the mixture with Cu(2+) ions and subsequent chromatography of these chelates over the acetate form of diethylaminoethylcellulose or triethylaminoethylcellulose. 2. The amino acid fraction is obtained by elution with 0.01m-collidine-acetate buffer, pH8.0. 3. Peptide fractions are eluted with 0.01m-collidine-acetate buffer, pH4.5, 0.17n-acetic acid and 0.1n-hydrochloric acid respectively. 4. With the exception of aspartic acid and glutamic acid, which are partly found in the acidic peptide fraction, the amino acids are completely separated from the peptides. 5. Contamination of the acidic peptide fraction with glutamic acid and aspartic acid can be largely avoided by previous addition of an excess of arginine. 6. Copper is removed from the eluates by extraction with 8-hydroxyquinoline in chloroform.     

A yeast screening method to decipher the interaction between the adenosine A2B receptor and the C-terminus of different G protein α-subunits

The expression of human G protein-coupled receptors (GPCRs) in Saccharomyces cerevisiae containing chimeric yeast/mammalian G_α subunits provides a useful tool for the study of GPCR activation. In this study, we used a one-GPCR-one-G protein yeast screening method in combination with molecular modeling and mutagenesis studies to decipher the interaction between GPCRs and the C-terminus of different α-subunits of G proteins. We chose the human adenosine A_2B receptor (hA_2BR) as a paradigm, a typical class A GPCR that shows promiscuous behavior in G protein coupling in this yeast system. The wild-type hA_2BR and five mutant receptors were expressed in 8 yeast strains with different humanized G proteins, covering the four major classes: G_αi, G_αs, G_αq, and G_α12. Our experiments showed that a tyrosine residue (Y) at the C-terminus of the G_α subunit plays an important role in controlling the activation of GPCRs. Receptor residues R103^3.50 and I107^3.54 are vital too in G protein-coupling and the activation of the hA_2BR, whereas L213^IL3 is more important in G protein inactivation. Substitution of S235^6.36 to alanine provided the most divergent G protein-coupling profile. Finally, L236^6.37 substitution decreased receptor activation in all G protein pathways, although to a different extent. In conclusion, our findings shed light on the selectivity of receptor/G protein coupling, which may help in further understanding GPCR signaling.     

Dissociation-reconstitution experiments support the presence of two catalytic β-subunits in mitochondrial F1

Mitochondrial F₁, inactivated to various extents with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), was dissociated with LiCl and reconstituted after removal of the salt. This procedure resulted in a reactivation that corresponded with a reactivation theoretically expected on the basis of the assumption that the reassociation of β-subunits into native F₁ molecules is random and that two out of the three β-subunits are directly involved in catalysis. Repeated inactivation of such reactivated F₁, followed by the same dissociation-association procedure, resulted in similar data. After inactivation of F₁ by covalent binding of 2-N-AT(D)P to one catalytic site, no reactivation upon dissociation-reassociation was obtained due to the fact that such modified F₁ did not dissociate under the experimental conditions used.     

Association of α-subunits with nucleotide-modified β-subunits induces asymmetry in the catalytic sites of the F1-ATPase α3β3

The photoaffinity spin-labeled ATP analog, 2-N₃-SL-adenosine triphosphate (ATP), was used to covalently modify isolated β-subunits from F₁-ATPase of the thermophilic bacterium PS3. Approximately 1.2 mol of the nucleotide analog bound to the isolated subunit in the dark. Irradiation leads to covalent incorporation of the nucleotide into the binding site. ESR spectra of the complex show a signal that is typical for protein-immobilized radicals. Addition of isolated α-subunits to the modified β-subunits results in ESR spectra with two new signals indicative of two distinctly different environments of the spin-label, e.g., two distinctly different conformations of the catalytic sites. The relative ratio of the signals is approx 2∶1 in favor of the more closed conformation. The data show for the first time that when nucleotides are bound to isolated β-subunits, binding of α-subunits induces asymmetry in the catalytic sites even in the absence of the γ-subunit. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to PDV.     

Characterization of glycerol dehydratase expressed by fusing its α- and β-subunits

The gdh and gdhr genes, encoding B₁₂-dependent glycerol dehydratase (GDH) and glycerol dehydratase reactivase (GDHR), respectively, in Klebsiella pneumoniae, were cloned and expressed in E. coli. Part of the β-subunit was lost during GDH purification when co-expressing α, β and γ subunit. This was overcome by fusing the β-subunit to α- or γ-subunit with/without the insertion of a linker peptide between the fusion moieties. The kinetic properties of the fusion enzymes were characterized and compared with wild type enzyme. The results demonstrated that the fusion protein GDHALB/C, constructed by linking the N-terminal of β-subunit to the C-terminal of α subunit through a (Gly₄Ser)₄ linker peptide, had the greatest catalytic activity. Similar to the wild-type enzyme, GDHALB/C underwent mechanism-based inactivation by glycerol during catalysis and could be reactivated by GDHR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.