Soybean Seed Storage Protein Research Articles

Generation of New β-Conglycinin-Deficient Soybean Lines by Editing the lincRNA lincCG1 Using the CRISPR/Cas9 System.

Soybean β-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in β-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean β-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the β-conglycinin α-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans-acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic α'-, α-, and β-subunits of soybean β-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1, and consequently, the β-conglycinin-deficient phenotype associated with the lincCG1-gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and β-conglycinin-deficient homozygous mutant lines were obtained in the T1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein β-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the β-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.

Increased Accumulation of Recombinant Proteins in Soybean Seeds via the Combination Strategy of Polypeptide Fusion and Suppression of Endogenous Storage Proteins

Soybean seeds show great potential as a safe and cost-effective host for the large-scale production of biopharmaceuticals and industrially important macromolecules. However, the yields of desired recombinant proteins in soybean seeds are usually lower than the economic threshold for their potential commercialization. Our previous study demonstrated that polypeptide fusion such as maize γ-zein or elastin-like polypeptide (ELP) could significantly increase the accumulation of foreign proteins. In the present study, a recombination strategy of polypeptide fusions (γ-zein or ELP) and suppression of intrinsic storage proteins (glycinin or conglycinin) via RNA interference was further exploited to improve the yield of the target protein in soybean seeds. Transgenic soybean plants harboring both polypeptide-fused green fluorescent protein (GFP) and glycinin/conglycinin RNAi expression cassettes were generated and confirmed by molecular analysis. The results showed that on both the glycinin and conglycinin suppression backgrounds, the average accumulation levels of recombinant zein-GFP and GFP-ELP proteins were significantly increased as compared to that of their counterparts without such suppressions in our previous study. Moreover, zein-GFP and GFP-ELP accumulation was also remarkably higher than unfused GFP on the glycinin suppression background. However, no significant differences were detected in the glycinin or conglycinin suppression backgrounds for the same polypeptide fusion constructs, though suppression of one of the storage proteins in soybean seeds led to a significant increase in the other. Additionally, the increases in the recombinant protein yield did not affect the total protein content and the protein/oil ratio in soybean seeds. Taken together, the results indicate that both the fusion of the foreign protein with polypeptide tags together with the depletion of endogenous storage proteins contributed to a higher accumulation of the recombinant proteins without affecting the total protein content or the protein/oil ratio in soybean seeds.

Linkage and association study discovered loci and candidate genes for glycinin and β-conglycinin in soybean (Glycine max L. Merr.).

Linkage mapping and GWAS identified 67 QTLs related to soybean glycinin, β-conglycinin and relevant traits. Polymorphisms of the candidate gene Gy1 promoter were associated with the glycinin content in soybean. The major components of storage proteins in soybean seeds are glycinin and β-conglycinin, which play important roles in determining protein nutrition and soy food processing properties. Increasing the protein content while improving the ratio of glycinin to β-conglycinin is substantially important for soybean protein improvement. To investigate the genetic mechanism of storage protein subunits, 184 recombinant inbred lines (RILs) derived from a cross of Kefeng No. 1 and Nannong 1138-2 and 211 diverse soybean cultivars were used to detect loci related to glycinin (11S), β-conglycinin (7S), the sum of glycinin and β-conglycinin (SGC), and the ratio of glycinin to β-conglycinin (RGC). Sixty-seven QTLs and 11 hot genomic regions were identified as affecting the four traits. One genetic region (q10-1) on chromosome 10 was associated with multiple traits by both linkage and association analysis. Eight genes in 11 hot genomic regions might be related to soybean protein subunit. The candidate gene analysis showed that polymorphisms in Gy1 promoters were significantly correlated with the 11S content. The QTLs and candidate genes identified in the present study allow for further understanding the genetic basis of 11S and 7S regulation and provide useful information for marker-assisted selection (MAS) in soybean quality improvement.

Involvement of sulfur assimilation in the low β subunit content of soybean seed storage protein revealed by comparative transcriptome analysis

The β subunit of soybean [Glycine max (L.) Merr.] seed storage protein is of great significance in sulfur-containing amino acid balance and soybean processing properties. The objective of this study was to elucidate the relationship between the β subunit and sulfur-containing amino acid composition, and the potential regulatory mechanism. The β subunit was independently accumulated in comparison with other major subunits (α/α′, acidic, basic, and A3) during seed filling, and a low level of β subunit content (BSC) was formed during the accumulation process. In low-BSC mature seeds, crude protein, oil content, and fatty acid composition were not changed, but sulfur-containing amino acids (Cys + Met) in the low-BSC seeds increased significantly (by 31.5%), suggesting that an internal regulatory mechanism within seed might be responsible for the rebalance of seed protein composition and that sulfur assimilation might be deeply involved in β subunit accumulation. Transcriptomic analysis revealed that genes involved in anabolism of cysteine, methionine, and glutathione were up-regulated but those involved in the catabolism of these compounds were down-regulated, suggesting a relationship between the elevation of methionine and glutathione and low BSC. Our study sheds light on seed composition in low BSC lines and on the potential molecular regulatory mechanism of β subunit accumulation, broadening our understanding of soybean seed protein synthesis and its regulation.

Recombinant proteins in plants

Background Over the last few decades, several studies have shown that plants can be a viable option for producing functional recombinant proteins with a wide range of structural characteristics [1]. In addition, the potential benefit for developing countries is a prominent feature that we have recently addressed [2]. Plant-produced recombinant proteins can already be considered a novel component in sustainable food production [3]. A major reason for this optimism relates to cost. Indeed, it is widely recognized that plants used as bioreactors to produce recombinant proteins would enable a significant reduction in overall manufacturing costs [2]. Although recombinant proteins can be functionally expressed in different plant systems, it is imperative to determine the platform that offers the most advantageous conditions for the expression and recovery of a particular protein [1]. In addition, because plant pathogenic organisms cannot cause human disease, this opens the possibility of exploiting plants and edible fruits as potential candidates for the production of orally administered antigens [1]. Basically, there are three strategies for recombinant protein production in plant-based systems: (1) use of cell-culture-based systems that are equivalent to mammalian, microbial and insect cell systems; (2) transient expression of foreign genes in plant tissues that are transformed by either agroinjection or by viral infection and (3) development of transgenic plants carrying stably integrated transgenes [4,5]. Here, I will focus on some of our recent results on transient expression and soybean seed as bioreactor-based systems. Transient expression systems are very useful for research and are now being routinely used for the rapid production of valuable proteins. These systems allow high throughput production and straightforward manipulation, permitting the rapid validation of expression constructs and the production of large amounts of recombinant protein within a few weeks. As a direct consequence, the protein yields from transient expression in plants are normally higher than yields observed in other recombinant plant systems. Transient technology is based on the insertion of transgenes into plant cells using plant viruses, commonly the tobacco mosaic virus (TMV) and the potato virus × (PVX) as well as transgenic Agrobacterium tumefaciens. Transgene insertion occurs without stable chromosomal integration, resulting in non-permanent and non-inheritable gene expression. Because the transfer rates of Agrobacterium T-DNA and viral-carried genes can reach a very high number of plant cells after infection. Tobacco leaves are the dominant choice for the development of commercial platforms using transient expression [1]. Seeds as bioreactors also provide a potential economical platform for the large-scale production and storage of recombinant proteins [1,5]. Soybean seed storage proteins are of great interest for the development of regulated tissue-specific genes products of commercial interest through recombinant DNA technology. The 7S globulins are comprised of b-conglycinin subunits. b-conglycinin regulatory sequences are seed tissue-specific, temporally regulated and expressed in both the embryonic axis and cotyledons of developing seeds.

A Dominant Locus, qBSC-1, Controls β Subunit Content of Seed Storage Protein in Soybean (Glycine max (L.) Merri.)

Soybean seed storage protein is one of the most important plant vegetable proteins, and β subunit is of great significance to enhance soybean protein quality and processing property. F2 segregated population and residual heterozygous lines (RHL) derived from the cross between Yangyandou (low level of β subunit) and Zhonghuang 13 (normal level of β subunit) were used for mapping of β subunit content. Our results showed that β subunit content was controlled by a single dominant locus, qBSC-1 (β subunit content), which was mapped to a region of 11.9 cM on chromosome 20 in F2 population of 85 individuals. This region was narrowed down to 2.5 cM between BARCSOYSSR_20_0997 and BARCSOYSSR_20_0910 in RHL with a larger population size of 246 individuals. There were 48 predicted genes within qBSC-1 region based on the reference genome (Glyma 1.0, Williams 82), including the two copies of β subunit coding gene CG4. An InDel marker developed from a thymine (TT) insertion in one copy of CG4 promoter region in Yangyandou cosegregrated with BARCSOYSSR_20_0975 within qBSC-1 region, suggesting that this InDel marker maybe useful for marker-assisted selection (MAS).

Identification of epitopes of the A1aBx and A5A4B3 subunits of glycinin antigenic in three animal species

Soybean meal is commonly added to a variety of animal feeds to supplement protein sources and to optimise growth. While soybean protein is a valuable food supplement it has been recognised as an important food allergen. The soybean seed storage protein, glycinin, has been identified as an allergen. A tiled peptide array of the A1aBx and A5A4B3 subunits of glycinin was screened to identify the epitopes that bind antibodies from multiple species. We have identified four regions in these two glycinin subunits that are antigenic in most or all of the species tested. One region is implicated in an allergic response in dogs by the dog's ability to bind IgE. Three regions overlap or abut regions that are similar to allergenic epitopes in peanut. It will be critical to identify immunogenic regions able to cause allergies to soy in order to prioritise them for mitigation.

Seed storage proteins as a system for teaching protein identification by mass spectrometry in biochemistry laboratory

Mass spectrometry (MS) has become an important tool in studying biological systems. One application is the identification of proteins and peptides by the matching of peptide and peptide fragment masses to the sequences of proteins in protein sequence databases. Often prior protein separation of complex protein mixtures by 2D-PAGE is needed, requiring more time and expertise than instructors of large laboratory classes can devote. We have developed an experimental module for our Biochemistry Laboratory course that engages students in MS-based protein identification following protein separation by one-dimensional SDS-PAGE, a technique that is usually taught in this type of course. The module is based on soybean seed storage proteins, a relatively simple mixture of proteins present in high levels in the seed, allowing the identification of the main protein bands by MS/MS and in some cases, even by peptide mass fingerprinting. Students can identify their protein bands using software available on the Internet, and are challenged to deduce post-translational modifications that have occurred upon germination. A collection of mass spectral data and tutorials that can be used as a stand-alone computer-based laboratory module were also assembled.

High-level production of lactostatin, a hypocholesterolemic peptide, in transgenic rice using soybean A1aB1b as carrier

Hypercholesterolemia, a form of cardiovascular disease, is one of the leading causes of deaths worldwide. Lactostatin (Ile-Ile-Ala-Glu-Lys), derived from β-lactoglobulin in cow's milk, is a bioactive peptide with hypocholesterolemic activity higher than sitosterol, a known anti-hypercholesterolemic drug. Here, we successfully developed a transgenic rice accumulating a much higher level of lactostatin by inserting 29 IIAEK sequences into the structurally flexible (nonconserved) regions of soybean seed storage protein, A1aB1b, and introducing it into LGC-1 (low glutelin content mutant 1) as host variety. A1aB1b containing 29 lactostatins was expressed in the endosperm of rice seed cells by using seed specific promoters and sorted into novel compartments distinct from normal PB-I (ER-derived protein body) and PB-II (protein storage vacuoles). Transgenic rice seeds accumulated approximately 2 mg of lactostatins/g of dry seeds, which is relatively high compared with previous reports. Our findings suggest that the introduction of a high copy number of bioactive peptide into seed storage proteins as carrier is one of the effective means in producing higher amounts of bioactive peptides in rice.

Silencing of soybean seed storage proteins results in a rebalanced protein composition preserving seed protein content without major collateral changes in the metabolome and transcriptome.

The ontogeny of seed structure and the accumulation of seed storage substances is the result of a determinant genetic program. Using RNA interference, the synthesis of soybean (Glycine max) glycinin and conglycinin storage proteins has been suppressed. The storage protein knockdown (SP-) seeds are overtly identical to the wild type, maturing to similar size and weight, and in developmental ontogeny. The SP- seeds rebalance the proteome, maintaining wild-type levels of protein and storage triglycerides. The SP- soybeans were evaluated with systems biology techniques of proteomics, metabolomics, and transcriptomics using both microarray and next-generation sequencing transcript sequencing (RNA-Seq). Proteomic analysis shows that rebalancing of protein content largely results from the selective increase in the accumulation of only a few proteins. The rebalancing of protein composition occurs with small alterations to the seed's transcriptome and metabolome. The selectivity of the rebalancing was further tested by introgressing into the SP- line a green fluorescent protein (GFP) glycinin allele mimic and quantifying the resulting accumulation of GFP. The GFP accumulation was similar to the parental GFP-expressing line, showing that the GFP glycinin gene mimic does not participate in proteome rebalancing. The results show that soybeans make large adjustments to the proteome during seed filling and compensate for the shortage of major proteins with the increased selective accumulation of other proteins that maintains a normal protein content.

A novel plant protein disulfide isomerase family homologous to animal P5 – molecular cloning and characterization as a functional protein for folding of soybean seed‐storage proteins

The protein disulfide isomerase is known to play important roles in the folding of nascent polypeptides and in the formation of disulfide bonds in the endoplasmic reticulum (ER). In this study, we cloned a gene of a novel protein disulfide isomerase family from soybean leaf (Glycine max L. Merrill. cv Jack) mRNA. The cDNA encodes a protein called GmPDIM. It is composed of 438 amino acids, and its sequence and domain structure are similar to that of animal P5. Recombinant GmPDIM expressed in Escherichia coli displayed an oxidative refolding activity on denatured RNase A. The genomic sequence of GmPDIM was also cloned and sequenced. Comparison of the soybean sequence with sequences from Arabidopsis thaliana and Oryza sativa showed significant conservation of the exon/intron structure. Consensus sequences within the promoters of the GmPDIM genes contained a cis-acting regulatory element for the unfolded protein response, and other regulatory motifs required for seed-specific expression. We observed that expression of GmPDIM was upregulated under ER-stress conditions, and was expressed ubiquitously in soybean tissues such as the cotyledon. It localized to the lumen of the ER. Data from co-immunoprecipitation experiments suggested that GmPDIM associated non-covalently with proglycinin, a precursor of the seed-storage protein glycinin. In addition, GmPDIM associated with the alpha' subunit of beta-conglycinin, a seed-storage protein in the presence of tunicamycin. These results suggest that GmPDIM may play a role in the folding of storage proteins and functions not only as a thiol-oxidoredactase, but also as molecular chaperone.

ダイズ種子貯蔵タンパク質β-コングリシニンαサブユニット遺伝子プロモーターの形質転換シロイヌナズナにおける時間的・空間的な発現制御

ダイズ種子貯蔵タンパク質β-コングリシニンαサブユニット遺伝子プロモーターの形質転換シロイヌナズナにおける時間的・空間的な発現制御

Improvement of protein quality in transgenic soybean plants

Glycinin is one of the abundant storage proteins in soybean seeds. A modified Gy1 (A1aB1b) proglycinin gene with a synthetic DNA encoding four continuous methionines (V3-1) was connected between the hpt gene and the modified green fluorescent protein sGFP(S65T) gene, and a resultant plasmid was introduced into soybean by particle bombardment in order to improve nutritional value of its seeds. After the selection with hygromycin, the efficiency of gene introduction was evaluated. More than 60 % of the regenerated plants tolerant to hygromycin yielded the hpt and V3-1 fragment by polymerase chain reaction (PCR) analysis, and the expression of sGFP was detected in about 50 % of putative transgenic soybeans. Southern hybridization confirmed the presence of transgenes in T0 plants and the transgenic soybeans hybridized with the hpt and V3-1 genes were analyzed showed different banding patterns. Most of the transgenic plants were growing, flowering normally and produced seeds. Analysis of seed obtained from transgenic soybean plants expressing hpt and V3-1 genes showed higher accumulation of glycinin compared with non-transgenic plants. In addition, protein expression in transgenic soybean plants was observed by using 2D-electrophoresis.

Optimization of extraction and isolation for 11S and 7S globulins of soybean seed storage protein

A new method was developed for extraction and isolation of 7S and 11S fractions from soybean seed, based on methods of Nagano et al., Thanh and Shibasaki [Nagano, T., Hirotsuka, M., & Mori, H. (1992). Dynamic viscoelastic study on the gelation of 7S globulin from soybeans. Journal of Agricultural and food chemistry 40, 941–944 and Thanh, V. H., & Shibasaki, K. (1976). Major proteins of soybean seeds. A straightforward fraction and their characterization. Journal of Agricultural and Food Chemistry 24, 1117–1121]. Optimization of the extraction and isolation of 11S and 7S globulins from soybean seed was investigated under various conditions by the Kjeldahl method and SDS-PAGE. The optimal conditions were as follows: 0.03–0.06 M Tris–HCl buffer (pH 8.5) containing 0.01 M sodium bisulfite as extract solution, extraction twice at 45 °C for 1 h, and with a 1:15 ratio ( w/v) of flour:Tris–HCl. The 11S fraction was precipitated at pH 6.4, and the supernatant, after centrifugation, was adjusted to pH 5.5 to remove the insoluble intermediate fraction by further centrifugation. The supernatant obtained was then adjusted to pH 4.8 to afford the 7S fraction as a precipitate by centrifugation. With the improvements, the protein contents and purities of the isolated 11S and 7S fractions were significantly increased. The contents of all subunits of the isolated 11S and 7S fraction were markedly higher than those by Thanh and Shibasaki method, while the contents of α, β and B 3 were also significantly higher than those by Nagano et al. method.

Molecular characterization of a β-conglycinin deficient soybean

The soybean seed storage protein β-conglycinin has a low amino acid score, shows lower functional gelling properties compared with glycinin and contains a major allergen. The wild soybean (Glycine soja Sieb et Zucc.) QT2 lacks all the subunits of β-conglycinin, and this deficiency is controlled by a single dominant gene Scg-1 (suppressor of β-conglycinin). Scg-1 was introduced into a soybean cultivar Fukuyutaka from QT2 and this near-isogenic line was designated as QY7-25. Segregation analyses of the progeny derived from a cross between QY7-25 and the wild type did not show any significant changes caused by Scg-1 in the germination ratio and seed weight. Low amounts of mRNAs for the α ’, α and β subunits of β-conglycinin were detected by RT-PCR in QY7-25. We revealed that an α subunit mRNA expressed from a region which replaced with mutant line in the near-isogenic line QY7-25 by single nucleotide polymorphisms analysis. In addition, an abnormal splicing event in a cDNA clone for the β subunit isolated from immature seed of QY7-25 was observed. Southern analysis using the coding region of α ’ subunit gene as a probe revealed a polymorphism between QY7-25 and wild type and this genotypes co-segregate with the deficiency of β-conglycinin subunits. These results suggest that the β-conglycinin deficiency might be controlled by a claster region of β-conglycinin subunit genes. In the present study, no agronomical disadvantage in QY7-25 was observed, confirming that Scg-1 is a valuable gene for soybean breeding.

High-level Expression of Basic Fibroblast Growth Factor in Transgenic Soybean Seeds and Characterization of its Biological Activity

The glycinin G1 gene encodes a soybean seed storage protein accumulating at a high level. We have used the G1 promoter to confer seed-specific expression of human basic fibroblast growth factor (bFGF) in transgenic soybeans. The coding region of 18 kDa bFGF was fused to the promoter or promoter-signal peptide sequence of G1 gene, and transferred into soybean. Analysis of transgenic plants demonstrated that bFGF transcript or protein was confined to the seeds. The highest level of bFGF accumulation in the seeds reached up to 2.3% of total soluble protein. The soybean-derived bFGF was biologically active as confirmed by its mitogenic activity on Balb/c 3T3 cells, and exhibited other properties identical to native bFGF. We also observed a seed-specific expression of beta-glucuronidase driven by the G1 promoter. These results indicated that the G1 promoter contains essential cis-elements for seed-specific expression, and thus can be used for expression of pharmaceutical proteins in soybean seeds.

A rice-based edible vaccine expressing multiple T cell epitopes induces oral tolerance for inhibition of Th2-mediated IgE responses

Peptide immunotherapy using multiple predominant allergen-specific T cell epitopes is a safe and promising strategy for the control of type I allergy. In this study, we developed transgenic rice plants expressing mouse dominant T cell epitope peptides of Cry j I and Cry j II allergens of Japanese cedar pollen as a fusion protein with the soybean seed storage protein glycinin. Under the control of the rice seed storage protein glutelin GluB-1 promoter, the fusion protein was specifically expressed and accumulated in seeds at a level of 0.5% of the total seed protein. Oral feeding to mice of transgenic rice seeds expressing the T cell epitope peptides of Cry j I and Cry j II before systemic challenge with total protein of cedar pollen inhibited the development of allergen-specific serum IgE and IgG antibody and CD4(+) T cell proliferative responses. The levels of allergen-specific CD4(+) T cell-derived allergy-associated T helper 2 cytokine production of IL-4, IL-5, and IL-13 and histamine release in serum were significantly decreased. Moreover, the development of pollen-induced clinical symptoms was inhibited in our experimental sneezing mouse model. These results indicate the potential of transgenic rice seeds in production and mucosal delivery of allergen-specific T cell epitope peptides for the induction of oral tolerance to pollen allergens.

Quantitative trait loci for β‐conglycinin (7S) and glycinin (11S) fractions of soybean storage protein

AbstractGlycinin (11S) and β‐conglycinin (7S) are important seed storage proteins in soybean [Glycine max (L.) Merr.]. A major limitation of soybean seed storage proteins is their low levels of the sulfur‐containing amino acids, methionine and cysteine, which are important nutritional components of protein mea. Glycinin contains significantly more S‐containing amino acids than does β‐conglycinin. Thus, detection of quantitative trait loci (QTL) that govern 11S may provide marker‐assisted selection (MAS) opportunities to improve soybean total S‐containing amino acids. The objective of this study was to detect and map QTL governing 7S and 11S fractions of soybean seed storage proteins. To achieve this objective, 101 F6‐derived recombinant inbred lines (RIL) developed from a cross of N87‐984‐16 ×TN93‐99 were used. Storage proteins were extracted from all RIL and separated in 10–20% linear gradient polyacrylamide gels. Dried gels were scanned for individual subunits of storage protein with a densitometer equipped with a He−Ne laser light source. Data were converted to concentration for each subunit component and analyzed using SAS software. A significant (P<0.05) difference among genotypes was found for glycinin and β‐conglycinin. A total of 94 polymorphic simple sequence repeat molecular genetic markers were used in screening all RIL. Three QTL for glycinin (Satt461, Satt292, and Satt156) were distributed on linkage group (LG) D2, I, and L, respectively, whereas two QTL for conglycinin (Satt461 and Satt249) were distributed on LG D2 and J. Phenotypic variation explained by individual QTL ranged from 9.5 to 22%. These QTL may provide useful MAS opportunities for improvement of nutritional quality in soybean.

Soy immunotherapy for peanut-allergic mice: Modulation of the peanut-allergic response

Allergen-specific immunotherapy (IT) is an effective therapeutic modality to prevent further anaphylactic episodes in patients with insect sting hypersensitivity and is being investigated for peanut allergy. So far, peanut-specific IT has been unsuccessful because of the side effects of therapy. Soybean seed storage proteins share significant homology with the respective peanut allergens. This study was undertaken in mice to investigate whether specific doses of soybean would desensitize peanut-allergic mice. C3H/HeJ mice were sensitized to peanut with 3 intraperitoneal (IP) injections of crude peanut extract. The mice were desensitized by IP injections with either crude peanut or soybean extract for 4 weeks, 3 times a week. Controls included placebo desensitization with PBS and naive mice. After 2 weeks of rest, mice were challenged IP with crude peanut extract. Thirty minutes later, symptom scores and body temperatures were recorded. Serum immunoglobulins, peanut-induced splenocyte proliferation, and secreted cytokines were measured before and after desensitization. The clinical symptoms in the soybean- and peanut-desensitized animals were markedly reduced compared with the placebo-treated mice. Specific IgG1 levels to crude peanut were significantly lower in the soy IT group than in the peanut IT group. The cellular response to crude peanut was also downregulated in the soy IT group, as shown by decreased peanut-specific stimulation indices and a cytokine profile skewed toward a T H 1 response. Soy IT can be used to desensitize/downregulate peanut-specific response in peanut-allergic mice and could provide a new therapeutic intervention for peanut allergy.

The composition of newly synthesized proteins in the endoplasmic reticulum determines the transport pathways of soybean seed storage proteins.

Glycinin (11S) and beta-conglycinin (7S) are major storage proteins in soybean (Glycine max L.) seeds and accumulate in the protein storage vacuole (PSV). These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the PSV by vesicles. Electron microscopic analysis of developing soybean cotyledons of the wild type and mutants with storage protein composition different from that of the wild type showed that there are two transport pathways: one is via the Golgi and the other bypasses it. Golgi-derived vesicles were observed in all lines used in this study and formed smooth dense bodies with a diameter of 0.5 to several micrometers. ER-derived protein bodies (PBs) with a diameter of 0.3-0.5 microm were observed at high frequency in the mutants containing higher amount of 11S group I subunit than the wild type, whereas they were hardly observed in the mutants lacking 11S group I subunit. These indicate that pro11S group I may affect the formation of PBs. Thus, the composition of newly synthesized proteins in the ER is important in the selection of the transport pathways.