Sucrose Binding Protein Research Articles

A sucrose-binding protein and β-conglycinins regulate soybean seed protein content and control multiple seed traits.

Expanded agriculture production is required to support the world's population but can impose substantial environmental and climate change costs, particularly with intensifying animal production and protein demand. Shifting from an animal- to a plant-based protein diet has numerous health benefits. Soybean (Glycine max [L.] Merr.) is a major source of protein for human food and animal feed; improved soybean protein content and amino acid composition could provide high-quality soymeal for animal feed, healthier human foods, and a reduced carbon footprint. Nonetheless, during the soybean genome evolution, a balance was established between the amount of seed protein, oil, and carbohydrate content, burdening the development of soybean cultivars with high proteins (HPs). We isolated 2 high-seed protein soybean mutants, HP1 and HP2, with improved seed amino acid composition and stachyose content, pointing to their involvement in controlling seed rebalancing phenomenon. HP1 encodes β-conglycinin (GmCG-1) and HP2 encodes sucrose-binding protein (GmSBP-1), which are both highly expressed in soybean seeds. Mutations in GmSBP-1, GmCG-1, and the paralog GmCG-2 resulted in increased protein levels, confirming their role as general regulators of seed protein content, amino acid seed composition, and seed vigor. Biodiversity analysis of GmCG and GmSBP across 108 soybean accessions revealed haplotypes correlated with protein and seed carbohydrate content. Furthermore, our data revealed an unprecedented role of GmCG and GmSBP proteins in improving seed vigor, crude protein, and amino acid digestibility. Since GmSBP and GmCG are present in most seed plants analyzed, these genes could be targeted to improve multiple seed traits.

Characterization of sucrose binding protein as a seed-specific promoter in transgenic tobacco Nicotiana tabacum L.

Seed-specific expression using appropriate promoters is a recommended strategy for the efficiently producing valuable metabolites in transgenic plants. In the present study, we investigated the sequence of sucrose binding protein (SBP) as a seed-specific promoter to find the cis-acting elements specific to gene expression in seeds. The 1860 bp SBP sequence was analyzed using Plant Care and PLACE databases to find cis-acting elements, which resulted in a finding of 22 cis-acting elements required for seed expression. In addition, we have discovered cis- acting elements that are indirectly involved in triacylglycerol synthesis (GATABOX, DOFCOREZM, CACGTGMOTIF). The seed specificity of SBP was analyzed by generating a stable transgenic tobacco plant harboring β-glucuronidase (GUS) reporter gene under the control of the SBP promoter. Histochemical analysis of these transgenic tobacco plants indicated decreasing GUS activity in the leaves during the vegetative stage. However, the mature seeds of transgenic plants showed GUS activity. Moreover, the SBP promoter function in the seed oil content was evaluated by the expression of DGAT1. The expression analysis of DGAT1 in SBP-DGAT1 transgenic tobacco seeds using quantitative real-time PCR revealed a 7.8-fold increase in DGAT1 than in non-transgenic plants. Moreover, oil content increased up to 2.19 times more than in non-transgenic plants. And the oil content of the SBP-DGAT1 transgenic tobacco leaves did not change compared to the control plant. Therefore, we suggested that the SBP promoter could be used as a seed-specific promoter for targeted expression of desired genes in the metabolite engineering of oilseed crops.

Deleterious effects of Schinus terebinthifolius Raddi seed flour on cowpea weevil, Callosobruchus maculatus (F.), larval development

Schinus terebinthifolius, Raddi, has been extensively studied due to its anti-inflammatory and antibiotic properties. S. terebinthifolius was also toxic to some insects, however little has been explored about the nature of its insecticide compounds or the toxicity of this plant to insect species. In this work, we investigate the toxicity of S. terebinthifolius seed flour against the insect C. maculatus. S. terebinthifolius seed flour interfered with the post hatch development of the C. maculatus larvae, decreasing larval survival, mass and length. Using DEAE-cellulose chromatography, five protein fractions were isolated, a non-retained fraction (NRF) and four retained fractions, eluted with 0.25, 0.5, 0.7 and 1.0 M NaCl. Proteins with varying molecular masses were observed in all fractions. The majority protein bands were identified by mass spectrometry analysis and among the main identified proteins are 11S globulins (such glycinin), lipoxygenase, chitinases, 7S globulins (vicilins, canavalin and β conglycinin), annexin, catalase and sucrose binding protein. All DEAE-protein fractions were toxic to the insect, interfering with the post hatch larval development and survival. Decreases greater than 90% were observed in the larval mass and length at 20 days after oviposition (DAO) for larvae raised on diet containing 0.5% of some fractions. Alterations in the level of proteins, glucose and in the activity of the enzymes lipases and cysteine proteases were also detected in these larvae. Our results show that seeds of S. terebinthifolius have an arsenal of toxic proteins with potential for the control of the insect C. maculatus.

Effect of Heat Stress on Seed Protein Composition and Ultrastructure of Protein Storage Vacuoles in the Cotyledonary Parenchyma Cells of Soybean Genotypes That Are Either Tolerant or Sensitive to Elevated Temperatures.

High growth temperatures negatively affect soybean (Glycine max (L.) Merr) yields and seed quality. Soybean plants, heat stressed during seed development, produce seed that exhibit wrinkling, discoloration, poor seed germination, and have an increased potential for incidence of pathogen infection and an overall decrease in economic value. Soybean breeders have identified a heat stress tolerant exotic landrace genotype, which has been used in traditional hybridization to generate experimental genotypes, with improved seed yield and heat tolerance. Here, we have investigated the seed protein composition and ultrastructure of cotyledonary parenchyma cells of soybean genotypes that are either susceptible or tolerant to high growth temperatures. Biochemical analyses of seed proteins isolated from heat-tolerant and heat-sensitive genotypes produced under 28/22 °C (control), 36/24 °C (moderate), and 42/26 °C (extreme) day/night temperatures revealed that the accumulation in soybean seeds of lipoxygenase, the β-subunit of β-conglycinin, sucrose binding protein and Bowman-Birk protease inhibitor were negatively impacted by extreme heat stress in both genotypes, but these effects were less pronounced in the heat-tolerant genotype. Western blot analysis showed elevated accumulation of heat shock proteins (HSP70 and HSP17.6) in both lines in response to elevated temperatures during seed fill. Transmission electron microscopy showed that heat stress caused dramatic structural changes in the storage parenchyma cells. Extreme heat stress disrupted the structure and the membrane integrity of protein storage vacuoles, organelles that accumulate seed storage proteins. The detachment of the plasma membrane from the cell wall (plasmolysis) was commonly observed in the cells of the sensitive line. In contrast, these structural changes were less pronounced in the tolerant genotype, even under extreme heat stress, cells, for the most part, retained their structural integrity. The results of our study demonstrate the contrasting effects of heat stress on the seed protein composition and ultrastructural alterations that contribute to the tolerant genotype’s ability to tolerate high temperatures during seed development.

Combining 2-DE immunoblots and mass spectrometry to identify putative soybean (Glycine max) allergens

Soybean is recognized as a commonly allergenic food, but the identity of important allergens is not well studied. Recently, some global regulatory agencies started requiring quantitative analysis of individual allergens, including unproven allergens, as part of the risk assessment for genetically engineered (GE) soybeans. We sought to identify soybean proteins that bind IgE from any of 10 individual soybean-sensitized subjects. Soybean IgE binding proteins were identified by 2-DE immunoblots using sera from four soy-allergic and plasma from six soy-sensitized human subjects. Corresponding spots were excised from stained gels, digested, and analyzed using a quadrupole TOF Synapt G2-S tandem mass spectrometer. Results showed the major IgE binding proteins were subunits of either β-conglycinin (Gly m 5) or glycinin (Gly m 6). Soybean Kunitz trypsin inhibitor (SKTI) was a significant IgE binding protein for four subjects. Soybean agglutinin, seed biotinylated protein (SBP) of 65 kDa, late embryogenesis protein (LEP), and sucrose-binding protein were identified as IgE binding only for soy-sensitized subjects. We conclude that the major soybean allergens are isoforms of Gly m 5, Gly m 6, and possibly SKTI and that requirements for quantitative measurement of proteins that are not clear allergens is not relevant to safety.

Proteomic and metabolomic analysis of minimax and Williams 82 soybeans grown under two different conditions

MiniMax was developed as a model soybean for research because of its small stature, limited planting space, and rapid generation time. In the present study, the proteins, isoflavones, and fatty acid methyl esters (FAMEs) were compared between dwarf (MiniMax) and commercially cultivated (Williams 82) soybean genotypes under two growing conditions (greenhouse and field). The protein profile of the soybean extracts by 2D-PAGE showed at least 16 of 716 spots that were differently expressed in MiniMax and Williams 82. These spots were primarily six proteins namely, seed biotin, β-conglycinin, glycinin, seed-maturation protein, glutathione-S-transferase, and sucrose binding protein. In both genotypes, higher content of isoflavone aglycones was observed in greenhouse grown samples as compared to the field grown samples. However, only field grown Willams 82 had higher levels of FAMEs as compared to other three groups. This study shows that both growing conditions and cultivars significantly influence the compositional content of metabolites. Practical applications The global demand of soybean and its products has increased due to its high protein and isoflavones content. Researchers are actively developing new genotypes with improved yields, greater drought and disease tolerance, and reduced growth time. MiniMax, a dwarf genotype of soybean showed early flowering, seed setting, and maturation traits due to its multiple dwarfing genes. This characteristic feature enables researchers to study effects of various treatments on soybean in a shorter period with multiple replications in a single growing season.

Detection of IgE-reactive proteins in hydrolysed dog foods.

Commercial hydrolysed diets are used for the diagnosis of food allergy in dogs. The cleaved parent proteins are presumed to be too small to elicit an allergic response by reacting with allergen-specific immunoglobin E (IgE). To evaluate three commercial hydrolysed dog diets for proteins. Sera were collected from dogs with suspected food allergy. Two batches of each hydrolysed diet were examined by electrophoresis and visualized by Coomassie blue, silver nitrate staining and IgE immunoblotting. From two to five proteins, ranging from 21 to 67 kDa, were detected in all three diets evaluated. Circulating IgE antibodies targeting these proteins were detected by immunoblotting of dog sera. Six different carbohydrate proteins were identified by mass spectrometry; maize/potato granule-bound starch synthase-1, soybean glycinin, soybean β-conglycinin α chain, potato aspartic protease inhibitor, rice glutelin type B1 and soybean sucrose-binding protein. Four of these proteins have been described as allergens in humans. Some commercial hydrolysed diets contain carbohydrate proteins. Some dogs have circulating IgE antibodies targeting these proteins. The clinical significance of these findings is unknown.

Proteomics Changes in Filling Seeds of Vegetable Soybean

Vegetable soybean is an important economic and nutritious crop. In this study, 48 differentially expressed proteins were identified from filling seeds of soybean (Glycine max) cv. Mindou 6 by using two-dimensional electrophoresis (2-DE) combined with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Among them, 25% were related to protein destination and storage, 42% to energy and metabolism, 15% to disease/defense, 6% to transporters, 4% to secondary metabolism, 4% to transcription, 2% to protein synthesis, and 2% to cell growth/division. Along with the maturity of seeds, the number of unchanged abundance proteins decreased, while that of both upregulated and downregulated proteins increased. Both downregulated expression of caffeic acid O-methyltransferase (COMT) and upregulated expression of sucrose-binding protein (SBP) 2 precursor may contribute to increase in digestibility, nutritional value, and eating quality of vegetative seeds at suitable picking period. The pattern of unchanged proteins during the whole seed-filling stage may be also beneficial to the quality of vegetable soybean.

Proteomic profiling of the coagulation of soymilk proteins induced by magnesium chloride

The coagulation of soymilk proteins induced by magnesium chloride at 30 °C was investigated using the SDS-PAGE and two-dimensional electrophoresis. Approximately 88.4% of the soymilk proteins were coagulated into the soymilk pellet fraction (SPF) by 5 mM magnesium chloride, and the total protein in the soymilk supernatant fraction (SSF) decreased from 7.80 ± 0.12 mg mL−1 to 0.90 ± 0.18 mg mL−1. SDS-PAGE showed that the total intensities of the protein bands corresponding to the 7S α′ subunit, the 7S α subunit, the 7S β subunit and the 11S A3 subunit were decreased to 10.5 ± 0.3%, 11.8 ± 1.1%, 6.6 ± 0.2% and 15.1 ± 2.2%, respectively. Two-dimensional electrophoresis indicated that most of the soymilk proteins in the SSF, including the 7S subunits, 11S subunits, β-amylase, sucrose binding protein 2, Bd 30K, lectin and trypsin inhibitor A, were coagulated into the SPF by 5 mM magnesium chloride.

Assessment of the natural variation of low abundant metabolic proteins in soybean seeds using proteomics

Using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, we investigated the distribution of the low abundant proteins that are involved in soybean seed development in four wild and 12 cultivated soybean genotypes. We found proteomic variation of low abundant proteins involved in soybean seed metabolism within and between groups. The major proteomic variation among these 16 genotypes was observed in the seed maturation proteins, which consisted of 4–6 protein spots. All genotypes showed 3 spots of sucrose-binding protein except one wild genotype which had 2 spots. Two protein spots were detected for a triacylglycerol (TAG) accumulation factor protein in 13 genotypes, while one spot was detected in one ancestral and one wild genotype, and no spot was detected in one of the wild genotypes. All genotypes showed 3 spots of alcohol dehydrogenase except one Asian landrace and one wild genotype that had only one spot. Minor proteomic variation was detected in 16 genotypes for an elongation factor (EF2) and formate dehydrogenase, with apparent absence of EF2 in one landrace and formate dehydrogenase in another landrace genotype. No differences in the proteomic distribution of dehydrin-like protein, seed biotin-containing protein, glyceraldehyde 3-phosphate dehydrogenase, and fructose bisphosphate aldolase were detected. This information will be useful in selecting genotypes for breeding programs, and forms a baseline to which variation in transgenic varieties can be compared.

Composition, Vigor, and Proteome of Mature Soybean Seeds Developed under High Temperature

The effects of high temperature treatment on soybean [Glycine max (L.) Merr.] seed composition, vigor, and proteome were investigated using mature dry seeds harvested from plants grown in environment‐controlled chambers. High day/night temperatures (37/30°C) from stages R5 through R8 altered ratios of individual fatty acids to total fatty acid compared to the control (27/18°C). Concentration of sugars decreased, but total protein and phytic acid concentration were unchanged. High temperature resulted in a greater proportion of abnormal seeds, but normal‐appearing seed exhibited reduced germination and vigor. Proteomic analysis detected 20 protein identities whose accumulations were changed by the high temperature. Fourteen spots were identified as seven subunits of seed storage proteins. The remaining six proteins were identified as those responding to abiotic stresses or having a function in respiration: (i) sucrose binding protein, (ii) Class III acidic endochitinase, (iii) heat shock protein (HSP22), (iv) late embryo abundant protein, (v) Bowman–Birk proteinase inhibitor, and (vi) formate dehydrogenase. High temperature during seed development changed soybean seed composition and decreased seed vigor, but also changed seed protein expression profiles.

A 64 kDa sucrose binding protein is membrane-associated and tonoplast-localized in developing mung bean seeds

Sucrose binding proteins (SBPs) were predicted to be membrane-associated, but have been shown to localize in the lumen of protein storage vacuoles of various seeds. In this study, a new 64 kDa SBP has been identified from developing mung bean (Vigna radiata) seeds (here termed VrSBP1) via MS/MS analysis and N-terminal amino acid sequencing analysis and specific antibodies were generated using purified VrSBP1 proteins. Western blot analysis with the new VrSBP1 antibodies showed that, similar to most seed storage proteins, VrSBP1 proteins accumulated during seed development and were subsequently mobilized once the mung bean seeds germinated. Immunogold electron microscope (EM) studies on ultra-thin sections of high-pressure freezing/frozen substituted developing mung bean cotyledons demonstrated that VrSBP1 was localized specifically to the tonoplast of the protein storage vacuole and to the limiting membrane of a novel putative prevacuolar compartment. Biochemical and subcellular fractionation studies further demonstrated that VrSBP1 proteins were membrane-associated in developing mung beans, consistent with their tonoplast localization. This study thus shows convincing evidence of tonoplast-localization of a plant SBP for its future functional characterization and provides a model of studying non-integral membrane proteins associated with the tonoplasts in plant cells.

Distinct repressing modules on the distal region of the SBP2 promoter contribute to its vascular tissue-specific expression in different vegetative organs

The Glycine max sucrose binding protein (GmSBP2) promoter directs vascular tissue-specific expression of reporter genes in transgenic tobacco. Here we showed that an SBP2-GFP fusion protein under the control of the GmSBP2 promoter accumulates in the vascular tissues of vegetative organs, which is consistent with the proposed involvement of SBP in sucrose transport-dependent physiological processes. Through gain-of-function experiments we confirmed that the tissue-specific determinants of the SBP2 promoter reside in the distal cis-regulatory domain A, CRD-A (position -2000 to -700) that is organized into a modular configuration to suppress promoter activity in tissues other than vascular tissues. The four analyzed CRD-A sub-modules, designates Frag II (-1785/-1508), Frag III (-1507/-1237), Frag IV (-1236/-971) and Frag V (-970/-700), act independently to alter the constitutive pattern of -92pSBP2-mediated GUS expression in different organs. Frag V fused to -92pSBP2-GUS restored the tissue-specific pattern of the full-length promoter in the shoot apex, but not in other organs. Likewise, Frag IV confined GUS expression to the vascular bundle of leaves, whereas Frag II mediated vascular specific expression in roots. Strong stem expression-repressing elements were located at positions -1485 to -1212, as Frag III limited GUS expression to the inner phloem. We have also mapped a procambium silencer to the consensus sequence CAGTTnCaAccACATTcCT which is located in both distal and proximal upstream modules. Fusion of either repressing element-containing module to the constitutive -92pSBP2 promoter suppresses GUS expression in the elongation zone of roots. Together our results demonstrate the unusual aspect of distal sequences negatively controlling tissue-specificity of a plant promoter.

Expression of the sucrose binding protein from soybean: Renaturation and stability of the recombinant protein

The sucrose binding protein (SBP) belongs to the cupin family of proteins and is structurally related to vicilin-like storage proteins. In this investigation, a SBP isoform (GmSBP2/S64) was expressed in E. coli and large amounts of the protein accumulated in the insoluble fraction as inclusion bodies. The renatured protein was studied by circular dichroism (CD), intrinsic fluorescence, and binding of the hydrophobic probes ANS and Bis-ANS. The estimated content of secondary structure of the renatured protein was consistent with that obtained by theoretical modeling with a large predominance of β-strand structure (42%) over the α-helix (9.9%). The fluorescence emission maximum of 303 nm for SBP2 indicated that the fluorescent tryptophan was completely buried within a highly hydrophobic environment. We also measured the equilibrium dissociation constant ( K d) of sucrose binding by fluorescence titration using the refolded protein. The low sucrose binding affinity ( K d = 2.79 ± 0.22 mM) of the renatured protein was similar to that of the native protein purified from soybean seeds. Collectively, these results indicate that the folded structure of the renatured protein was similar to the native SBP protein. As a member of the bicupin family of proteins, which includes highly stable seed storage proteins, SBP2 was fairly stable at high temperatures. Likewise, it remained folded to a similar extent in the presence or absence of 7.6 M urea or 6.7 M GdmHCl. The high stability of the renatured protein may be a reminiscent property of SBP from its evolutionary relatedness to the seed storage proteins.

Conversion of a Putative Agrobacterium Sugar-binding Protein into a FRET Sensor with High Selectivity for Sucrose

Glucose is the main sugar transport form in animals, whereas plants use sucrose to supply non-photosynthetic organs with carbon skeletons and energy. Many aspects of sucrose transport, metabolism, and signaling are not well understood, including the route of sucrose efflux from leaf mesophyll cells and transport across vacuolar membranes. Tools that can detect sucrose with high spatial and temporal resolution in intact organs may help elucidate the players involved. Here, FRET sensors were generated by fusing putative sucrose-binding proteins to green fluorescent protein variants. Plant-associated bacteria such as Rhizobium and Agrobacterium can use sucrose as a nutrient source; sugar-binding proteins were, thus, used as scaffolds for developing sucrose nanosensors. Among a set of putative sucrose-binding protein genes cloned in between eCFP and eYFP and tested for sugar-dependent FRET changes, an Agrobacterium sugar-binding protein bound sucrose with 4 mum affinity. This FLIPsuc-4mu protein also recognized other sugars including maltose, trehalose, and turanose and, with lower efficiency, glucose and palatinose. Homology modeling enabled the prediction of binding pocket mutations to modulate the relative affinity of FLIPsuc-4mu for sucrose, maltose, and glucose. Mutant nanosensors showed up to 50- and 11-fold increases in specificity for sucrose over maltose and glucose, respectively, and the sucrose binding affinity was simultaneously decreased to allow detection in the physiological range. In addition, the signal-to-noise ratio of the sucrose nanosensor was improved by linker engineering. This novel reagent complements FLIPs for glucose, maltose, ribose, glutamate, and phosphate and will be used for analysis of sucrose-derived carbon flux in bacterial, fungal, plant, and animal cells.

Combinatorial regulation modules on GmSBP2 promoter: A distal cis-regulatory domain confines the SBP2 promoter activity to the vascular tissue in vegetative organs

The Glycine max sucrose binding protein ( GmSBP2) promoter directs phloem-specific expression of reporter genes in transgenic tobacco. Here, we identified cis-regulatory domains (CRD) that contribute with positive and negative regulation for the tissue-specific pattern of the GmSPB2 promoter. Negative regulatory elements in the distal CRD-A (−2000 to −700) sequences suppressed expression from the GmSBP2 promoter in tissues other than seed tissues and vascular tissues of vegetative organs. Deletion of this region relieved repression resulting in a constitutive promoter highly active in all tissues analyzed. Further deletions from the strong constitutive −700 GmSBP2 promoter delimited several intercalating enhancer-like and repressing domains that function in a context-dependent manner. Histochemical examination revealed that the CRD-C (−445 to −367) harbors both negative and positive elements. This region abolished promoter expression in roots and in all tissues of stems except for the inner phloem. In contrast, it restores root meristem expression when fused to the −132pSBP2-GUS construct, which contains root meristem expression-repressing determinants mapped to the 44-bp CRD-G (−136 to −92). Thus, the GmSBP2 promoter is functionally organized into a proximal region with the combinatorial modular configuration of plant promoters and a distal domain, which restricts gene expression to the vascular tissues in vegetative organs.

Directed evolution of protein switches and their application to the creation of ligand-binding proteins

We describe an iterative approach for creating protein switches involving the in vitro recombination of two nonhomologous genes. We demonstrate this approach by recombining the genes coding for TEM1 beta-lactamase (BLA) and the Escherichia coli maltose binding protein (MBP) to create a family of MBP-BLA hybrids in which maltose is a positive or negative effector of beta-lactam hydrolysis. Some of these MBP-BLA switches were effectively "on-off" in nature, with maltose altering catalytic activity by as much as 600-fold. The ability of these switches to confer an effector-dependent growth/no growth phenotype to E. coli cells was exploited to rapidly identify, from a library of 4 x 10(6) variants, MBP-BLA switch variants that respond to sucrose as the effector. The transplantation of these mutations into wild-type MBP converted MBP into a "sucrose-binding protein," illustrating the switches potential as a tool to rapidly identify ligand-binding proteins.

The soybean sucrose binding protein gene family: genomic organization, gene copy number and tissue-specific expression of the SBP2 promoter.

The sucrose binding protein (SBP) from soybean has been implicated as an important component of the sucrose uptake system. Two SBP genomic clones, gsS641.1 and gsS641.2, which correspond to allelic forms of the GmSBP2/S64 gene, have been isolated and characterized. As a member of the seed storage protein superfamily, it has been shown that the SBP gene structure is similar to vicilin genes with intron/exon boundaries at conserved positions. Fluores cence in situ hybridization (FISH) suggested that the soybean SBP gene family is represented by at least two non-allelic genes corresponding to the previously isolated GmSBP1 and GmSBP2/S64 cDNAs. These two cDNAs share extensive sequence similarity but are located at different loci in the soybean genome. To investigate transcriptional activation of the GmSBP2 gene, 2 kb 5'-flanking sequences of gsS641.1 and gsS641.2 were fused to the beta-glucuronidase (GUS) reporter gene and to the green fluorescent protein (GFP) reporter gene and inde pendently introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. The SBP2 promoter directed expression of both GUS and GFP reporter genes with high specificity to the phloem of leaves, stems and roots. Thus, the overall pattern of SBP-GUS or SBP-GFP expression is consistent with the involvement of SBP in sucrose translocation-dependent physiological processes.

Protein sorting and expression of a unique soybean cotyledon protein, GmSBP, destined for the protein storage vacuole.

The initial biochemical characterization of the soybean sucrose-binding protein, GmSBP, within our lab and others produced several incongruous characteristics that required a re-characterization of GmSBP via sequence homology, cell biology, immunolocalization, and semi-quantitative analysis. The GmSBP proteins share amino acid sequence homology as well as putative structural homology with globulin-like seed storage proteins. A comparison to the major soybean seed storage proteins, glycinin and beta-conglycinin established several storage protein-like characteristics for GmSBP. All three proteins were present in a prevacuolar compartment and protein storage vacuole. All three proteins increased in expression during seed development and are remobilized during germination. Quantitatively, the relative concentrations of GmSBP, beta-conglycinin (alpha/alpha' subunits), and glycinin (acidic subunits) indicated that GmSBP contributes 19-fold less to the stored nitrogen. The quantitative differences between GmSBP and glycinin may be attributed to the unconserved order and spacing of cis-acting regulatory elements present within the promoter regions. Ultimately, GmSBP is transported to the mature protein storage vacuole. The biological function of GmSBP within the protein storage vacuole remains uncertain, but its localization is a remnant of its evolutionary link to a globulin-like or vicilin-like ancestor that gave rise to the 7S family of storage proteins.

Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results

Background: The hazelnut major allergens identified to date are an 18-kd protein homologous to Bet v 1 and a 14-kd allergen homologous to Bet v 2. No studies have reported hazelnut allergens recognized in patients with positive double-blind, placebo-controlled food challenge (DBPCFC) results or in patients allergic to hazelnut but not to birch. Objective: We characterized the hazelnut allergens by studying the IgE reactivity of 65 patients with positive DBPCFC results and 7 patients with severe anaphylaxis to hazelnut. Methods: Hazelnut allergens were identified by means of SDS-PAGE and IgE immunoblotting. Further characterization was done with amino acid sequencing, evaluation of the IgE-binding properties of raw and roasted hazelnut with enzyme allergosorbent test inhibition, assessment of cross-reactivity with different allergens by means of immunoblotting inhibition, and purification by means of HPLC. Results: All the sera from the patients with positive DBPCFC results recognized an 18- and a 47-kd allergen; other major allergens were at molecular weights of 32 and 35 kd. Binding to the 18-kd band was inhibited by birch extract, indicating its homology with the birch major allergen, and abolished in roasted hazelnut. The 47-kd allergen is a sucrose-binding protein, the 35-kd allergen is a legumin, and the 32-kd allergen is a 2S albumin. Patients with severe anaphylactic reactions to hazelnut showed specific IgE reactivity to a 9-kd allergen, totally inhibited by purified peach lipid-transfer protein (LTP), which was heat stable and, when purified, corresponded to an LTP. Conclusions: The major allergen of hazelnut is an 18-kd protein homologous to Bet v 1, and the 9-kd allergen is presumably an LTP. Other major allergens have molecular weights of 47, 32, and 35 kd. (J Allergy Clin Immunol 2002;109:563-70.)