Legume Seed Storage Proteins Research Articles

Morphology, Formation Kinetics and Core Composition of Pea and Soy 7S and 11S Globulin Amyloid Fibrils.

Legume seed storage proteins can be induced to form amyloid fibrils upon heating at low pH, which could improve their functionality for use in food and materials. However, the amyloidogenic regions of legume proteins are largely unknown. Here, we used LC-MS/MS to determine the amyloid core regions of fibrils formed by enriched pea and soy 7S and 11S globulins at pH 2, 80 °C, and characterized their hydrolysis, assembly kinetics, and morphology. A lag phase was absent from the fibrillation kinetics of pea and soy 7S globulins, while 11S globulins and crude extracts displayed a similar lag time. Pea and soy protein fibrils differed in morphology, with most pea fibrils being straight and soy fibrils being worm-like. Pea and soy globulins were abundant in amyloid-forming peptides, with over 100 unique fibril-core peptides from pea 7S and around 50 unique fibril-core peptides identified from pea 11S, soy 7S, and soy 11S globulins. Amyloidogenic regions derive predominantly from the homologous core region of 7S globulins and the basic subunit of 11S globulins. Overall, pea and soy 7S and 11S globulins are rich in amyloidogenic regions. This study will help understand their fibrillation mechanism and engineer protein fibrils with specific structures and functionality.

A Novel Multipeptide Microarray for the Specific and Sensitive Mapping of Linear IgE-Binding Epitopes of Food Allergens

Background: The identification of B-cell epitopes of food allergens can possibly lead to novel diagnostic tools and therapeutic reagents for food allergy. We sought to develop a flexible, low-tech, cost-effective and reproducible multipeptide microarray for the research environment to enable large-scale screening of IgE epitopes of food allergens. Methods: Overlapping peptides (15-mer, 4 amino acid offset) covering the primary sequence of either peanut allergen Ara h 1 or all 3 subunits of the soybean allergen Gly m 5 were simultaneously synthesized in-house on a porous cellulose matrix. Identical peptide microarrays created with up to 384 duplicate peptide-cellulose microspots each were investigated for specificity and sensitivity in IgE immunodetection and in direct experimental comparison to the formerly established SPOT™ membrane technique. Results: The in-house microarray identified with 98% reproducibility the same IgE-binding peptides as the SPOT™ membrane technique. Additional IgE-binding peptides were identified using the microarray. While the sensitivity was increased between 2- and 20-fold, the amount of human serum required was reduced by at least two thirds over the SPOT™ membrane technique using the microarray. After subtraction of the potential background, we did not observe non-specific binding to the presented peptides on microarray. Conclusions: The novel peptide microarray allows simple and cost-effective screening for potential epitopes of large allergenic legume seed storage proteins, and it could be adapted for other food allergens as well, to study allergenic epitopes at the individual subject level in large paediatric and adult study groups of food allergic subjects.

Hypocholesterolaemic effect of rat-administered oral doses of the isolated 7S globulins from cowpeas and adzuki beans.

The role of seed proteins, especially soyabean 7S globulins, in controlling dyslipidaemia is widely acknowledged. Amino acid sequence homology among the proteins of this family could reflect similar biological functions in other species. The aim of the present study was to unveil a hypolipidaemic effect of the 7S globulins from cowpeas (7S-C) and adzuki beans (7S-A), administered orally to rats fed a hypercholesterolaemic (HC; high cholesterol and TAG) diet for 28 d. A total of forty-five rats were divided into five groups (nine rats per group): (1) standard (STD) diet; (2) HC diet; (3) HC diet+7S-C (300mg/kg per d); (4) HC diet+7S-A (300mg/kg per d); and (5) HC diet+simvastatin (SVT; 50mg/kg per d), as a control. Significant decreases in food intake and final body weight of rats receiving HC+7S-C and HC+7S-A diets compared with groups fed the HC and STD diets were observed. Significant decreases in serum total and non-HDL-cholesterol of 7S-C, 7S-A and SVT groups were also observed. HDL-cholesterol levels increased in the 7S-C, 7S-A and SVT groups, while hepatic cholesterol and TAG concentrations were significantly lower than in the HC diet group for the 7S-C-supplemented group only. Faecal excretions of fat and cholesterol in HC diet groups were considerably higher in animals consuming the 7S globulins. The results show that cowpea and adzuki bean 7S globulins promote cholesterol-decreasing effects in hypercholesterolaemic rats even at low dosages, as already observed for other legume seed storage proteins of this family. This main effect is discussed in relation to the possible mechanisms of action.

Molecular biology and plant breeding

The use of modern molecular biological techniques has played a significant role in elucidating various aspects of the molecular organization of plant genes. Most of the information gained concerns characters and phenomena that are of relevance to plant breeding. It has been shown at the molecular level that cereal and legume seed storage proteins are controlled by complex multigenic nuclear loci. The analysis of movable nuclear genes, exemplified by the Ds-Ac transposable elements in maize, has given insight into the nature of these elements and the genetic changes that occur at their insertion and deletion sites. The characterization of the heterogeneous mitochondrial DNA and the uniform chloroplast DNA has helped to elucidate the molecular basis of cytoplasmic male sterility, RuBP carboxylase enzyme synthesis and herbicide resistance, In N2 fixation, the emphasis has been put on the molecular dissection of N2-fixing genes in bacteria and the interactions between symbiotic and host DNAs. It has been possible to develop vectors of Agrobacterium tumefaciens Ti-plasmids which lack oncogenes for DNA transfer. Cauliflower and gemini viruses as well as liposomes are also potential systems for DNA transport to protoplasts which can be regenerated into plants. Furthermore, it has been shown that certain molecular biological techniques are useful for screening purposes in genetic analysis and plant breeding work.

Self-aggregation of legume seed storage proteins inside the protein storage vacuoles is electrostatic in nature, rather than lectin-mediated

Conglutins are multisubunit, glycosylated, major storage proteins present in Lupinus seeds that self-aggregate in a calcium/magnesium-dependent manner. Two of these globulins exhibit lectin activity. The 210 kDa globulin derived from β-conglutin that accumulates in Lupinus cotyledons during germination was used as a model protein to establish whether the self-aggregation process is electrostatic in nature or lectin-mediated. This protein binds in a very strong manner to chitin and recognizes a variety of glycoproteins including immunoglobulins G. Several compounds were tested for their inhibitory effect on the cation-dependent self-aggregation process. Sialic acid and phytin were the most effective whereas chitin and mucin were totally ineffective. The inability of the oligosaccharidic side chains of the 210 kDa protein, β-conglutin and immunoglobulin G to interfere with the aggregation strongly supports the view that Ca/Mg are electrostatically involved in the in vitro self-aggregation of Lupinus globulins. The results suggest that calcium and magnesium ions are also electrostatically involved in vivo in the macromolecular aggregation of legume seed storage proteins, ensuring their efficient packing inside the protein storage vacuoles. This mechanism is responsible for the typical insolubility of legume globulins in water.

Evidence that the N-terminal extension of the Vicieae convicilin genes evolved by intragenic duplications and trinucleotide expansions

This study was aimed to identify lentil (Lens culinaris subsp. culinaris) convicilin genes and to carry out a comparative analysis of these genes in the tribe Vicieae. Convicilins differ from vicilins, a related group of plant seed storage proteins, mainly by the presence of an additional sequence of amino acids in the sequence corresponding to the first exon, referred as the N-terminal extension. A single gene for convicilin, a component of legume seed storage proteins, was identified in the cultivated lentil. In this species, the N-terminal extension is formed by a stretch of 126 amino acids of which 59.2% are charged amino acids: 29.6% glutamic acid, 3.2% aspartic acid, 14.4% arginine, 8.8% lysine, and 3.2% histidine. This lentil convicilin sequence is similar to the sequence of convicilins in other species of the tribe Vicieae. However, the size of the N-terminal extension clearly differs among convicilins. Sequence comparison and phylogenetic analyses including convicilin and vicilin of Vicieae species indicated that the differentiation between vicilins and convicilins predated the differentiation of the two vicilin gene families (47- and 50-kDa vicilins), and that the N-terminal extension evolved mainly by a series of duplications of short internal sequences and triplet expansions, the predominant one being GAA.

Evidence that the N-terminal extension of the Vicieae convicilin genes evolved by intragenic duplications and trinucleotide expansions

This study was aimed to identify lentil (Lens culinaris subsp. culinaris) convicilin genes and to carry out a comparative analysis of these genes in the tribe Vicieae. Convicilins differ from vicilins, a related group of plant seed storage proteins, mainly by the presence of an additional sequence of amino acids in the sequence corresponding to the first exon, referred as the N-terminal extension. A single gene for convicilin, a component of legume seed storage proteins, was identified in the cultivated lentil. In this species, the N-terminal extension is formed by a stretch of 126 amino acids of which 59.2% are charged amino acids: 29.6% glutamic acid, 3.2% aspartic acid, 14.4% arginine, 8.8% lysine, and 3.2% histidine. This lentil convicilin sequence is similar to the sequence of convicilins in other species of the tribe Vicieae. However, the size of the N-terminal extension clearly differs among convicilins. Sequence comparison and phylogenetic analyses including convicilin and vicilin of Vicieae species indicated that the differentiation between vicilins and convicilins predated the differentiation of the two vicilin gene families (47- and 50-kDa vicilins), and that the N-terminal extension evolved mainly by a series of duplications of short internal sequences and triplet expansions, the predominant one being GAA.Key words: convicilin, evolution by duplications, Lens culinaris Medik., lentil, legumes, trinucleotide expansion.

Calcium- and magnesium-dependent aggregation of legume seed storage proteins.

The solubility characteristics and sedimentation behavior of total or individual globulins from legume seeds [Lupinus albus L., Pisum sativum L., and Glycine max (L.) Merr.] were investigated. The typical insolubility of globulins detected during their extraction seems to be due to the presence of a low molecular weight factor(s) in the seed extract. The solubility of the purified globulins decreases with increasing concentrations of calcium and/or magnesium, but not of other cations, showing minimum values at concentrations that vary with the particular globulin considered. Ultracentrifugation analyses revealed that the Ca(2+)- and/or Mg(2+)-induced insolubilization of the globulins involves the formation of high-order aggregates of molecules of the same or of different globulins. These macromolecular structures are dissociated under conditions of high ionic strength, suggesting the involvement of electrostatic interactions in the aggregation process. The degree of association relies heavily on the amount of Ca(2+) and/or Mg(2+) available, on the presence of chelating agents for these divalent cations, and on the ionic strength of the surrounding medium. The possible physiological significance of the findings is discussed.

Improvement of the nutritional quality of legume seed storage proteins by molecular breeding

Legume seeds contain a large amount of proteins and are one of the essential protein sources for humans and animals. However, the protein, in legume seeds is usually poor in sulfur-containing amino acids, and its nutritional value is lower than the protein from animal sources. Recently plant breeding has become available by the introduction of molecular biology, and a technique, called molecular breeding, was applied to the production of legume seeds that contain proteins with high nutritional quality. This review describes the expression of legume seed protein genes and the transformation of legume plants. Approaches to improve the legume seed storage protein will be discussed.

Correct post-translational modification and stable vacuolar accumulation of phytohemagglutinin engineered to contain multiple methionine residues.

Most legume seed storage proteins are deficient in sulfur amino acids. In this study, we demonstrate that replacing specific amino acid residues of a seed protein with methionine residues at positions known to be occupied by methionine residues in homologous proteins, is an effective strategy to create methionine-enriched seed proteins. Mutant phytohemagglutinin polypeptides with three or four methionine residues were found to undergo correct post-translational modifications in transformed cultured tobacco cells and to accumulate stably in the protein storage vacuoles of transgenic tobacco seeds.

Plant Food Protein Engineering

Protein engineering is a technique to modify protein primary sequences by gene manipulation. The primary sequences of a target protein can be systematically modified, thus protein engineering has the potential to become a method for enhancing the nutritional value and functional properties of food proteins. The final aim of plant food protein engineering is to breed protein-producing crops that have high food qualities. The chapter reviews the subject of plant food protein engineering, focusing on cereal and legume seed storage proteins. Protein engineering to improve the quality of food proteins and elucidate the relationships between the structure and the functional properties of food proteins requires the isolation of genomic DNAs or complementary DNAs (cDNAs) encoding the target proteins. A procedure of genomic DNA and cDNA cloning is presented in the chapter. The recent development of techniques, enzymes, and vectors related to cloning make the procedure are outlined in the chapter.

5003045 Modified 7S legume seed storage proteins: Leslie Hoffman assigned to Lubrizol Genetics Inc

5003045 Modified 7S legume seed storage proteins: Leslie Hoffman assigned to Lubrizol Genetics Inc

Evolution of legume seed storage proteins--a domain common to legumins and vicilins is duplicated in vicilins.

We examined the primary sequence of canavalin, the major storage protein of jack beans, and found that an ancient sequence duplication accounts for 80% of the amino acid residues. Evidence for such a duplication was also found in the orthologous proteins phaseolin and pea vicilin. This sequence duplication presumably accounts for a structural duplication in the canavalin monomer observed by crystallographic analysis. One copy of this repeat was found in a second storage-protein family, the legumins, where it encompasses almost the entire B-chain of the mature molecule. We propose that the vicilin and legumin families of legume seed proteins evolved from a common precursor, which consisted of one copy of the repeat in the vicilins.

Isolation and Characterization of Alfalfa 7S and 11S Seed Storage Protein

This paper reports the extraction, characterization and partial purification of the major seed proteins of alfalfa (Medicago sativa L.). Alfalfa seeds were extracted at a variety of pHs or with low and high salt. The major seed proteins extracted have sedimentation coefficients of 7S and 11S. The 7S protein is soluble at low salt while the 11 S protein is soluble at pH 3 or 9 in low salt or at pH 7 at 1 M N aCl. The 7S protein can be purified by gel filtration and DEAE ion exchange. It is composed of peptides of 52, 36, 32 and 20 kD. The 11S protein is purified by extracting contaminating proteins at pH 7, 1 M N aCI followed by solubilization at pH 9. It is composed of a triplet of peptides at 49, 47 and 45 kD, as well as peptides at 39, 22 and 20 kD. This is the first report of separation and characterization of the major alfalfa seed proteins. These results are discussed with reference to other legume seed storage proteins and with respect to gene expression in somatic embryos.

The subunit structure and stability of conglutin δ, a sulphurrich protein from the seeds of Lupinus angustifolius L.

AbstractThe oligomeric structure and stability of conglutin δ, the 2S sulphur‐rich lupin seed protein, has been studied. Molecular weights were determined by sedimentation equilibrium methods in the analytical ultracentrifuge. Conglutin δ2 (Mr 14 000, 2S), the most abundant form, was composed of one large (˜9600) and one small polypeptide chain linked by disulphide bonds. The minor oligomeric form, conglutin δ1 (Mr 28 000, 2.8S), was a disulphide‐linked dimer of conglutin δ2. Each form was capable of reversible association and at low ionic strength (neutral pH), the conglutin δ1 momomer associated to a homogeneous dimer (Mr 56 000, 4.1S). Calculated frictional ratios (f/f0 ˜ 1.28–1.49) suggested that the three different forms were asymmetric. Spectral studies (ORD/CD) showed that conglutins δ1 and δ2 were rich in alpha‐helix (˜38%) unlike the major 7S and 11S legume seed storage proteins. The helical structure was unusually stable both to heat and chemical denaturants; below 60°C (at neutral pH) the helix remained unaffected and only partial denaturation occurred at higher temperatures. Nevertheless, complete denaturation was achieved (at 20°C) in high concentrations of guanidine hydrochloride (greater than 6.5 M). The stability was due to the presence of disulphide cross‐links; with the addition of reducing agent, as little as 1 M guanidine hydrochloride (GuHCl) was sufficient for denaturation of the helical structure. Titration experiments showed a single buried tyrosine (pK 11.4) which could be exposed (pK 10.2) in 6 M GuHCl.

Convicilin mRNA from pea (Pisum sativum L.) has sequence homology with other legume 7S storage protein mRNA species.

Nucleotide-sequence analysis of a complementary-DNA clone for convicilin, one of the storage proteins from pea (Pisum sativum L.) seeds, shows it to be homologous with the 7S legume seed storage proteins vicilin, conglycinin and phaseolin. Convicilin is more similar to vicilin than to phaseolin or to conglycinin. Significant areas of sequence difference are discussed.

Characterization and Complexity of Wheat Developing Endosperm mRNAs

Free and membrane-bound (MB) polysomes and the corresponding polyadenylated RNAs (polyA(+) RNAs) have been isolated from developing wheat endosperm (Triticum aestivum L.) Free and MB poly(A)(+) RNAs, analyzed on isokinetic sucrose gradient with [(3)H]polyuridylic acid [poly(U)] hybridization detection, appear to be 11S to 12S in size with a 7% poly(A) tail for MB RNAs. cDNAs synthesized using both of these mRNA populations in presence of a potent RNase inhibitor (RNasin), have been used for hybridization kinetics experiments. The mean square fitting analysis of the hybridization kinetics between MB cDNA and its template reveals the presence of two abundance classes representing roughly (2/3) and (1/3) of the MB poly(A)(+) RNAs and containing the information for approximately 75 superabundant species (21,000 copies per cell) and 750 intermediate species (530 copies per cell), respectively. The mRNA population extracted from free polysomes is divided into three abundance classes. The first one is composed of superabundant sequences which would correspond to the MB superabundant mRNAs. The free mRNAs consist of about 11,000 diverse sequences, most of them being rare sequences. Heterologous hybridizations of MB cDNAs to free mRNAs have shown that some mRNAs are common to both populations. This could be explained either by a partial contamination or by free polysomes en route to their membrane destination. Contrary to the low number of diverse mRNAs corresponding to the legume seed storage proteins, the wheat endosperm superabundant mRNAs consist of about 75 different sequences which would encode most of the seed storage proteins, especially gliadins.