Isolation and computer analysis of the 5'-regulatory region of the seed storage protein gene from buckwheat (Fagopyrum esculentum Moench)

Using the modified rapid amplification of cDNA ends (5'-RACE) approach, a fragment containing the 955 bp long 5'-regulatory region of the buckwheat storage globulin gene (FeLEG1) has been amplified from the genomic DNA of buckwheat. The entire fragment was sequenced, and the sequence was analyzed by computer prediction of cis-regulatory elements possibly involved in tissue-specific and developmentally controlled seed storage protein gene expression. The promoter obtained might be interesting not only for fundamental research but also as a useful tool for biotechnological application.     

Characterization of the soluble allergenic proteins of cashew nut (Anacardium occidentale L.)

The allergens associated with cashew food allergy have not been well-characterized. We sought to identify the major allergens in cashew nut by performing IgE immunoblots to dissociated and reduced or nonreduced cashew protein extracts, followed by sequencing of the peptides of interest. Sera from 15 subjects with life-threatening reactions to cashews and 8 subjects who tolerate cashews but have life-threatening reactions to other tree nuts were compared. An aqueous cashew protein extract containing albumin/globulin was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to IgE immunoblotting using patient sera. Selected IgE reactive bands were subjected to N-terminal amino acid sequencing. Each of the 15 sera from cashew-allergic subjects showed IgE binding to the cashew protein extract. The dominant IgE-binding antigens in the reduced preparations included peptides in the 31-35 kD range, consistent with the large subunits of the major storage 13S globulin (legumin-like protein). Low-molecular-weight polypeptides of the 2S albumin family, with similarity to the major walnut allergen Jug r 1, also bound IgE. The sera from eight patients who tolerate cashew but displayed allergies to other tree nuts showed only minimal or no IgE binding to cashew. Cashew food allergy is associated with the presence of IgE directed against the major seed storage proteins in cashew, including the 13S globulin (legumin group) and 2S albumins, both of which represent major allergen classes in several plant seeds. Thus, the legumin-group proteins and 2S albumins are again identified as major food allergens, which will help further research into seed protein allergenicity.     

Molecular cloning of 11S globulin and 2S albumin, the two major seed storage proteins in sesame

Insoluble 11S globulin and soluble 2S albumin, conventionally termed alpha-globulin and beta-globulin, are the two major storage proteins and constitute 80-90% of total seed proteins in sesame. Two full-length cDNA clones were sequenced and deduced to encode sesame 11S globulin and 2S albumin precursors, respectively. Deduced amino acid composition reveals that 2S albumin, but not 11S globulin, is a sulfur-rich protein. Three abundant polypeptides of 50-60 kDa were resolved on SDS-PAGE when seed-purified 11S globulin was prepared in nonreducing conditions. Immunological analysis suggests that these three polypeptides are encoded by homologous genes. Immunodetection on the overexpressed protein of the 11S globulin clone in Escherichia coli indicates that this clone encodes the precursor protein of one of the three purified 11S globulin polypeptides.     

Effect of environmental stress during grain filling on the soluble proteome of wheat (Triticum aestivum) dough liquor

The influence of genotype and environment on a soluble wheat dough liquor proteome was studied for four cultivars grown under field conditions and under hot/dry and cool/wet regimes by two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole time-of-flight mass spectrometry. Although the four cultivars had similar patterns, differences in the relative abundances of some components were observed. Similarly, some differences were observed between the control samples and the samples grown under cool/wet and hot/dry conditions. These included differences in the abundances of storage proteins belonging to the 7S globulin (vicilin-like) and alpha-globulin families and of protective proteins including members of the serpin, described as allergens, and chitinase families. A number of novel annotations were made as compared to previous work on the dough liquor of cv. Hereward, including two 19 kDa alpha-globulins, precursors of endochitinases A and C, and several polypeptides belonging to the 7S globulin (vicilin-like) family.     

Gene families encoding isoforms of two major sesame seed storage proteins, 11S globulin and 2S albumin

Sesame (Sesamum indicum L.) seed has been recognized as a nutritional protein source owing to its richness in methionine. Storage proteins have been implicated in allergenic responses to sesame consumption. Two abundant storage proteins, 11S globulin and 2S albumin, constitute 60-70 and 15-25% of total sesame proteins, respectively. Two gene families separately encoding four 11S globulin and three 2S albumin isoforms were identified in a database search of 3328 expressed sequence tag (EST) sequences from maturing sesame seeds. Full-length cDNA sequences derived from these two gene families were completed by PCR using a maturing sesame cDNA library as the template. The amino acid compositions of these deduced storage proteins revealed that the richness in methionine is attributed mainly to two 2S albumin isoforms and partly to one 11S globulin isoform. The presence of four 11S globulin and three 2S albumin isoforms resolved in SDS-PAGE was confirmed by MALDI-MS analyses. The abundance of these isoforms was in accord with the occurrence frequency of their EST sequences in the database. A comprehensive understanding of these storage proteins at the molecular level may also facilitate the identification of allergens in crude sesame products that have caused severe allergic reactions increasingly reported in the past decade.     

Overexpression, purification, and in vitro refolding of the 11S globulin from amaranth seed in Escherichia coli

An amarantin 11S globulin cDNA encoding one of the most important storage proteins of amaranth seeds, with a high content of essential amino acids, was expressed in Escherichia coli. A good level of expression of recombinant amarantin with a molecular weight of 59 kDa was obtained. The recombinant protein was extracted by ammonium sulfate precipitation and purified to homogeneity using ion-exchange chromatography and reversed phase high-performance liquid chromatography. The expressed protein exhibited electrophoretic, immunochemical, and surface hydrophobicity properties similar to those of native amarantin from amaranth seed. Also, the recombinant protein was refolded in vitro using two different methods.     

Comparative proteomical analysis of zygotic embryo and endosperm from Coffea arabica seeds

During coffee seed development, proteins are predominantly deposited in cotyledons and in the endosperm. Reserve proteins of the 11S family are the most abundant globulins in coffee seeds, acting as a nitrogen source during roasting and guaranteeing flavor and aroma. The aim of the present study was to compare the protein profiles of endosperm and zygotic embryos of coffee seeds. Proteins were extracted from whole seed as well as from embryo and endosperm, separately. Total proteins were analyzed by two-dimensional electrophoresis (2-DE) followed by identification by mass spectrometry (MS). The most abundant spots observed in the gels of coffee seeds were excised, digested with trypsin, and identified by MS as subunits of the 11S globulin. Spots with identical pI and molecular masses were also observed in the protein profiles of coffee endosperm and embryo, indicating that 11S protein is also highly expressed in those tissues. Peptide sequence coverage of about 20% of the entire 11S globulin was obtained. Three other proteins were identified in the embryo and endosperm 2-DE profiles as a Cupin superfamily protein, an allergenic protein (Pru ar 1), exclusive to the endosperm 2D map, and a hypothetical protein, observed only in the zygotic embryo profile.     

Characterization of globulins from common vetch (Vicia sativa L.)

The proteins from Vicia sativa L. (common vetch) seeds were investigated. Protein comprises approximately 11.4% of the seed fresh weight, >50.8% of which is composed by globulins and 43.6% by albumins. The globulins may be fractionated into two main components, which were named alpha-vicinin (comprising 73% of the total globulin fraction, and hence >37% of the total seed protein) and beta-vicinin. Two minor globulin components are also present, gamma-vicinin and delta-vicinin. alpha-Vicinin, the legumin-like globulin, with a sedimentation coefficient of 10.6 S, is a nonglycosylated, disulfide-bond-containing globulin, composed of a group of subunits with molecular masses ranging from 50 to 78 kDa. Upon reduction, each of these subunits releases a heavy polypeptide chain (34-66 kDa) and a light polypeptide chain (21-23 kDa). beta-Vicinin, the vicilin-like globulin, with a sedimentation coefficient of 7.7 S, is a nonglycosylated globulin that contains no disulfide bonds and consists of two major polypeptides with molecular masses of 58 and 66 kDa. gamma-Vicinin is a minor, glycosylated, disulfide-bond-containing globulin. In the reduced form, it comprises six polypeptide chains with molecular masses of 12, 19, 21, 22, 23, and 31 kDa. Finally, delta-vicinin is a minor, highly glycosylated globulin that exhibits hemagglutinating activity. It is composed of a major 47 kDa polypeptide and two minor (33 and 38 kDa) polypeptides. N-terminal sequencing of the delta-vicinin 47 kDa polypeptide revealed no homology to any other known storage protein.     

Imbibition of soybean seeds in warm water results in the release of copious amounts of Bowman-Birk protease inhibitor, a putative anticarcinogenic agent

Protease inhibitors play a protective role against pathogenic microorganisms and herbivorous insects. The two predominant protease inhibitors of soybean seeds are the Kunitz trypsin inhibitor (KTI) and Bowman-Birk protease inhibitor (BBI). In this study, we report that soybean seeds incubated in warm water release large amounts of proteins into the surrounding media. Two-dimensional gel electrophoresis analysis of the seed exudates resulted in the separation of 93 distinct protein spots out of which 90 spots were identified by LC-MS/MS. The basic 7S globulin and the BBI are the two predominant proteins found in the soybean seed exudates. In addition to 7S and 11S seed storage proteins, others known to protect the seeds against pathogens and pests including KTI, peroxidase, α-galactosidase, and endo-1.3-β-glucanase were also identified in the seed exudates. Soybean seed exudate obtained by incubating the seeds in warm water was also able to inhibit the growth of human breast cancer cell line MCF-7. Since soybean seeds release large amounts of enzymatically active BBI when immersed in warm water, our procedure could be exploited as a simplified alternative method for the preparation of BBI concentrate which is being used as a cancer chemoprotective agent.     

Bowman-Brik胰蛋白酶抑制剂在水稻叶片生长和种子萌发过程中的表达

Bowman-Birk胰蛋白酶抑制剂(BBTI)是一类富含半胱氨酸的丝氨酸蛋白酶抑制剂,广泛分布于豆科、禾本科和菊科植物中,在蛋白质存储和植物防御中具有重要作用。水稻(Oryza sativa L.ssp.indica)BBTI家族具有13个成员,其中BBTI-9和BBTI-10都为假基因。其余BBTI成员都存在数量不同的保守Bowman-Birk结构域。为了解BBTI的表达信息,本研究主要通过设计多肽序列的方法,制备了11个BBTI的多克隆抗体,利用Western blot技术检测其在叶片生长发育和种子萌发过程中的表达。结果表明BBTI在叶片生长过程中呈现不同的时空特异性表达,BBTI-1、-2、-11、-12和-13伴随叶片生长含量增加,BBTI-5、-6和-8在苗期表达含量高,而BBTI-3、-4、-7在苗早期1cm时表达量达到峰值,以修饰或多聚体的形式在叶片生长过程中表达。在种子萌发过程中发现BBTI-8在种子中表达量较高,萌发后2h降到最低,24h后一条分子量略小的降解带开始表达,其余抗体在种子萌发阶段未发现有明显的变化。本研究还统计了苗期叶片、分蘖期叶片和萌发期的种子的MPSStags数据,试图比较转录与蛋白质水平对应关系。本研究结果证明,BBTI蛋白质在水稻生长发育过程中存在差异表达,为深入了解其功能提供了重要线索。     

Proteomic analysis of high protein soybean (Glycine max) accessions demonstrates the contribution of novel glycinin subunits

Limited biochemical information is available on soybean accessions that have seed protein content greater than 45% of the seed dry weight. SDS-PAGE analysis of seed proteins from nine soybean accessions revealed significantly higher amount of seed storage proteins in these accessions when compared with that of soybean cultivar Williams 82. High-resolution two-dimensional gel electrophoretic analysis of seed proteins revealed significant differences among several seed storage protein components in these accessions. A total of 51 protein spots were identified using peptide mass fingerprinting (MALDI-TOF MS). The contribution of these proteins to the overall protein content of the accessions was quantified using Delta2D image analysis software. Results showed that among the majority of the nine accessions, the largest difference in higher protein quantity was within the seed 11S storage globulins. The high protein trait from PI407788A was successfully transferred to an experimental line, LG99-469, demonstrating that this trait was transferable and robust.     

Molecular cloning, recombinant expression, and immunological characterization of a novel allergen from tartary buckwheat

Buckwheat is generally regarded as a nutritionally rich food source. However, earlier studies prove that it also causes allergies to subjects. Allergenic proteins with a strong IgE-binding activity have been identified in common buckwheat (CB) and a 24 kDa allergen (rTBa) in tartary buckwheat (TB). The objective of this research was to clone and express a novel allergen in tartary buckwheat and to evaluate its structure and immunological activity. The 1773 bp full-length cDNA was amplified and cloned from the total RNA of TB by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods. Its nucleotide sequence had high similarity with legume-like 13S storage protein mRNA in CB. The deduced amino acid sequence included a putative signal peptide and 18 fragments as its epitope sites. The predicted full-length TB allergen sequence was found to have two domains, and the recombinant protein reacted with sera from patients with positive IgE binding to buckwheat and had a lower binding ability than the recombinant TBa and recombinant TBb (C- and N-terminal amino acid sequence of TBt codes for protein). This fact suggests that full-length TB allergen may hydrolyze to two domains in vivo, decreasing the IgE-binding ability.     

Purification of an ACE inhibitory peptide after hydrolysis of sunflower (Helianthus annuus L.) protein isolates

Sunflower protein isolates and the proteases pepsin and pancreatin were used for the production of protein hydrolysates that inhibit angiotensin-I converting enzyme (ACE). Hydrolysates obtained after 3 h of incubation with pepsin and 3 h with pancreatin were studied. An ACE inhibitory peptide with the sequence Phe-Val-Asn-Pro-Gln-Ala-Gly-Ser was obtained by G-50 gel filtration chromatography and high-performance liquid chromatography C18 reverse phase chromatography. This peptide corresponds to a fragment of helianthinin, the 11S globulin from sunflower seeds, which is the main storage protein in sunflower. These results show that sunflower seed proteins are a potential source of ACE inhibitory peptides when hydrolyzed with pepsin and pancreatin.     

Electrophoretic analysis of some <Emphasis Type="Italic">Lathyrus</Emphasis> L. species based on seed storage proteins

Present study reported the variation in electrophoretic seed subfraction (albumins, globulin A, globulin B, glutelins) patterns in some Lathyrus species collected from their natural habitats different localities in Turkey. Electrophoretic data were documented by using a gel documentation system (Bio-Rad, USA) and analysed by using Quantity 1-D analysis software and also the dendograms were formed with 4.0% tolerance in UPGAMA (Unweighed Pair-Group Arithmetic Mean). The differences among species were observed and all seven species were clearly identifiable from the protein patterns. The dendograms obtained from cluster analysis of seed storage proteins data showed examined species have displayed a number of distinct relationships. According to our results, it seems justified to recommend a wider use of electrophoretic analysis of seed storage proteins in taxonomic investigations of leguminous plants.     

Storage proteins from Lathyrus sativus seeds

The proteins from Lathyrus sativus Linn. (chickling vetch or grass pea) seeds were investigated. Protein constitutes approximately 20% of the seed dry weight, >60% of which is composed by globulins and 30% by albumins. A single, 24 kDa polypeptide comprises more than half of the protein present in the albumin fraction. The globulins may be fractionated into three main components, which were named alpha-lathyrin (the major globulin), beta-lathyrin, and gamma-lathyrin. alpha-Lathyrin, with a sedimentation coefficient of approximately 18S, is composed of three main types of unglycosylated subunits (50-66 kDa), each of which produce, upon reduction, a heavy and a light polypeptide chain, by analogy with 11S. beta-Lathyrin, with a sedimentation coefficient of 13S, is composed by a relatively large number of subunits (8-66 kDa). Two major polypeptides are glycosylated and exhibit structural similarity with beta-conglutin from Lupinus albus. One of these possesses an internal disulfide bond. gamma-Lathyrin, with a sedimentation coefficient of approximately 5S, contains two interacting, unglycosylated polypeptides, with no disulfide bonds: the major 24 kDa albumin and the heavier (20 kDa) polypeptide chain of La. sativus lectin.     

Surface physicochemical properties of globulin-P amaranth protein

Globulin-P, the polymerized 11S amaranth globulin, is composed of 280 kDa unitary molecules (UM, 23%) and aggregates larger than 500 kDa (A, 70%). Antibodies against these proteins were prepared to study their surface characteristics and to assess their homology with other storage proteins. Results showed that globulin-P unitary molecules and aggregates had similar reactive surfaces. A polypeptide of 56 kDa was found to be the most reactive to the antibodies assayed, followed by the acidic polypeptides. Such results support previous information, according to which these polypeptides appeared to be the most exposed on the molecule surface. Globulin-P fraction presented cross-reactivity with the remaining amaranth protein fractions: 11S-globulin, glutelins, and albumins. Globulin-P and 11S-globulin showed similar reactive surfaces whereas glutelin and albumins presented a lower cross-reactivity. The reactivity of the glutelin fraction depended on its sequence. Globulin-P fraction presented cross-reactivity with quinoa globulins, and to a lesser extent with globulins of sunflower and rice. Moreover, the anti-Gp serum was unable to detect either conformational or sequence epitopes in globulins of soybean, wheat, buckwheat, rice, and rye.     

Expression, cloning, and immunological analysis of buckwheat (Fagopyrum esculentum Moench) seed storage proteins

cDNA of buckwheat (Fagopyrum esculentum Moench) was isolated from immature seeds harvested 14 days after pollination. Two genes, designated FA02 and FA18, were found to encode legumin-like proteins and were expressed during seed development. The deduced amino acid sequence of FA02 was identical to the N-terminal amino acid domain of BW24KD, which was believed to be a major buckwheat allergen (Urisu, A.; Kondo, Y.; Morita, Y.; Yagi, E.; Tsuruta, M.; Yasaki, T.; Yamada, K.; Kuzuya, H.; Suzuki, M.; Titani, K.; Kurosawa, K. Isolation and characterization of a major allergen in buckwheat seeds. In Current Advances in Buckwheat Research; Shinshu University Press: Matsumoto, Japan, 1995; pp 965--974). It was predicted that FA02 would be cleaved to generate two separate components, a 41.3 kDa alpha-subunit and a 21 kDa beta-subunit. Antiserum was raised against the deduced FA02 beta-subunit, and immunoblotting of total protein from buckwheat seeds (F. esculentum M. and Fagopyrum tartaricum Gaertn.) revealed that several groups of proteins reacted with the antiserum. Polypeptides in the 23--25 kDa range displayed the greatest reactivity.