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.     

Environmental conditions during vegetative growth determine the major proteins that accumulate in mature grapes

Despite recent reports in the literature that chitinases comprise approximately 50% of the soluble proteins present in mature Vitis vinifera L. (cv. Moscatel) grapes, with the other major proteins being thaumatin-like proteins, a careful inspection of the published data reveals inconsistencies as to which proteins actually accumulate in mature grapes. Mature Moscatel grapes were harvested in the same vineyard in two consecutive years, 1999 and 2000. The grapes exhibited widely distinct polypeptide patterns when analyzed by either FPLC cation exchange chromatography or two-dimensional electrophoresis: whrereas the 2000 grapes possessed a much higher protein content (1.96 versus 1.11 mg g(-)(1) of fresh weight), the 1999 grapes contained a greater heterogeneity of polypeptides. In addition, highly specific antibodies that recognize the pathogenesis-related proteins present in the grapes demonstrated that the 2000 harvest grapes had a wider variety of pathogenesis-related polypeptides. N-Terminal sequencing of the major polypeptides revealed differences in the relative abundance and amino acid sequence of thaumatin-like and osmotin-like polypeptides present in the 1999 and 2000 harvest Moscatel grapes and did not detect the presence of chitinase. As a whole, the data indicate that the expression and accumulation of the major proteins in grapes is essentially determined by the environmental and/or pathological conditions prevailing during grape development and maturation. The potential physiological and technological implications are discussed. The results of the present work suggest that it is not possible to base varietal differentiation of grapes on the profile of the pathogenesis-related polypeptides present in the mature berries.     

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.     

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.     

Heat-Induced Fragmentation of the Maize Waxy Protein During Protein Extraction from Starch Granules

The starch granule of maize contains a characteristic set of tightly bound polypeptides. Granule-associated polypeptides are typically extracted from starch granules by heating starch granule suspensions at 90–100°C in a detergent such as SDS. Solubilized proteins are recovered by centrifugation and analyzed by gel electrophoresis. Previously identified tightly bound granule intrinsic proteins consist of the 85-kDa starch-branching enzyme IIb, the 76-kDa starch synthase I, and the 60-kD waxy (Wx) protein, also known as granule-bound starch synthase I. However, SDS extracts from starch granules of maize also contain a cluster of proteins ranging in mass between 47 and 32 kDa In this study, we analyzed this group of granule-associated proteins and found that each was recognized by the Wx antibody. A 15 amino acid N-terminal sequence from the 47-kDa polypeptide was identical to the predicted N-terminus of the Wx protein. Further analysis revealed that each immunoreactive polypeptide between 47 and 32 kDa was a heat-induced fragmentation product of the Wx protein. Conditions for the extraction of granule proteins were evaluated. Our results demonstrate that granule proteins are effectively released by mild extraction (10-min incubation at 72°C). Relative to the Wx protein, starch synthase I and starch branching enzyme IIb were less susceptible to thermal fragmentation. These results demonstrate that the 85-, 76-, and 60-kDa polypeptides are authentic granule-intrinsic proteins, and that the majority of polypeptides between 47 and 32 kDa are artifacts of high-temperature granule extraction procedures.     

Purification and glycosylation analysis of an acidic pectin methylesterase in jelly fig (Ficus awkeotsang) achenes

An acidic pectin methylesterase (PME) is responsible for the gelation of water extract from jelly fig (Ficus awkeotasang) achenes. A new, fast and efficient, method has been developed to purify this acidic PME. The method includes preparing jelly curd by traditional hand washing, extracting proteins from the curd, and separating PME by anion-exchanger. The purified PME exists as a monomer of 38 kDa determined by gel filtration, and exerts enzymatic activity over a broad pH range, particularly in acidic environments where most known PME enzymes from various species are inactivated. Chemical staining and enzymatic cleavage suggest that the jelly fig PME is an N-linked glycoprotein. Fluorophore-assisted carbohydrate electrophoresis reveals that the polysaccharide of this glycoprotein putatively consists of 22 hexoses including 16 mannose, 4 N-acetylglucosamine, and 2 galactose residues.     

Cloning and expression of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

Pectin methylesterase (PME) is the key enzyme responsible for the gelation of jelly curd in the water extract of jelly fig (Ficus awkeotasang) achenes. The jelly fig PME extracted from achenes was isoelectrofocused at pH 2.5 and subjected to N-terminal amino acid sequencing. A cDNA fragment encoding the mature protein of this acidic PME was obtained by PCR cloning using a poly(T) primer and a degenerate primer designed according to the N-terminal sequence of the purified PME. The complete cDNA sequence of its precursor protein was further obtained by PCR using the same strategy. The PME clone was overexpressed in Escherichia coli, and its expressed protein was immunologically recognized as strongly as the original antigen using antibodies against purified PME. Fractionation analysis revealed that the overexpressed PME was predominantly present in the pellet and thus presumably formed insoluble inclusion bodies in E. coli cells.     

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.     

Characterization of methanol-soluble and methanol-insoluble proteins from developing peanut seed

The 85% methanol-soluble proteins are known to specifically contribute to the production of flavor of roasted peanut. To determine the nature of the 85% methanol-soluble proteins, they were isolated from the peanut seed, and the 85% methanol-soluble (MS) and 85% methanol-insoluble (MIS) fractions were characterized using polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. The results showed that the 85% MS fraction contained lower amounts (9-10%) of protein than the MIS fraction (15-33%). Protein content of the MIS fraction increased more significantly during seed maturation than it did in the MS fraction. Unlike the protein, free amino acids and soluble sugars levels of the MS fraction decreased significantly during seed maturation. The 85% MS fraction contained predominantly low molecular weight (<20 kDa) proteins/polypeptides, whereas the MIS fraction contained a mixture of polypeptides with molecular weight between 14 kDa and 90 kDa. SDS-PAGE showed no major changes in the polypeptide composition of the MS fraction during seed maturation. Capillary electrophoretic analysis revealed major qualitative and quantitative changes in the protein and polypeptide composition of the MS and MIS fractions during seed maturation. Fatty acid analysis of these fractions indicated that the MS fraction is lipoprotein in nature and rich in oleic and linoleic acids.     

两蜂种蜂王浆蛋白质组分析

采用蛋白质组双向电泳技术和已鉴定蜂王浆蛋白质组的比对,对王浆高产蜜蜂（浆蜂）和中华蜜蜂（中蜂）的蜂王浆蛋白质组进行了差异比较。结果表明,浆蜂蜂王浆的总蛋白质点数（93个）显著高于中蜂蜂王浆总蛋白质点数（70个）,它们的等电点主要集中在5-8之间,分子量在50-80kDa之间,其中共有蛋白质点为30个;通过与已鉴定的浆蜂蜂王浆蛋白质组比较,这些共有蛋白质点推断为蜂王浆主蛋白1、2、3和葡萄糖氧化酶。同时两蜂种蜂王浆的蛋白质丰度也存在较大差异,共有点中有4个蛋白质的丰度中蜂显著高于浆蜂,7个浆蜂显著高于中蜂中。结果表明浆蜂和中蜂蜂王浆蛋白质种类及丰度均有较大差异。     

Two-dimensional electrophoresis and western-blotting analyses with anti Ara h 3 basic subunit IgG evidence the cross-reacting polypeptides of Arachis hypogaea, Glycine max, and Lupinus albus seed proteomes

The allergenicity of seed storage proteins, the major components of edible legume seeds, may cause serious reactions in both children and adult population. Updated methodologies for evaluation of the activity of these proteins are needed. In this paper we used two-dimensional (2D) electrophoretic techniques to investigate the immuno-cross-reactivities of anti Ara h 3 basic subunit IgG to the seed proteomes of three legume species, namely, peanut, soybean, and lupin. The seed proteins, extracted with two different procedures, were separated by 2D electrophoresis, and the electrophoretic maps were analyzed by Western blot. In peanut proteome the antibodies strongly reacted with the 23 kDa polypeptides, corresponding to Ara h 3 basic isoforms, the antigen they were raised to, and three unidentified acidic polypeptides near 45 kDa. Remarkable cross-reactivities with lupin and soybean Ara h 3 homologous polypeptides and nonrelated proteins, namely, lupin conglutin gamma and soybean Bg7S, were detected. Therefore, these proteins may be regarded as new putative allergens. The present findings show the potentiality of 2D electrophoresis in the identification of food allergens and open the way to the traceability of the new cross-reacting proteins in the food chain.     

Characterization of the proteins from Vigna unguiculata seeds

The proteins from Vigna unguiculata (L.) Walp. (cowpea) seeds were investigated. Globulins constitute over 51% of the total seed protein, with albumins composing approximately 45%. The globulins may be fractionated by native electrophoresis or anion exchange chromatography into three main components, which were termed (in decreasing order of anodic mobility) alpha-vignin, beta-vignin, and gamma-vignin. alpha-Vignin, with a sedimentation coefficient of 16.5S, is a major, nonglycosylated globulin, composed of a major 80 kDa subunit, which upon reduction, produces two polypeptides (20 and 60 kDa). beta-Vignin, with a sedimentation coefficient of 13S, is a major, glycosylated globulin, composed of two main polypeptides (55 and 60 kDa) with no disulfide bonds. Finally, gamma-vignin, a minor globulin, is composed by one main type of subunit (22 kDa), which upon reduction, is converted into a single, apparently heavier polypeptide chain (30 kDa) due to the presence of an internal disulfide bond. Immunological analyses revealed structural homology between beta-vignin and beta-conglutin (the vicilin from Lupinus seeds) but not between alpha- or gamma-vignins and their Lupinus counterparts. Haemagglutination activity toward trypsinized rabbit erythrocytes was found exclusively in the albumin fraction and was strongly inhibited by N-acetylglucosamine or chitin.     

Immunochemical approach to detection of adulteration in honey: physiologically active royal jelly protein stimulating TNF-alpha release is a regular component of honey

The presence of royal jelly (RJ) proteins in honey collected from nectars of different plants, origin, and regions and in honeybee's pollen was detected by Western-blot analysis using polyclonal antibodies raised against water-soluble RJ-proteins. The most abundant RJ-protein in honeybee products corresponded to a 55 kDa protein. The N-terminal amino acid sequence of 55 kDa protein was N-I-L-R-G-E. This sequence is identical to the apalbumin-1, the most abundant protein of RJ. Apalbumin-1 is a regular component of honeybee products and thus is a suitable marker tool for proving adulteration of honey by means of immunochemical detection. Its presence in all tested samples of honeys and honeybee pollen was confirmed also by Western-blot analysis using polyclonal antibodies raised against recombinant apalbumin-1. It has been found that major RJ-proteins, apalbumin-1, and apalbumin-2, stimulate mouse macrophages to release TNF-alpha, which demonstrates that physiologically active proteins of honey could be used for its biological valuation.     

Stability of white wine proteins: combined effect of pH, ionic strength, and temperature on their aggregation

Protein haze development in white wines is an unacceptable visual defect attributed to slow protein unfolding and aggregation. It is favored by wine exposure to excessive temperatures but can also develop in properly stored wines. In this study, the combined impact of pH (2.5-4.0), ionic strength (0.02-0.15 M), and temperature (25, 40, and 70 °C) on wine protein stability was investigated. The results showed three classes of proteins with low conformational stability involved in aggregation at room temperature: β-glucanases, chitinases, and some thaumatin-like protein isoforms (22-24 kDa). Unexpectedly, at 25 °C, maximum instability was observed at the lower pH, far from the protein isoelectric point. Increasing temperatures led to a shift of the maximum haze at higher pH. These different behaviors could be explained by the opposite impact of pH on intramolecular (conformational stability) and intermolecular (colloidal stability) electrostatic interactions. The present results highlight that wine pH and ionic strength play a determinant part in aggregation mechanisms, aggregate characteristics, and final haze.     

Isolation and characterization of 2S cocoa seed albumin storage polypeptide and the corresponding cDNA

The amine pool of cocoa is known to be an essential component for the development of the typical cocoa flavor. To better understand and to produce an intense in vitro cocoa flavor, identification of the polypeptides that are the source of the amine flavor precursor pool is essential. Chromatographic analysis of the polypeptide profile of unfermented cocoa resulted in identification of a novel storage polypeptide of M(r) 8515. The N-terminal sequence of the first 34 residues of the purified polypeptide shows similarity to 2S storage albumins of cotton and Brazil nut and sweet protein, Mabinlin. To identify the corresponding cDNA of the putative cocoa 2S albumin, 18 randomly chosen clones from the cDNA library of immature Theobroma cacao seed mRNA were sequenced, and a full-length cDNA clone encoding a protein harboring the N-terminal sequence of the novel polypeptide was selected. The open reading frame of the clone encodes a polypeptide of M(r) 17125. Comparison of the translated amino acid sequence of the precursor protein or the mature polypeptide against the Swiss-Prot and TrEMBL databases shows high sequence similarity (>52%) and identity (>38%) to many plant 2S albumins. Tryptic peptide mass fingerprinting of the purified polypeptide by high-performance liquid chromatography-electrospray ionization mass spectrometry shows 10 masses that match the expected tryptic peptides of the deduced sequence. Together with the published work on plant 2S albumin processing, the results presented here suggest that post-translational processing yields a 73-residue polypeptide (residue positions 78-150) corresponding to the 9 kDa subunit of the mature cocoa 2S albumin protein.     

NsLTP1 and NsLTP2 isoforms in soft wheat (Triticum aestivum Cv. Centauro) and farro (Triticum dicoccon Schrank) bran

Isoforms of nonspecific lipid-transfer protein 1 (nsLTP1) and nonspecific lipid-transfer protein 2 (nsLTP2) were investigated in bran tissues isolated from caryopses of two cereal crops quite relevant for the Italian market, the cultivar Centauro of soft wheat (Triticum aestivum) and Italian emmer or farro (Triticum dicoccon Schrank). By sequential separation of the bran extracts on cation-exchange and gel filtration chromatographies, fractions containing only proteins belonging to the nsLTP1 and nsLTP2 classes were obtained. The proteins were roughly identified by SDS-PAGE and by immunoreactions in Western blotting experiments. By MALDI-MS and RP-HPLC/ESI-MS analyses we were able to show the presence of several LTP1 and LTP2 isoforms in the investigated species. Bioinformatic searches based on the determined Mr indicated that (i) two nsLTP1s already identified in T. aestivum have Mr and number of Cys residues identical to that of a 9.6 kDa protein present both in soft wheat cv. Centauro and in farro; (ii) two isoforms of nsLTP2 detected in T. aestivum have the same Mr and number of Cys residues of two 7 kDa proteins found in Centauro; and (iii) a nsLTP1 detected in Ambrosia artemisiifolia has Mr and number of Cys residues coincident to that of a 9.9 kDa protein found both in soft wheat cv. Centauro and in farro.     

Expression of apalbumin1 of Apis cerana cerana in the larvae of silkworm, Bombyx mori

Royal jelly (RJ) is a thick, milky material produced by both the hypopharyngeal and the mandibular glands of nurse honeybees. The main proteins of RJ, named apalbumins or major royal jelly proteins (MRJPs), have multiple biological functions. Apalbumin1 is the most abundant glycoprotein of RJ. In this study, Bacmid- apalbumin1 was constructed for Apis cerana cerana using the newly established Bac-to-Bac/BmNPV baculovirus expression system (BES). This procedure allowed us to obtain the recombinant A. cerana cerana ( Acc) apalbumin1 (r Accapalbumin1) from the hemolymph of silkworm larvae through the BmNPV bacmid system, 96 h postinfection. The r Accapalbumin1 was then purified by Ni-NTA spin columns and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. A 55 kDa protein with good solubility was then obtained. The peptide Ile-Phe was identified from trypsin production of r Accapalbumin1. Such a peptide has been reported to have an antihypertensive ability. Our results have therefore potential applications in biomedical research and open new perspectives for the study of apalbumins.     

Functional expression in pichia pastoris of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

A cDNA fragment encoding an acidic pectin methylesterase (PME) of jelly fig achene was successfully expressed in Pichia pastoris under the control of the glyceraldehydes-3-phosphate dehydrogenase promoter. The recombinant PME was produced as a secretory protein by N-terminal fusion of a cleavable prepropeptide for signal trafficking, and thus easily harvested from the culture medium. Compared with native N-glycosylated PME (38 kDa) purified from jelly fig achenes, this recombinant PME (45 kDa) appeared to be hyperglycosylated. Activity staining indicated that the recombinant PME was functionally active. Yet the hyperglycosylated recombinant PME possessed thermostability and enzymatic capability over a broad pH range equivalent to those of the native PME. The success of functional production of this acidic jelly fig PME in P. pastoris has significantly broadened its applications in industry.     

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.     

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.