Biochemical safety evaluation of transgenic rice seeds expressing T cell epitopes of Japanese cedar pollen allergens

Transgenic rice seeds, which express a hybrid peptide comprising seven predominant human T cell epitopes (7Crp) derived from Japanese cedar pollen allergens, have been shown to function as an effective edible vaccine for the control of pollen allergen-induced responses. In this study, we characterized biochemical properties of transgenic seeds expressing the 7Crp peptide. The levels of chemical compositions, such as carbohydrate, protein, lipid, amino acid, fatty acid, mineral, and vitamin, were substantially equivalent between transgenic 7Crp and its nontransgenic counterpart seeds. The contents of three major allergenic proteins in transgenic seeds were not enhanced by expression of the 7Crp peptide when compared with those of nontransgenic seeds. The 7Crp peptide expressed in seeds was susceptible to simulated gastric/intestinal fluids. N-Glycosylation was not observed in the 7Crp peptide sequence. These results indicate that transgenic 7Crp seeds are substantially equivalent to nontransgenic parental seeds except for the presence of the 7Crp peptide. Keywords: Food safety assessment; transgenic rice seed; edible vaccine; peptide-based immunotherapy; Japanese cedar pollinosis.     

Beta-lactoglobulin structure and retinol binding changes in presence of anionic and neutral detergents

Bovine beta-lactoglobulin (beta-LG) in vivo (in milks) has been found in complexes with lipids such as butyric and oleic acids. To elucidate the still unknown structure-function relationship in this protein, the structural changes of beta-lactoglobulin variant A (beta-LG A) in the presence of anionic surfactant such as sodium n-dodecyl sulfate (SDS) and in the presence of nonionic surfactant such as Triton X-100 have been investigated. Subsequently, the retinol binding by beta-LG has been investigated in the presence of various amounts of these surfactants as its binding indicator. The results of UV-vis and fluorescence studies show a higher denaturating effect of SDS at acid pH that can be due to greater positive charges of beta-LG at this pH indicating also the nonspecific hydrophobic interactions of Triton X-100 with beta-LG at all studied pHs. Isothermal titration calorimetry (ITC) measurements indicate the endothermic nature of beta-LG/SDS interactions and the exothermic nature of Triton X-100/beta-LG interactions. The analysis of the binding data demonstrates the absence of considerable changes in retinol binding properties of beta-LG in the presence of various amounts of these surfactants. This implies that surfactant binding does not change the conformation of beta-LG in the regions defining the retinol-binding site.     

Bioactive peptides in amaranth (Amaranthus hypochondriacus) seed

Amaranth seeds are rich in protein with a high nutritional value, but little is known about their bioactive compounds that could benefit health. The objectives of this research were to investigate the presence, characterization, and the anticarcinogenic properties of the peptide lunasin in amaranth seeds. Furthermore, to predict and identify other peptides in amaranth seed with potential biological activities. ELISA showed an average concentration of 11.1 microg lunasin equivalent/g total extracted protein in four genotypes of mature amaranth seeds. Glutelin fraction had the highest lunasin concentration (3.0 microg/g). Lunasin was also identified in albumin, prolamin and globulin amaranth protein fractions and even in popped amaranth seeds. Western blot analysis revealed a band at 18.5 kDa, and MALDI-TOF analysis showed that this peptide matched more than 60% of the soybean lunasin peptide sequence. Glutelin extracts digested with trypsin, showed the induction of apoptosis against HeLa cells. Prediction of other bioactive peptides in amaranth globulins and glutelins were mainly antihypertensive. This is the first study that reports the presence of a lunasin-like peptide and other potentially bioactive peptides in amaranth protein fractions.     

Protein components of low-density lipoproteins purified from hen egg yolk

To identify apoproteins present in purified low-density lipoproteins from hen egg yolk in relation with their emulsifying properties, they have been separated by SDS-PAGE. We identified two different proteins by liquid chromatography-tandem mass spectrometry analysis of the peptides obtained by the trypsin digestion of protein gel bands. Apovitellenin I was identified as a monomer and a dimer. Its amino acid sequence was totally confirmed, and molecular mass determination by liquid chromatography-mass spectrometry showed that it did not present post-translational modifications but only a slight heterogeneity by the loss of one or two amino acids at the C-terminal part of the protein. Apolipoprotein B was identified into seven bands corresponding to fragments resulting of a processing of the hen blood apo-B protein. The identity of the fragments was determined by the observation of the sequence coverage by trypsin peptides and the sequence alignment with homologous human blood apolipoprotein B-100.     

Effects of hydration,lipids, and temperature on the binding of the volatile aroma terpenes by beta-lactoglobulin powders

The binding properties of dry proteins are relatively poorly known. Many proteins are present in emulsions and suspensions and also in dry forms. This is particularly true of dairy proteins, which are often stored and sold in powdered form. In the present work, the binding of three terpenes (alpha-terpinene, gamma-terpinene, and terpinolene), which belong to the basic aroma components, and of decane by powdered beta-lactoglobulin (BLG) was studied at different hydration levels (0.05-0.40 g of H(2)O/g of protein) and temperatures (298 and 309.5 K), in the presence or absence of lipids and small concentrations of ethanol. Vapor sorption isotherms were determined for these systems by a static method of headspace gas chromatographic analysis. A cooperative effect of hydrophobic hydration was observed for the binding of aroma terpenes and decane by the solid BLG. The temperature increase from 298 to 309.5 K reduced the observed hydration threshold of BLG by 0.05-0.08 g of H(2)O/g of protein. Lipids (1.2% w/w) in hydrated BLG gave at least a 2-fold increase in its binding affinity for the hydrocarbons studied, and synergic effects of the hydration and lipid on this affinity were observed.     

Cloning of a novel L-amino acid oxidase from Trichoderma harzianum ETS 323 and bioactivity analysis of overexpressed L-amino acid oxidase

L-amino acid oxidases (L-AAOs) have been isolated from many organisms, such as snake, and are known to have antibacterial activity. To the best of the authors' knowledge, this is the first report of the cloning of cDNA encoding a novel Trichoderma harzianum ETS 323 L-amino acid oxidase (Th-L-AAO). The protein was overexpressed in Escherichia coli and purified to homogeneity. Comparisons of its deduced amino acid sequence with the sequence of other L-AAOs revealed the similarity to be between 9 and 24%. The molecular mass of the purified protein was 52 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme substrate specificity was highest for L-phenylalanine, and its optimal pH and temperature for activity were 7 and 40 °C, respectively; exogenous metal ions had no significant effect on activity. Circular dichroism spectroscopy indicated that the secondary structure of Th-L-AAO is composed of 17% α-helices, 28% β-sheets, and 55% random coils. The bacterially expressed Th-L-AAO also mediated antibacterial activity against both gram-positive and gram-negative food spoilage microorganisms. Furthermore, a three-dimensional protein structure was created to provide more information about the structural composition of Th-L-AAO, suggesting that the N-terminal sequence of Th-L-AAO may have contributed to the antibacterial activity of this protein.     

Novel protein from Labramia bojeri A. DC. seeds homologue to Kunitz-type trypsin inhibitor with lectin-like properties

This study starts by isolating and characterizing the first protein from Labramia bojeri seeds, which belong to the Sapotaceae family. The purified lectin analyzed by SDS-PAGE with and without beta-mercaptoethanol shows two protein bands (M(r) = 19 and 20 kDa), which cannot be resolved. Protein bands have shown similar characteristics as molecular masses, determined by gel filtration and native gel; N-terminal sequences presented a difference in their isoelectric points. We have suggested that those protein bands might be variants of the protein named Labramin. The sequence database search has shown that the N-terminal sequence of Labramin presented a high degree of homology to Kunitz-type trypsin inhibitor (82-52%) despite no trypsin inhibition activity detection. The lectin-like form from Labramin was better inhibited by glycoproteins and has also presented growth inhibition of the fungus Colletotrichum lindemuthianum and the yeast Saccharomyces cerevisiae, but it has not presented an apparent effect on Fusarium oxysporum.     

Primary structure of the abundant seed albumin of Theobroma cacao by mass spectrometry

The most abundant albumin present in seeds of Theobroma cacao was purified to apparent homogeneity as judged by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NH(2)-terminal sequence analysis. Tryptic peptide mass fingerprinting of the purified protein by HPLC/ESI-MS showed the presence of 16 masses that matched the expected tryptic peptides corresponding to 95% of the translated amino acid sequence from the cDNA of the 21 kDa cocoa albumin. Collision-induced dissociation MS/MS analysis of the C-terminal peptide isolated from the CNBr cleavage products provided unequivocal evidence that the mature cocoa albumin protein is nine amino acid residues shorter than expected from the reported cDNA of its corresponding gene. The experimentally determined M(r) value of 20234 was in excellent agreement with the truncated version of the amino acid sequence. The purified cocoa albumin inhibited the catalytic activities of bovine trypsin and chymotrypsin. The inhibition was stoichiometric with 1 mol of trypsin or chymotrypsin being inhibited by 1 mol of inhibitor with apparent dissociation constants (K(i)) of 9.5 x 10(-8) and 2. 3 x 10(-6) M, respectively, for inhibitor binding at pH 8.5 and 37 degrees C. No inhibition of the catalytic activities of subtilisin, papain, pepsin, and cocoa endoproteases was detected under their optimal reaction conditions.     

Binding of the pepper alkaloid piperine to bovine beta-lactoglobulin: circular dichroism spectroscopy and molecular modeling study

The pepper alkaloid piperine is a nontoxic, natural dietary compound with a broad range of physiological activity. The present work is the first demonstration of its interaction with a mammalian protein. Circular dichroism (CD) spectroscopy was used to reveal and analyze the binding of piperine to a lipocalin protein. Induced CD spectra measured in pH 7.7 phosphate buffer at 37 degrees C demonstrated reversible, non-covalent association of piperine with bovine beta-lactoglobulin (BLG), the major whey protein in milk. The binding parameters (K(a) approximately 8 x 10(4) M(-1), n = 0.8) determined from the CD titration data showed no significant differences between the piperine binding properties of the two main genetic variants of BLG (A and B). The vanishing extrinsic CD signal obtained upon acidification of the piperine-BLG sample solution (Tanford transition) suggested that the ligand binds in the central hydrophobic cavity of the beta-barrel. The cavity binding concept was further supported by a CD displacement experiment using palmitic acid, the well-known hydrophobic ligand of BLG. Molecular docking calculations showed that piperine can be efficiently accommodated within the calyx of BLG. Additional molecular modeling calculations indicated that the beta-barrel of human tear lipocalin, human serum retinol binding protein, and human neutrophil gelatinase associated lipocalin might also accommodate a piperine molecule.     

Characterization of a salt-independent pectin methylesterase purified from valencia orange peel

The pectin methylesterase (PME; EC 3.1.1.11) present in a commercial orange peel enzyme preparation was characterized to establish its identity among the multiple PME isozymes present in Valencia orange (Citrus sinensis L.) peel. We show the commercial enzyme corresponds to the major peak 2 PME previously separated by heparin-Sepharose chromatography (Cameron et al., J. Food Sci. 1998, 63, 253). Both PMEs have comparable elution profiles on cation-exchange and hydrophobic-interaction perfusion chromatography columns, molecular weights (ca. 34 kDa) and pI (pH 9.2), and biochemical properties, including a broad pH activity range and activity in the absence of added cations. An identical partial amino terminal peptide sequence was also obtained for the PMEs, which further demonstrated a structural identity with other plant PMEs. The biochemical and structural properties readily distinguish this Valencia orange PME from salt-dependent isozymes and further suggest that it is an ortholog to the salt-independent fruit-specific isozyme of tomato. This work provides a well-defined, enzymatically homogeneous, salt-independent (type 1) plant PME isozyme that is suitable for studying details of the enzyme's mode of action and for use in modifying methylester patterns for studying the structure-functional property relationships in pectin.     

Phase model interpretation of the structural properties of two molecular soy protein fractions

The structural properties of mixtures of two molecular soy fractions, 11S (glycinin) and 2S, were investigated in the presence of glucono-delta-lactone (GDL) during an isothermal run at 25 degrees C for 1500 min. Analytical methodology included small-deformation dynamic oscillation, visual observations, scanning electron microscopy, and blending-law modeling. The aim of the work was to identify the state of phase separation and the pattern of solvent distribution between the two constituent polymers. It was found that the high molecular weight distribution of 11S supported rapid kinetics of structure formation, with this continuous matrix being concentrated and, hence, further reinforced in the presence of small additions of 2S. Blending-law modeling suggested that 2S was able to retain disproportionate volumes of solvent within its phase. A consequence of such property was that high additions of 2S leading to equal concentrations of the two molecular fractions in the blend resulted in a catastrophic drop in the values of the overall network strength. This behavior is rather unexpected for the structural properties of a phase-separated system, and it has been rationalized on the basis of the high water-holding capacity of the small molecular weight fraction. 2S entraps in its phase the polymeric segments of glycinin, which are then unable to become structural knots of a cohesive three-dimensional morphology observed in single 11S preparations.     

Shining light on the differences in molecular structural chemical makeup and the cause of distinct degradation behavior between malting- and feed-type barley using synchrotron FTIR microspectroscopy: a novel approach

The objective of this study was to use advanced synchrotron-sourced FTIR microspectroscopy (SFTIRM) as a novel approach to identify the differences in protein and carbohydrate molecular structure (chemical makeup) between these two varieties of barley and illustrate the exact causes for their significantly different degradation kinetics. Items assessed included (1) molecular structural differences in protein amide I to amide II intensities and their ratio within cellular dimensions, (2) molecular structural differences in protein secondary structure profile and their ratios, and (3) molecular structural differences in carbohydrate component peak profile. Our hypothesis was that molecular structure (chemical makeup) affects barley quality, fermentation, and degradation behavior in both humans and animals. Using SFTIRM, the protein and carbohydrate molecular structural chemical makeup of barley was revealed and identified. The protein molecular structural chemical makeup differed significantly between the two varieties of barleys. No difference in carbohydrate molecular structural chemical makeup was detected. Harrington was lower than Valier in protein amide I, amide II, and protein amide I to amide II ratio, while Harrington was relatively higher in model-fitted protein alpha-helix and beta-sheet, but lower in the others (beta-turn and random coil). These results indicated that it is the molecular structure of protein (chemical makeup) that may play a major role in the different degradation kinetics between the two varieties of barleys (not the molecular structure of carbohydrate). It is believed that use of the advanced synchrotron technology will make a significant step and an important contribution to research in examining the molecular structure (chemical makeup) of plant, feed, and seeds.     

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.     

Binding between bixin and whey protein at pH 7.4 studied by spectroscopy and isothermal titration calorimetry

Bixin is the major coloring component of annatto used in manufacturing colored cheeses, but its presence in liquid whey causes undesirable quality of the recovered whey protein ingredients. The objective of this work was to study molecular binding between bixin and three major whey proteins (β-lactoglobulin, α-lactalbumin, and bovine serum albumin) at pH 7.4 using UV-vis absorption spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and circular dichroism. These complementary techniques illustrated that the binding is a spontaneous complexation process mainly driven by hydrophobic interactions. The complexation is favored at a lower temperature and a higher ionic strength. At a lower temperature, the binding is entropy-driven, while it changes to an enthalpy-driven process at higher temperatures. The binding also increases the percentage of unordered secondary structures of proteins. Findings from this work can be used to develop whey protein recovery processes for minimizing residual annatto content in whey protein ingredients.     

葡萄籽中水溶性钙结合蛋白的分离和鉴定

为了开发植物源葡萄籽补钙制剂,该研究通过亚细胞定位试验表明,葡萄籽的胚乳中含有大量的钙元素。通过电泳分析发现,葡萄籽的水溶性蛋白包括2种主要成分,其中一种是11 S球蛋白(蛋白质B),也是最主要的钙结合蛋白,另一种是表观分子量为670 k Da的蛋白质A。在蛋白质组成相同的情况下,用传统的碱溶酸沉法来分离葡萄籽蛋白会导致大量的钙流失。但用30%~50%硫酸铵沉淀法得到的蛋白质得率是(22.5±0.02)g/kg,蛋白质中钙质量分数(3.47%)是碱溶酸沉法(1.11%)的3倍。该研究结果为食品工业中矿物结合蛋白质的分离纯化提供参考。     

Dimeric inhibitors of human salivary alpha-amylase from emmer (Triticum dicoccon Schrank) seeds

The proteins belonging to the cereal trypsin/alpha-amylase inhibitor family are abundant water/salt-soluble flour proteins active against alpha-amylases from several seed parasites and pests and inactive against endogenous alpha-amylases. Three alpha-amylase inhibitor families have been described in cereals that vary in size and are differently expressed among Triticeae seeds. The present work investigates the presence of human salivary alpha-amylase inhibitors in emmer (Triticum dicoccon Schrank) flour. The isolation was obtained by a series of chromatography steps, and the purification progress was monitored through the inhibition of human salivary alpha-amylase activity. The purified fraction was subjected to protein sequencing by tandem mass spectrometry (MSMS) of the tryptic digests obtained after the sample separation on 2-DE. MSMS data indicated that the emmer alpha-amylase inhibitory fraction was composed of two newly identified proteins [emmer dimeric inhibitor 1 (EDI-1) and emmer dimeric inhibitor 2 (EDI-2)] sharing very high identity levels with related proteins from Triticum aestivum.     

Reveal protein molecular structural-chemical differences between two types of winterfat (forage) seeds with physiological differences in low temperature tolerance using synchrotron-based Fourier transform infrared microspectroscopy

Winterfat (Krascheninnikovia lanata) (forage seed) is a long-lived native shrub with superior forage quality for livestock and wildlife. The objectives of this study were to use advanced synchrotron technology [S-Fourier transform infrared microspectroscopy (FTIR)] as a novel approach to reveal protein molecular structural-chemical differences in terms of protein secondary structures between the two types of winterfat (forage) seeds, which show physiological differences in low-temperature tolerances. This experiment was performed at beamline U10B at the National Synchrotron Light Source (NSLS) in Brookhaven National Laboratory (BNL), U.S. Department of Energy (NSLS-BNL, New York). The results showed that with the synchrotron analytical technique (S-FTIR), the molecular structural-chemical makeup and characteristics of the winterfat seed tissues could be imaged and revealed. The protein secondary structures differed between the large and the small seed tissues. By using the multicomponent peaks modeling method, the results show that the large seeds contained no significant differences (P > 0.05) in percentage of beta-sheet (average 37.0%) and alpha-helix (average 24.1%). However, the large seeds contained a lower (P < 0.05) percentage of beta-turns (18.1 vs 20.1%) and a lower (P < 0.05) ratio of beta-turns to alpha-helices (0.8 vs 0.9) and beta-turns to beta-sheets (0.5 vs 0.6). Our results demonstrate the potential of highly spatially resolved synchrotron-based FTIR microspectroscopy to reveal differences of structural molecular chemistry and protein secondary structures, which are associated with seed size variation and may affect germination behaviors.     

Identification of an IgE reactive peptide in hen egg riboflavin binding protein subjected to simulated gastrointestinal digestion

Riboflavin binding protein (RfBP) is a minor protein in hen egg; its potential involvement in egg allergy has seldom been studied. The aim of this work was to investigate the IgE binding capacity of RfBP before and after simulated gastrointestinal digestion. It was shown that digestion of RfBP mainly occurred during the gastric phase. The protein fragments resulting from the subsequent duodenal phase remained linked through disulfide bonds. Both the intact protein and its digests were subjected to inhibition ELISA with sera obtained from patients allergic to egg. The results revealed significant IgE binding to intact RfBP, whereas the digests showed reduced but substantial IgE binding levels, with serum-to-serum variability. The RfBP digests were then subjected to immunoblot with allergic patients' sera, and the IgE-reactive peptides were further analyzed by MALDI-TOF/TOF mass spectrometry for sequence determination. The RfBP sequence 41-84 was identified as a novel IgE binding peptide in patients allergic to egg.     

Characterization of royal jelly proteins in both Africanized and European honeybees (Apis mellifera) by two-dimensional gel electrophoresis

In this study, the proteins contained in royal jelly (RJ) produced by Africanized honeybees and European honeybees (Apis mellifera) haven been analyzed in detail and compared using two-dimensional gel electrophoresis, and the N-terminal amino acid sequence of each spot has been determined. Most spots were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular MRJP3, in terms of molecular weights and isoelectric points between the two species of RJ. Furthermore, during the determination of the N-terminal amino acid sequence of each spot, for the first time, MRJP4 protein has been identified, the existence of which had been only implied by cloning of its cDNA sequence. The presence of heterogeneous bands of glucose oxidase was also identified. Thus, the results suggest that two-dimensional gel electrophoresis provides a suitable method for the qualitative analysis of the proteins contained in RJ derived from different honeybee species.     

Characterization of Thiamin‐Binding Protein from Wheat Germ

A thiamin‐binding protein was isolated from wheat germ (Triticum aestivum). Its molecular mass was estimated as 120,000 Da consisting of two 56,000‐subunits. The protein contained a large amount of glutamine or glutamic acid (15.4 mol%), arginine (12.5 mol%), and glycine (12.0 mol%). The levels of tyrosine, methionine, tryptophan, and cysteine in the protein were low. Optimum pH for its thiamin‐binding activity was pH 8.0. It bound free thiamin specifically, not thiamin phosphates. The apparent dissociation and maximum bound values for the thiamin‐binding were 2.52 μM and 9.34 nmol/mg of protein, respectively. Properties of the thiamin‐binding protein from wheat germ were similar to those of the thiamin‐binding protein from rice seeds, but not from buckwheat, sesame, or sunflower seeds.