Polypeptide modification: an improved proglycinin design to stabilise oil-in-water emulsions.

ß-Conglycinin and glycinin are soybean major seed storage proteins. Previous studies have shown that adding the extension region of ß-conglycinin α subunit improves the emulsifying properties of proglycinin and confers more favourable characteristics than fusing the extension region of ß-conglycinin α' subunit or the hypervariable regions (A4IV) of glycinin A1aB1b subunit. To evaluate the polypeptide properties, we designed mutants of A1aB1b subunits fused with truncated versions of A4IV (A4IVcut), α (αcut) or α' (α'cut) extension regions lacking the C-terminus 25 or 31 residues (A4IVC25, αC25 or α'C31), and also A4IVcut and α'cut with αC25 residues added (A4IVcut-αC25 and α'cut-αC25). All the modified proteins displayed conformations similar to the wild type. With good solubilities, the emulsion properties of the modified proteins were much better at ionic strength µ = 0.08 than at µ = 0.5. The modified A1aB1bαcut and A1aB1bα'cut showed poorer emulsion properties than those of A1aB1bα and A1aB1bα'. Replacing the hydrophobic A4IVC25 region of A1aB1bA4IV with hydrophilic αC25 created A1aB1bA4IVcut-αC25, which had the best emulsion stability among these proglycinin mutants. We found that addition of αC25 improves the emulsifying properties of two C-terminally truncated proglycinin variants, thereby illustrating its potential general utility. Our investigation showed that in order to improve the emulsifying ability and emulsion stability of a globular protein, the introduced polypeptide should (i) be highly hydrophilic, (ii) consist of multiple hydrophobic-strong hydrophilic regions comprising at least two alpha helixes, (iii) harbour a terminal α-helix at the end of the C-terminus and (iv) have properties similar to those of αC25.

Development of a novel transgenic rice with hypocholesterolemic activity via high-level accumulation of the α' subunit of soybean ß-conglycinin.

Soybean 7S globulin, known as ß-conglycinin, has been shown to regulate human plasma cholesterol and triglyceride levels. Furthermore, the α' subunit of ß-conglycinin has specifically been shown to possess low-density lipoprotein (LDL)-cholesterol-lowering activity. Therefore, accumulation of the α' subunit of ß-conglycinin in rice seeds could lead to the production of new functional rice that could promote human health. Herein, we used the low-glutelin rice mutant 'Koshihikari' (var. a123) and suppressed its glutelins and prolamins, the major seed storage proteins of rice, by RNA interference. The accumulation levels of the α' subunit in the lines with suppressed glutelin and prolamin levels were >20 mg in 1 g of rice seeds, which is considerably higher than those in previous studies. Oral administration of the transgenic rice containing the α' subunit exhibited a hypocholesterolemic activity in rats; the serum total cholesterol and LDL cholesterol levels were significantly reduced when compared to those of the control rice (var. a123). The cholesterol-lowering action by transgenic rice accumulating the α' subunit induces a significant increase in fecal bile acid excretion and a tendency to increase in fecal cholesterol excretion. This is the first report that transgenic rice exhibits a hypocholesterolemic activity in rats in vivo by using the ß-conglycinin α' subunit.

Purification, crystallization and preliminary crystallographic analysis of soybean mature glycinin A1bB2.

Glycinin is one of the most abundant storage-protein molecules in soybean seeds and is composed of five subunits (A1aB1b, A1bB2, A2B1a, A3B4 and A5A4B3). A1bB2 was purified from a mutant soybean cultivar containing glycinin composed of only A5A4B3 and A1bB2. At 281 K the protein formed hexagonal, rectangular and rod-shaped crystals in the first [0.1 M imidazole pH 8.0, 0.2 M MgCl2, 35%(v/v) MPD], second [0.1 M sodium citrate pH 5.6, 0.2 M ammonium acetate, 30%(v/v) MPD] and third (0.1 M phosphate-citrate pH 4.2, 2.0 M ammonium sulfate) crystallization conditions, respectively. X-ray diffraction data were collected to resolutions of 1.85, 1.85 and 2.5 Å from crystals of the three different shapes. The crystals belonged to space groups P6322, P21 and P1, with unit-cell parameters a = b = 143.60, c = 84.54 Å, a = 114.54, b = 105.82, c = 116.67 Å, ß = 94.99° and a = 94.45, b = 94.96, c = 100.66 Å, α = 107.02, ß = 108.44, γ = 110.71°, respectively. One, six and six subunits of A1bB2 were estimated to be present in the respective asymmetric units. The three-dimensional structure of the A1bB2 hexamer is currently being determined.

High-level production of lactostatin, a hypocholesterolemic peptide, in transgenic rice using soybean A1aB1b as carrier.

Hypercholesterolemia, a form of cardiovascular disease, is one of the leading causes of deaths worldwide. Lactostatin (Ile-Ile-Ala-Glu-Lys), derived from ß-lactoglobulin in cow's milk, is a bioactive peptide with hypocholesterolemic activity higher than sitosterol, a known anti-hypercholesterolemic drug. Here, we successfully developed a transgenic rice accumulating a much higher level of lactostatin by inserting 29 IIAEK sequences into the structurally flexible (nonconserved) regions of soybean seed storage protein, A1aB1b, and introducing it into LGC-1 (low glutelin content mutant 1) as host variety. A1aB1b containing 29 lactostatins was expressed in the endosperm of rice seed cells by using seed specific promoters and sorted into novel compartments distinct from normal PB-I (ER-derived protein body) and PB-II (protein storage vacuoles). Transgenic rice seeds accumulated approximately 2 mg of lactostatins/g of dry seeds, which is relatively high compared with previous reports. Our findings suggest that the introduction of a high copy number of bioactive peptide into seed storage proteins as carrier is one of the effective means in producing higher amounts of bioactive peptides in rice.

Crystal structure of a major seed storage protein, 11S proglobulin, from Amaranthus hypochondriacus: insight into its physico-chemical properties.

Amaranth is a crop known for its high quality proteins. 11S Globulin is one of the most abundant and important storage proteins of the amaranth grain. Here, we report the crystal structure of amaranth 11S proglobulin at a final resolution of 2.28 Å. It belonged to the space group P6(3) with cell dimensions a=b=96.6, c=75.0 Å. It contains one asymmetric unit consisting of 372 residues and 100 water molecules. Disordered regions in the model approximately correspond to the variable regions of the 11S globulins. The structure has an extended α-helix and ß-barrel domains at both N-terminal and C-terminal regions, which are characteristic of the 11S and 7S globulins. The three dimensional structure suggests that its high thermal stability is due to the cumulative effects of many factors and its good emulsifying property depended on the balance between its surface hydrophobicity and hydrophilicity.

Molecular design of seed storage proteins for enhanced food physicochemical properties.

Seed storage proteins such as soybean globulins have been nutritionally and functionally valuable in the food industry. Protein structure-function studies are valuable in modifying proteins for enhanced functionality. Recombinant technology and protein engineering are two of the tools in biotechnology that have been used in producing soybean proteins with better gelling property, solubility, and emulsifying ability. This article reviews the molecular basis for the logical and precise protein designs that are important in obtaining the desired improved physicochemical properties.

Crystal structure of the major peanut allergen Ara h 1.

Ara h 1, a 7S globulin, is one of the three major peanut allergens. We previously reported the crystallization of the core region of recombinant Ara h 1. Here, we present the crystal structure of the Ara h 1 core at a resolution of 2.43 Å. We also assayed the Ara h 1 core thermal stability and compared its final structure against other 7S globulins. The Ara h 1 core has a thermal denaturation temperature of 88.3°C and a structure that is very similar to other 7S globulins. Previously identified linear IgE epitopes were also mapped on the three-dimensional structure. Most linear epitopes were found in the extended loop domains and the coils between the N- and C-terminal modules, while others were found in the less accessible ß-sheets of the C-terminal core ß-barrel domain of each monomer. Most of these epitopes become either slightly or significantly buried upon trimer formation, implying that allergen digestion in the gut is required for these epitopes to be accessible to immunoglobulins. Our findings also suggest that both intact and partially degraded allergens should be employed in future diagnostic and immunotherapeutic strategies.

Expression and purification of peanut oleosins in insect cells.

Oleosins contain a unique hydrophobic domain which is inserted into the oil matrix and are involved in the formation and stability of plant oil bodies. These proteins have also been reported to possess some allergenic properties. Therefore, knowledge of its three-dimensional structure is vital for further structural and immunological characterization. However, due to the difficulty of soluble recombinant expression in Escherichia coli, no studies have been done in line with this goal. Here, we have developed a novel expression and purification system for three peanut oleosin isoforms (14 k, 16 k, and 18 kDa oleosins). Oleosin cDNAs were cloned and subsequently expressed in soluble form in insect cell-baculovirus system. Recombinant proteins can be purified to homogeneity using only Ni Sepharose affinity chromatography. Thermal denaturation midpoint temperatures of recombinant oleosins were also assayed and found to be very similar to that of native oleosins, indicating proper structural conformation of the recombinant proteins.

The development of transgenic crops to improve human health by advanced utilization of seed storage proteins.

Seed storage proteins are a major component of mature seeds. They are utilized as protein sources in foods. We designed seed storage proteins containing bioactive peptides based on their three-dimensional structures. Furthermore, to create crops with enhanced food qualities, we developed transgenic crops producing seed storage proteins with bioactive peptides. This strategy promises to prevent lifestyle-related diseases by simple daily food consumption. In this review, we discuss a strategy to develop transgenic crops to improve human health by advanced utilization of seed storage proteins.

Crystallization and preliminary X-ray analysis of the major peanut allergen Ara h 1 core region.

Peanuts contain some of the most potent food allergens known to date. Ara h 1 is one of the three major peanut allergens. As a first step towards three-dimensional structure elucidation, recombinant Ara h 1 core region was cloned, expressed in Escherichia coli and purified to homogeneity. Crystals were obtained using 0.1 M sodium citrate pH 5.6, 0.1 M NaCl, 15% PEG 400 as precipitant. The crystals diffracted to 2.25 A resolution using synchrotron radiation and belonged to the monoclinic space group C2, with unit-cell parameters a=156.521, b=88.991, c=158.971 A, beta=107.144 degrees. Data were collected at the BL-38B1 station of SPring-8 (Hyogo, Japan).

Expression, purification and preliminary crystallization of amaranth 11S proglobulin seed storage protein from Amaranthus hypochondriacus L.

11S globulin is one of the major seed storage proteins in amaranth. Recombinant protein was produced as up to approximately 80% of the total bacterial protein using Escherichia coli Rosetta-gami (DE3) containing pET21d with amaranth 11S globulin cDNA. The best expression condition was at 302 K for 20 h using LB medium containing 0.5 M NaCl. The recombinant protein was easily separated from most of the Escherichia coli proteins by precipitation with 0-40% ammonium sulfate solution. It formed aggregates at low temperature and at low salt concentrations. This behaviour may imply that it has a more hydrophobic nature than other 11S seed globulins. The crystals diffracted to 6 A resolution and belonged to space group P6(3), with unit-cell parameters a=b=97.6, c=74.8 A, gamma=120.0 degrees. One subunit of a trimer was estimated to be present in the asymmetric unit, assuming a Vsol of 41%. To obtain the complete structure solution, experiments to improve crystallization and flash-cooling conditions are in progress.

Soybean basic 7S globulin: subunit heterogeneity and molecular evolution.

Basic 7S globulin, a cysteine-rich protein from soybean seeds, consists of subunits containing 27 kD and 16 kD chains linked by disulfide bonding. Three differently sized subunits of the basic 7S globulin were detected and partially separated by SP Sepharose chromatography. The basic 7S globulin was characterized as a member of a superfamily of structurally related but functionally distinct proteins descended from a specific group of plant aspartic proteinases.

Development of transgenic rice containing a mutated beta subunit of soybean beta-conglycinin for enhanced phagocytosis-stimulating activity.

Improving the nutraceutical value of rice would positively impact the health and well-being of rice consumers worldwide. Based on the three-dimensional structure of soybean beta-conglycinin, we designed a beta subunit with a strong phagocytosis-stimulating activity (mbeta subunit). Here, we describe the genetic modification and production of rice seeds containing the mbeta subunit as part of our aim to develop a food material that promotes human health. The mbeta subunit folded correctly and was accumulated in the protein body II of rice seeds at a level similar to wild-type beta subunit. Mutant beta subunit purified from transgenic rice seeds exhibited high phagocytosis-stimulating activity, demonstrating its potential value in enhancing the nutritional value of rice.

Expression, purification, cross-reactivity and homology modeling of peanut profilin.

Plant profilins are known pan-allergens involved in the cross-reactions between pollen and plant foods. Peanut profilin, Ara h 5, is one of the important peanut allergens. Presently, most immunological, biochemical and structural studies on peanut allergens have focused on the three major allergens (Ara h 1, 2 and 3). Here Ara h 5 was cloned, expressed in Escherichia coli, Rosetta2(DE3) (Novagen), purified using a combination of ammonium sulfate fractionation and size-exclusion chromatography and yielded a total of 29 mg/l of culture. IgE reactivity was assayed using multiplexed microarray with other peanut allergens (Ara h 1, 2, 3, and 8) and birch (Bet v 2) and timothy (Phl p 2) profilin using sera from peanut allergic Swedish patients. Using homology modeling, Ara h 5 structure was also generated, compared against other profilins and utilized to predict surface-exposed residues potentially forming epitopes. The allergen was recognized by 3 out of 33 sera (9.1%). IgE reactivity to Ara h 5 also coincided with that of two other profilins, Phl p 12 and Bet v 2, confirming cross-reactivity. Interestingly, IgE reactivity to Ara h 5 was higher than above-mentioned profilins which may be indicating specificity of sera towards peanut profilin. Eight surface-exposed epitopes were predicted and verified against experimentally validated sequential epitopes. Three epitopes (#1, 5 and 7) mostly located at the accessible loops and neutral to relatively electropositive sites were found common among profilins, which should be involved in cross-reactivity. A specific putative epitope (#4) was also found which may explain the relative high IgE reactivity to Ara h 5 as compared to the other profilins. Due to its close relation to other allergenic profilins, Ara h 5 could be used as a model and allergen of choice for profilin allergy diagnosis.

Conservation and divergence on plant seed 11S globulins based on crystal structures.

The crystal structures of two pro-11S globulins namely: rapeseed procruciferin and pea prolegumin are presented here. We have extensively compared them with the other known structures of plant seed 11S and 7S globulins. In general, the disordered regions in the crystal structures among the 11S globulins correspond to their five variable regions. Variable region III of procruciferin is relatively short and is in a loop conformation. This region is highly disordered in other pro-11S globulin crystals. Local helical and strand variations also occur across the group despite general structure conservation. We showed how these variations may alter specific physicochemical, functional and physiological properties. Aliphatic hydrophobic residues on the molecular surface correlate well with Tm values of the globulins. We also considered other structural features that were reported to influence thermal stability but no definite conclusion was drawn since each factor has additive or subtractive effect. Comparison between proA3B4 and mature A3B4 revealed an increase in r.m.s.d. values near variable regions II and IV. Both regions are on the IE face. Secondary structure based alignment of 11S and 7S globulins revealed 16 identical residues. Based on proA3B4 sequence, Pro60, Gly128, Phe163, Phe208, Leu213, Leu227, Ile237, Pro382, Val404, Pro425 and Val 466 are involved in trimer formation and stabilization. Gly28, Gly74, Asp135, Gly349 and Gly397 are involved in correct globular folding.

Carbohydrate moieties contribute significantly to the physicochemical properties of french bean 7s globulin phaseolin.

We have previously reported that the solubility of French bean 7S globulin (phaseolin) at low ionic strength and its emulsifying stability are remarkably high compared with those of 7S globulins prepared from other plant species, including soybean (Kimura et al. J. Agric. Food Chem. 2008, 56, 10273-10279). In this study, we examined the role of carbohydrate moieties in the properties of phaseolin. Three preparations of phaseolin were analyzed: (i) N7S, prepared from defatted seed meal and having intact carbohydrate moieties; (ii) R7S, expressed in E. coli and lacking N-linked glycans; and (iii) EN7S, having partial N-linked glycans after treatment with Endo H. The solubilities of N7S and EN7S were much higher than that of R7S at a low ionic strength (micro = 0.08). N7S exhibited good emulsifying ability under the conditions examined, but R7S did not. In terms of emulsion stability, an emulsion of R7S separated into two phases after 1 h at micro = 0.01, 0.08, and 0.5, whereas the emulsion of N7S was stable for 5 days at micro = 0.01 and for at least 10 days at micro = 0.08 and 0.5. The emulsion stability of EN7S was comparable to that of N7S under most conditions examined. These results indicate the carbohydrate modifications are necessary for the good solubility, emulsifying ability, and emulsion stability of phaseolin. Further, a structural analysis of the carbohydrate moieties indicates that truncated carbohydrate moieties are sufficient for conferring these physicochemical properties to phaseolin.

Co-expression of alpha' and beta subunits of beta-conglycinin in rice seeds and its effect on the accumulation behavior of the expressed proteins.

A transgenic rice that produces both the alpha' and beta subunits of beta-conglycinin has been developed through the crossing of two types of transgenic rice. Although the accumulation level of the alpha' subunit in the alpha'beta-transgenic rice was slightly lower than that in the transgenic rice producing only the alpha' subunit, the accumulation level of the beta subunit in the alpha'beta-transgenic rice was about 60% higher than that in the transgenic rice producing only the beta subunit. Results from sequential extraction and gel-filtration experiments indicated that part of the beta subunit formed heterotrimers with the alpha' subunit in a similar manner as in soybean seeds and that the heterotrimers interacted with glutelin via cysteine residues. These results imply that the accumulation level of the beta subunit in the alpha'beta-transgenic rice increases by an indirect interaction with glutelin. Immunoelectron microscopy revealed that the alpha' and beta subunits are localized in a low electron-dense region of protein body-II (PB-II) and that alpha' homotrimers in the alpha'beta-transgenic rice seeds seem to accumulate outside of this low electron-dense region.

{alpha}' Subunit of soybean {beta}-conglycinin forms complex with rice glutelin via a disulphide bond in transgenic rice seeds.

The alpha' and beta subunits of soybean beta-conglycinin were expressed in rice seeds in order to improve the nutritional and physiological properties of rice as a food. The alpha' subunit accumulated in rice seeds at a higher level than the beta subunit, but no detectable difference in mRNA transcription level between subunits was observed. Sequential extraction results indicate that the alpha' subunit formed one or more disulphide bonds with glutelin. Electron microscopic analysis showed that the alpha' subunit and the beta subunit were transported to PB-II together with glutelin. In mature transgenic seeds, the beta subunit accumulated in low electron density regions in the periphery of PB-II, whereas the alpha' subunit accumulated together with glutelin in high-density regions of the periphery. The subcellular localization of mutated alpha' subunits lacking one cysteine residue in the N-terminal mature region (alpha'DeltaCys1) or five cysteine residues in the pro and N-terminal mature regions (alpha'DeltaCys5) were also examined. Low-density regions were formed in PB-II in mature seeds of transgenic rice expressing alpha'DeltaCys 5 and alpha'DeltaCys1. alpha'DeltaCys5 was localized only in the low-density regions, whereas alpha'DeltaCys1 was found in both low- and high-density regions. These results suggest that the alpha' subunit could make a complex via one or more disulphide bonds with glutelin and accumulate together in PB-II of transgenic rice seeds.

Production of a bioactive peptide (IIAEK) in Escherichia coli using soybean proglycinin A1ab1b as a carrier.

To produce large amounts of a peptide of fewer than 10 amino acid residues, construction of a gene encoding multimers of the small peptide is necessary. For this study a method was developed to facilitate the gene construction of high multimers of a small peptide with one step of cloning. A hypocholesterolemic peptide, IIAEK, from cow's milk beta-lactoglobulin was used as a model peptide for the construction of a gene encoding multimers of IIAEK and for the production of the peptide. Two systems for direct expression of 28-mers of IIAEK sequences (28IIAEK) and expression of 34 IIAEK sequences (4 IIAEK sequences in each of the disordered regions I, II, and III and 14 and 8 IIAEK sequences in disordered regions IV and V, respectively) in a mutant of soybean proglycinin A1aB1b lacking 31 residues in disordered region IV [A1aB1b(Delta31)-34IIAEK] were used. The protein produced from both systems formed inclusion bodies. The expression level of A1aB1b(Delta31)-34IIAEK was 29.9% of the total cell proteins and that of the 28IIAEK was 2.0%. The insoluble A1aB1b(Delta31)-34IIAEK was digested by trypsin without any help from urea or chemicals, and the produced IIAEK was purified using an octadecyl silica column. The yield of IIAEK was 58.6%. The results showed that A1aB1b as a carrier of multiple peptides and use of an Escherichia coli expression system are suitable for production of bioactive peptide.

Vacuolar sorting behaviors of 11S globulins in plant cells.

Plant seed cells amass storage proteins that are synthesized on the endoplasmic reticulumn (ER) and then transported to protein storage vacuoles (PSVs). Many dicotyledonous seeds contain 11S globulin (11S) as a major storage protein. We investigated the accumulation behaviors of pea and pumpkin 11S during seed maturation and compared them with soybean 11S biogenesis (Mori et al., 2004). The accumulation of pea 11S in seeds was very similar to that of soybean 11S at all the development stages we examined, whereas pumpkin 11S condensed in the ER. The determinant of accumulation behavior might be the surface hydrophobicity of 11S. Further, we examined the accumulation of 11Ss in tobacco BY-2 cells to analyze behavior in the same environment. 11Ss expressed in BY2 cells were all observed in precursor form (pro11S). Pro11S with high surface hydrophobicity might be transported to vacuoles in a multivesicular body-mediated pathway when the expression level remains low.