Shigeru Utsumi

Design and production of genetically modified soybean protein with anti-hypertensive activity by incorporating potent analogue of ovokinin(2–7)

The potent anti‐hypertensive peptide, RPLKPW, has been designed based on the structure of ovokinin(2–7). The sequence encoding this peptide was introduced into three homologous sites in the gene for soybean β‐conglycinin α′ subunit. The native α′ subunit as well as the modified, RPLKPW‐containing α′ subunit were expressed in Escherichia coli, recovered from the soluble fraction and then purified by ion‐exchange chromatography. The RPLKPW peptide was released from recombinant RPLKPW‐containing α′ subunit after in vitro digestion by trypsin and chymotrypsin. Moreover, the undigested RPLKPW‐containing α′ subunit given orally at a dose of 10 mg/kg exerted an anti‐hypertensive effect in spontaneously hypertensive rats, unlike the native α′ subunit. These results provide evidence for the first time that a physiologically active peptide introduced into a food protein by site‐directed mutagenesis could practically function in vivo even at a low dose.

Identification of the Bile Acid-binding Region in the Soy Glycinin A1aB1b Subunit

Soy glycinin has five major subunits which are classified into two groups according to their homology in amino acid sequences (group I, A1aB1b, A1bB2 and A2B1a; group II, A3B4 and A5A4B3). It has been reported that the peptide fragments derived from the A1a and A2 chains of the A1aB1b and A2B1a subunits had bile acid-binding ability and that the region of 114-161 residues of the A1a chain was responsible for this bile acid-binding ability. In this study, we constructed A1a, A3 and 9 deletion mutants of A1a lacking various numbers of residues at the C-terminus, and evaluated their bile acid-binding ability by a cholic acid-conjugated column and fluorescence analysis. The bile acid-binding ability of A1a was higher than that of A3 and there was a remarkable decrease in the bile acid-binding ability between the Δ[138-291] and Δ[130-291] mutants. The 130-138 region is rich in hydrophobic residues. In this regard, when we constructed the Δ[129-134] mutant lacking six contiguous hydrophobic residues (VAWWMY) and evaluated its bile acid-binding ability, a similar remarkable decrease in the bile acid-binding ability was observed. These results indicate that the 129-134 residue region (VAWWMY) with high hydrophobicity was important for bile acid-binding of A1a.

Anti-hypertensive activity of genetically modified soybean seeds accumulating novokinin

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp), which has been designed based on the structure of ovokinin (2-7), significantly reduces the systolic blood pressure at a dose of 100 microg/kg after oral administration in spontaneously hypertensive rats (SHRs). In this study, we generated a transgenic soybean which accumulates novokinin. A vector encoding a modified beta-conglycinin alpha subunit (4novokinin-alpha) in which four novokinin sequences have been incorporated by site-directed mutagenesis was introduced into somatic embryos by whisker-mediated gene transformation to produce a transgenic soybean. The 4novokinin-alpha occupied 0.5% of total soluble protein and 5% of the beta-conglycinin alpha subunit in the transgenic soybean seeds. Protein extracted from the transgenic soybean reduced systolic blood pressure after single oral administration in SHRs at a dose of 0.15 g/kg. Defatted flour from the transgenic soybean also reduced the systolic blood pressure at a dose of 0.25 g/kg. Thus, the 4novokinin-alpha produced in soybean exhibited an anti-hypertensive activity in SHRs after oral administration.

Design of genetically modified soybean proglycinin A1aB1b with multiple copies of bioactive peptide sequences.

The peptide IIAEK derived from beta-lactoglobulin has a hypocholesterolemic activity greater than that of beta-sitosterol. To create food proteins with multiple copies of this valuable peptide sequence, we introduced tandem multimers of the nucleotide sequence encoding the peptide into DNA regions corresponding to the five variable regions of soybean glycinin A1aB1b subunit, and expressed the mutants in Escherichia coli. The expression level and solubility of the five mutants, each containing four IIAEK sequences in each of the variable regions, were compared. Overall, the expression level and solubility of the mutants with four IIAEK sequences in the variable regions IV and V were the best followed by II > III > I. Further, introduction of the fifth IIAEK sequence to the variable region IV did not decrease expression level and solubility. Increasing the number of IIAEK to 7 and 10 slightly decreased expression level, while their solubility decreased to as low as 40 and 1%, respectively. Various mutations were combined to get a mutant containing as many IIAEK sequences as possible. Some of the resulting mutants were expressed in the soluble form. The mutant containing eight IIAEK from the combination of variable regions IV and V (IV-4 + V-4) showed the best balance of the expression level and solubility, followed by the combination of variable regions II and III (II-4 + III-4). The soluble fractions of these mutants were purified by hydrophobic, gel filtration and ion-exchange column chromatography. Yields of IIAEK peptide released by in vitro digestion with trypsin from both mutants were around 80%. This is the first report that a large amount of a physiologically active peptide could be introduced into food protein.

Structure-function relationships of soybean proteins revealed by using recombinant systems

Abstract Glycinin and β-conglycinin are the major storage proteins of soybean and determine the functional properties of soybean proteins. Structure-function relationships of glycinin and β-conglycinin were investigated by using Escherichia coli expression systems. Examination of functional properties of various modified versions of proglycinin A1aB1b suggests that the hydrophobicity of the C-terminal region is probably important for a high emulsifying ability, that the topology of free SH residues is closely related to the heat-induced gel forming ability and that the structural factors suitable for gelation and emulsification properties are quite different. Mutual comparison of functional properties of β-conglycinin constituent subunits (α, α′ and β) and the core regions of α and α′ indicate that the core regions determine thermal stability and surface hydrophobicity, that the extension regions of α and α′ contribute to high solubility and emulsifying abilities and that the carbohydrate moieties inhibit the formation of heat-induced aggregates. Analyses by chimerization of glycinin and β-conglycinin suggest that structure-function relationships are different between glycinin and β-conglycinin.

Soybean Glycinin A1aB1b Subunit Has a Molecular Chaperone-like Function to Assist Folding of the Other Subunit Having Low Folding Ability

Soybean (Glycine max L.) glycinin is composed of five subunits which are classified into two groups (group I: A1aB1b, A1bB2, and A2B1a; group II: A3B4 and A5A4B3). All the common soybean cultivars contain both group I and II subunits (Maruyama, N. et al., Phytochemistry, 64, 701–708 (2003)). The biosynthesis of group I starts earlier compared with that of the A3B4 subunit during seed development (Meinke, D.W. et al., Planta, 153, 130–139 (1981)). We have revealed that group I A1aB1b was mostly expressed as a soluble protein, but that A3B4 was expressed mainly as an insoluble protein in Escherichia coli under the same expression conditions; namely, A1aB1b had higher folding ability than A3B4. We therefore assumed that A1aB1b assists folding of group II subunits like a molecular chaperone does. In order to ascertain this, A1aB1b and A3B4 were co-expressed in E. coli. All of the expressed proteins of A3B4 were recovered in a soluble fraction. To confirm this result, we also co-expressed A1aB1b with modified A3B4 versions having extremely low folding ability. All expressed modified A3B4 versions were soluble. These results clearly suggest that A1aB1b has a molecular chaperone-like function in their folding.