Takako Fukuda

Comparison of Physicochemical Properties of 7S and 11S Globulins from Pea, Fava Bean, Cowpea, and French Bean with Those of Soybean-French Bean 7S Globulin Exhibits Excellent Properties

Legume seeds contain 7S and/or 11S globulins as major storage proteins. The amino acid sequences of them from many legumes are similar to each other in the species but different from each other, meaning that some of these proteins from some crops exhibit excellent functional properties. To demonstrate this, we compared protein chemical and functional properties (thermal stability, surface hydrophobicity, solubility as a function of pH, and emulsifying properties) of these proteins from pea, fava bean, cowpea, and French bean with those of soybean as a control at the same conditions. The comparison clearly indicated that the 7S globulin of French bean exhibited excellent solubility (100%) at pH 4.2-7.0 even at a low ionic strength condition (mu = 0.08) and excellent emulsion stability (a little phase separation after 3 days) at pH 7.6 and mu = 0.08, although the emulsions from most of the other proteins separated in 1 h. These results indicate that our assumption is correct.

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.

Molecular and structural analysis of electrophoretic variants of soybean seed storage proteins

Soybean (Glycine max L.) storage proteins are composed mainly of two major components, beta-conglycinin and glycinin. Electrophoretic variants of the beta subunit of beta-conglycinin and the A3 polypeptide of glycinin were detected on SDS-PAGE, and designated them as beta* and A3*, respectively. beta* and A3* exhibited higher and lower mobilities, respectively, than the common beta subunit and A3 polypeptide. The N-terminal nine and 10 amino acid sequences of beta* and A3* were completely identical to the previously reported sequences of the beta subunit and the A3 polypeptide, respectively. Analysis using concanavalin A-horseradish peroxidase and treatment with N-glycosidase indicated that glycans were not responsible for the difference in electrophoretic mobility of beta* or A3*. Furthermore, five clones of beta* or beta and three clones of A3*, respectively, were sequenced but we could not detect deletions and insertions except for a single or a few amino acid substitutions as compared with the common beta subunit and A3 polypeptide. These results indicate that a single or a few amino acid substitution affects the electrophoretic mobilities of beta* and A3*.

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.

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.

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.

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

Soybean mature glycinin was purified and crystallized and its preliminary crystallographic analysis is also reported.