β-Conglycinin, one of the dominant storage proteins of soybean, is a trimer composed of three subunits, A, A′ and β. All subunits are N-glycosylated and A and A′ contain extension regions in addition to the core regions common to all subunits. Non-glycosylated individual subunits and deletion mutants (A c and A′ c ) lacking the extension regions of A and A′ were expressed in Escherichia coli. All recombinant proteins were purified to near homogeneity and appeared to have the correct conformation, as judged by CD, density-gradient centrifugation and gel-filtration profiles, indicating that the N-linked glycans and extension regions are not essential for the folding and the assembly into trimers of β-conglycinin. Densitygradient centrifugation, gel-filtration and differential scanning calorimetry profiles of the recombinant proteins and the native β-conglycinin indicated that the N-linked glycans and extension regions contribute to the dimension of β-conglycinin but not to the density and the thermal stability. Comparing the solubilities of the individual subunits with those of deletion mutants, only the A and A′ subunits were soluble at lower ionic strength (µ Ͻ 0.25) at around the pH value of the endoplasmic reticulum. This suggests that the extension regions play an important role in the prevention of aggregation in the endoplasmic reticulum in analogy with the N-linked glycans.

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Two references cited in the text were omitted from the Literature Cited: Reddy, S.; Aggarwal, B. B. Curcumin is a non-competitive and selective inhibitor of phosphorylase kinase. FEBS Lett. 1994, 341, 19-22. Yamamoto, H.; Hanada, K.; Kawasaki, K.; Nishijima, M. Inhibitory effect of curcumin on mammalian phos-pholipase D activity.

The crystal structures of recombinant and native b homotrimers of soybean b-conglycinin were determined by X-ray crystallography at 2.7 and 2.8 A Ê resolutions, respectively. The crystals of the recombinant and native b homotrimers belong to space group P2 1 with cell parameters a 80. Keywords: b-conglycinin; crystal structure; N-linked glycan; soybean; vicilin.Soybean proteins are one of the most attractive plant food proteins for man, as they exhibit a hypocholesterolemic effect [1,2] and have good nutritional and physicochemical properties (such as gel-forming and emulsifying abilities required for food systems) [3,4]. Soybean proteins are composed of two major components, b-conglycinin (vicilin class) and glycinin (legumin class), which account for about 30% and 40% of the total seed proteins, respectively. These two components are responsible for the nutritional, physicochemical and physiological properties of soybean proteins.b-Conglycinin has a trimeric structure analogous to that of other 7S globulins [4,5] and is composed of three kinds of subunit a, a H and b. Many molecular species with different subunit compositions, including homotrimers with almost random combinations, are present in soybean seeds [6,7]. The amino-acid sequences of these subunits deduced from their nucleotide sequences indicate that the a and a H subunits contain extension regions in addition to core regions common to all subunits, and that the core regions exhibit high absolute homologies (90.4, 76.2 and 75.5% between a and a H , a and b, and a H and b, respectively [8]). However, it is known that the contributions of individual subunits to physicochemical and physiological properties and allergenicity differ as follows: (a) the orders of thermal stabilities, which are one of the structural features related to food processing and conferred by the core regions, are b . a H . a [8]; (b) b-conglycinin mainly contributes to the up-regulation of low-density lipoprotein receptors, which is one of the mechanisms of the hypocholesterolemic effect of soybean proteins [2,9], and only the a H subunit is responsible for the up-regulation activity of b-conglycinin [10]; (c) b-conglycinin is the third main allergen responsible for soybean allergy, and only the a subunit is identified as having a reactivity with soybean-sensitive-patients H sera [11].In order to investigate why individual subunits of b-conglycinin have different properties as described above, it is necessary to elucidate their crystal structures by means of X-ray crystallography. Although a large amount of homogeneous homotrimers is required for X-ray crystallography, they are very difficult to obtain from natural soybean seeds because of molecular heterogeneity [6,7] and the high homologies of their amino-acid sequences [8]. Recently, a mutant soybean cultivar has been developed which contains b-conglycinin composed of only the b subunit [12]. Therefore, b homotrimers can be purified easily using the mutant soybean. Alternatively, the Escherichia coli expression systems we cons...