7S Storage Globulins Research Articles

Formation of Amyloid Fibrils from Kidney Bean 7S Globulin (Phaseolin) at pH 2.0

The amyloid fibrils formed by heating 1.0% (w/v) kidney bean phaseolin (7S globulin) solution at pH 2.0 with an ionic strength of 20 mM at 85 degrees C were characterized using transmission electron microscopy (TEM), atomic force microscopy (AFM), binding of thioflavin T (Th T) and Congo Red dyes, and circular dichroism spectroscopy. The morphology of the formed fibrils was closely dependent upon heating time from 15 to 720 min. The diameters of the fibrils formed at various times were similar, but the mean contour length progressively increased with heating time. The Th T maximum fluorescence also progressively increased with heating time. The heating process caused remarkable changes in secondary, tertiary, and quaternary conformations of the phaseolin, but the extents of the changes were closely related to the heating time. With a short heating time (e.g., 15 min), the beta-strand content decreased from 38.7 to 22.9%, but further heating resulted in recovery of beta-strand structure. The tertiary and quaternary conformations gradually became flexible and unfolded upon heating. Gel electrophoresis analysis indicated that heating disrupted the polypeptides of phaseolin, leading to the formation of fragments with lower molecular mass (e.g., <10 kDa after 360 min). The results suggest that the amyloid fibril formation of phaseolin (7S globulin) involved the disruption of its polypeptides, as well as conformational changes at secondary, tertiary, and quaternary structural levels. This appears to be the first direct observation of amyloid fibrils from legume 7S storage globulin.

Presence of the storage seed protein vicilin in internal organs of larval Callosobruchus maculatus (Coleoptera: Bruchidae)

Variant vicilins (7S storage globulins) of cowpea seeds ( Vigna unguiculata) are considered as the main resistance factor present in some African genotypes against the bruchid Callosobruchus maculatus. It has been suggested that the toxic properties of vicilins may be related to their recognition and interaction with glycoproteins and other membrane constituents along the digestive tract of the insect. However, the possibility of a systemic effect has not yet been investigated. The objective of this work was to study the fate of 7S storage globulins of V. unguiculata in several organs of larvae of the cowpea weevil C. maculatus. Results demonstrated binding of vicilins to brush border membrane vesicles, suggesting the existence of specific receptors. Vicilins were detected in the haemolymph, in the midgut, and in internal organs, such as fat body and malpighian tubules. There is evidence of accumulation of vicilins in the fat body of both larvae and adults. The absorption of vicilins and their presence in insect tissues parallels classical sequestration of secondary compounds.

The seed coat of Phaseolus vulgaris interferes with the development of the cowpea weevil [Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)

We have confirmed here that the seeds of the common bean (Phaseolus vulgaris, L.) do not support development of the bruchid Callosobruchus maculatus (F.), a pest of cowpea [Vigna unguiculata (L.) Walp] seeds. Analysis of the testa (seed coat) of the bean suggested that neither thickness nor the levels of compounds such as tannic acid, tannins, or HCN are important for the resistance. On the other hand, we have found that phaseolin (vicilin-like 7S storage globulin), detected in the testa by Western blotting and N-terminal amino acid sequencing, is detrimental to the development of C. maculatus. As for the case of other previously studied legume seeds (Canavalia ensiformis and Phaseolus lunatus) we suggest that the presence of vicilin-like proteins in the testa of P. vulgaris may have had a significant role in the evolutionary adaptation of bruchids to the seeds of leguminous plants.

Multiple thiamine-binding proteins of legume seeds. Thiamine-binding vicilin of Vicia faba versus thiamine-binding albumin of Pisum sativum

Thiamine-binding proteins ubiquitously occur in plant seeds but they have not been characterised in legume seeds where, on the other hand, the general storage proteins are known in structural and functional detail. In this work, we show that thiamine-binding activity in seeds of several leguminous species is distributed between both globulin and albumin fractions. A thiamine-binding protein (TBP) with molecular properties typical of 7S storage globulin (vicilin) is isolated from broad bean ( Vicia faba L.) seeds. It has a molecular mass of 150–170 kDa and is an oligomer containing 50 and 27 kDa subunits. It binds thiamine with a dissociation constant of 1.3 μM comparable to that of other angiosperm thiamine-binding globulins. Garden pea ( Pisum sativum L.) seeds contain, in addition to a similar thiamine-binding vicilin, a smaller TBP which is a dimer of 24 kDa subunits. Its molecular characteristics are very similar to those of a well-characterised major pea albumin designated PA2. Specifically, the sequence of the first 35 amino acids from the N-terminus of TBP exactly matches that of PA2 albumin. This thiamine-binding protein binds thiamine with a low affinity (dissociation constant of 35 μM). Thiamine-binding vicilins of legume seeds probably represent a thiamine-storage function but a significance of the albumin-attributable thiamine binding remains to be determined.

Cowpea Vicilins: Fractionation of Urea Denatured Sub-Units and Effects on Callosobruchus maculatus F. (Coleoptera: Bruchidae) Development

Vicilins (7S storage globulins) isolated from cowpea (Vigna unguiculata L.) seeds which were susceptible (S) and resistant (R) to the cowpea weevil (Callosobruchus maculatus F., Coleoptera: Bruchidae) were denatured by urea and fractionated by ion-exchange chromatography. Isolated fractions were incorporated in artificial seeds for assessment of their toxicity to C. maculatus. The most acidic fractions of both susceptible (CE-31 cultivar) and resistant (IT81D-1045 line) seeds were shown to affect development and survival of the bruchid. Results indicated that vicilin polypeptides of toxic nature were expressed in both types of storage globulins although at different levels.

Selective Neoglycosylation Increases the Structural Stability of Vicilin, the 7S Storage Globulin from Pea Seeds

The effects of glycosylation on the stability and subunit interactions of vicilin, the major storage protein in pea seeds, were investigated. Glycosylated vicilin derivatives were prepared by alkylation of lysine ϵ-amino groups with various carbohydrates. Average modification levels of 13.4 ± 3.0, 11.1 ± 3.6, 7.5 ± 4.2, and 4.7 ± 0.3 moles of carbohydrate/mol of vicilin were obtained with glucose, galactose, galacturonic acid, and lactose, respectively. Nondenaturing polyacrylamide gel electrophoresis and size-exclusion chromatography indicated that the quaternary structure and hydrodynamic radius of vicilin were not affected by glycosylation at the levels used. We have previously shown that application of hydrostatic pressure causes dissociation of vicilin subunits [C. Pedrosa and S. T. Ferreira (1994) Biochemistry 33, 4046–4055]. Analysis of pressure dissociation data allowed determination of the Gibbs free energy change (ΔGdiss) and molar volume change (ΔVdiss) of dissociation of vicilin subunits. For unmodified vicilin, ΔGdiss = 18.2 kcal/mol and ΔVdiss = −102 ml/mol. Glycosylated vicilin derivatives were significantly stabilized against subunit dissociation, with ΔGdiss of 19.4, 19.2, 20.6, and 22.1 kcal/mol for glucose, galactose, lactose, and galacturonic acid derivatives, respectively. No changes in ΔVdiss were found for the glucose and galactose derivatives, whereas ΔVdiss of −128 and −135 ml/mol, respectively, were found for the lactose and galacturonic acid derivatives. The glycosylated derivatives also appeared more resistant to unfolding by guanidine hydrochloride than unmodified vicilin. Intrinsic fluorescence lifetime measurements showed that glycosylation caused a significant increase in heterogeneity of the fluorescence decay, possibly reflecting increased conformational heterogeneity of glycosylated derivatives relative to unmodified vicilin. These results indicate that the stability and subunit interactions of vicilin may be modulated by mild, selective glycosylation at low modification levels, an effect that may be of interest in the study of other oligomeric proteins.

Lima bean (Phaseolus lunatus) seed coat phaseolin is detrimental to the cowpea weevil (Callosobruchus maculatus).

The presence of phaseolin (a vicilin-like 7S storage globulin) peptides in the seed coat of the legume Phaseolus lunatus L. (lima bean) was demonstrated by N-terminal amino acid sequencing. Utilizing an artificial seed system assay we showed that phaseolin, isolated from both cotyledon and testa tissues of P. lunatus, is detrimental to the nonhost bruchid Callosobruchus maculatus (F) (cowpea weevil) with ED50 of 1.7 and 3.5%, respectively. The level of phaseolin in the seed coat (16.7%) was found to be sufficient to deter larval development of this bruchid. The expression of a C. maculatus-detrimental protein in the testa of nonhost seeds suggests that the protein may have played a significant role in the evolutionary adaptation of bruchids to legume seeds.

Legume vicilins (7S storage globulins) inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells

Vicilin (7S storage proteins) isolated from different legume seeds were shown to inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. The degree of growth inhibition varied with the origin of vicilins. It was more than 90% for vicilins from cowpea ( Vigna unguiculata, cultivar pitiuba) and equal to 65% for vicilins from Vigna radiata, in the case of Saccharomyces cerevisae. Vicilins from cowpea seeds inhibited the glucose stimulated acidification of the medium by S. cerevisae up to 60%. We have also observed that vicilins bind to yeast cells. We suggest that vicilins bind to chitin-containing structures of yeast cells and that such association could result in inhibition of H + pumping, cell growth and spore formation. A final consequence of the yeast growth inhibition by vicilins is (probably) the formation of spores.

Vicilin Storage Proteins from Vigna unguiculata (Legume) Seeds Inhibit Fungal Growth

Vicilin storage proteins (7S globulins) isolated from Vigna unguiculata (cowpea) seeds were shown to interfere with the germination of spores or conidia of the fungi Fusarium solani, Fusarium oxysporum, Colletotrichum musae, Phytophtora capsici, Neurospora crassa, and Ustilago maydis sporidia. Cowpea vicilins have been shown to bind to fungal structures, possibly chitin-containing structures of the cell wall, and can be desorbed by strong acid. The results presented in this paper are in agreement with data previously obtained on the chitin-binding properties of cowpea vicilins and the effect they exert on the development and survival of the storage pest insect Callosobruchus maculatus (Coleoptera: Bruchidae). Keywords: 7S storage globulins; cowpea seeds; Vigna unguiculata; chitin-binding proteins; fungi; spore germination

Deterministic pressure-induced dissociation of vicilin, the 7S storage globulin from pea seeds: effects of pH and cosolvents on oligomer stability.

A thermodynamic characterization of subunit association of vicilin, a storage protein from pea seeds, was performed using a combination of hydrostatic pressure and fluorescence spectroscopy. Application of pressure up to 2.4 kbar caused dissociation of vicilin subunits, as revealed by (1) size-exclusion PPLC of pressurized samples, (2) fluorescence anisotropy measurements of a dansyl-vicilin conjugate under pressure, and (3) quenching of the intrinsic fluorescence of vicilin. Pressure dissociation data were well described by a model for dissociation of a trimer. This enabled calculation of the standard molar volume change of association (delta Vo) and the equilibrium dissociation constant at atmospheric pressure (Ko); at pH 10 these were found to be delta Vo = 146 mL/mol and Ko = 2.2 x 10(-15) M2, respectively, corresponding to C1/2 (the concentration of protein at 50% dissociation at atmospheric pressure) = 18 nM and a stabilization free energy of -19.6 kcal/mol for the oligomer. Vicilin exhibited an anomalously low dependence on protein concentration for pressure dissociation. This appeared related to conformational changes in the dissociated subunits, which caused a loss of ca. 5 kcal/mol in the free energy of association and to structural/energetic heterogeneity in the population of oligomers. Pressure dissociation was markedly pH-dependent, with a stabilization free energy loss of 3.4 kcal/mol upon raising pH from 9 to 10. Circular dichroism and intrinsic fluorescence lifetime measurements at atmospheric pressure showed that the structure of vicilin was largely unaffected by pH in the range investigated. These results suggest that the effect of pH may involve deprotonation of lysine residues participating in salt bridges between vicilin subunits. Pressure dissociation of vicilin was significantly inhibited by addition of salts (NaCl, KCl, LiCl) or glycerol. Dissociation curves obtained in the presence of salts enabled calculation of the free energies of stabilization (ranging from ca. -1.2 to -2.4 kcal/mol) of the vicilin oligomers by these cosolvents. The similar effects of salts or glycerol suggest a common mechanism of stabilization of the oligomer involving exclusion of the cosolvents from the protein interface and preferential hydration of the protein.

Chromosomal location and RFLP utility in wheat and barley of a wheat gene with homology to a 7S storage-globulin sequence

Chromosomal location and RFLP utility in wheat and barley of a wheat gene with homology to a 7S storage-globulin sequence

Comparative protein accumulation patterns in soybean somatic and zygotic embryos

The relative maturity and competence of somatic embryos is often estimated on the basis of their morphologic similarity to various stages of immature zygotic embryo development. Morphologic abnormalities noted in soybean [Glycine max (L.) Merr.] somatic embryos are similar to those observed in zygotic embryos maturing in vitro and may reflect common interruptions of normal developmental processes. We provide here a more objective means of assessing the point(s) at which cultured embryos deviate from the normal embryogenical pathway by comparing the accumulation of the embryo-specific marker proteins (11S and 7S storage globulins, soybean agglutinin, and seed lipoxygenase) between somatic and immature zygotic embryos maturing in culture to zygotic embryos maturingin planta. Immature (heart-stage) soybean (cv. ‘McCall’) zygotic embryos were removed from the testa and cultured for 5, 15, or 45 days in nien modified Linsmaer-Skoog salts, 5% sucrose liquid medium. Somatic embryos were induced from immature cotyledon explants on a medium containing either naphthalene acetic acid or 2,4 dichlorophenoxyacetic acid (10 mg·liter−1). The measured level of the marker proteins present in cultured embryos never exceeded those observed in mature soybean seeds. During the culture period, immature zygotic embryos accumulated significant levels of all marker proteins except a 29 kDa soybean agglutinin associated with the final stages of seed maturationin planta. Somatic embryos of all morphologic classes exhibited similar levels of the marker proteins suggesting that morphology may not accurately represent the developmental state of the culture-derived embryos. Somatic embryos induced on naphthalene acetic acid-containing medium accumulated detectable levels of all maturation-specific marker proteins except the 7S β and 29-kD soybean agglutinin antigen and seemed similar in most respects to the cultured zygotic embryos. Embryos induced on 2,4-dichlorophenoxyacetic acid accumulated none of the mature 7S or 11S storage globulin subunits nor any soybean agglutinin antigen, and yet the synthesis of 7S and 11S precursor polypeptides was similar in both naphthalene acetic acid-and 2,4-dichlorophenoxyacetic acid-induced somatic embryos. These observations are consistent with the view that embryos induced on high 2,4-dichlorophenoxyacetic are arrested at a relatively earlier developmental stage than naphthalene acetic acid-induced embryos of similar morphology and may indicate that some external signal (e.g., abscisic acid or desiccation or both) is necessary for the transition to the late maturation stage of seed ontogeny.

Identification, Purification and Properties of the Precursor of Conglutin β, The 7S Storage Globulin ofLupinus albusL. Seeds

Lupinus albus L. developing cotyledons 35 d after flowering contained a major polypeptide of-average Mr 64000, immunologically related to conglutin β, the 7S storage globulin of this seed. This polypeptide decreased during seed maturation, without completely disappearing in the mature seed. This drop was accompanied by the formation of polypeptide fragments typical of the mature conglutin β. The 64000 polypeptide has been identified as the precursor polypeptide of conglutin β. Undenatured conglutin β precursor, purified by ion exchange chromatography and size exclusion chromatography, showed surface and association properties identical to the mature conglutin β molecule. The precursor oligomer, of Mr 190000, consisted of an association of three 64000 subunits. They strongly reacted with concanavalin A indicating the presence of covalently linked carbohydrate. Tryptic treatment of the undenatured conglutin β precursor led to the accumulation of a relatively stable 59000 polypeptide which was cleaved later on and produced three large polypeptide fragments differing from the mature conglutin β polypeptides.

Characterisation by two-dimensional gel electrophoresis of the protein components of protein bodies, isolated from the developing endosperm of wheat ( Triticum aestivum)

Protein bodies were extracted from the developing endosperm of two varieties of wheat and isolated by sucrose-gradient centrifugation. Their protein constituents were characterised by two-dimensional electrophoresis and compared with the proteins of the mature endosperm. It was concluded that for both varieties all the major proteins previously identified as prolamins, i.e. glutenin subunits and gliadins, occur in protein bodies. Also present in protein-body preparations are the ‘triplet-band’ proteins. In contrast, the ‘non-storage proteins’ occur in only trace amounts in protein-body preparations while the high molecular weight (HMW) albumins are absent. Also absent are the 7S storage globulins, which is probably due to their location in the embryo and aleurone layer rather than the starchy endosperm.