Non-identical subunits of 7S globulin (gossipulin-1) from cotton seeds]

7S globulin is isolated from cotton seeds and is designated as "Gossipulin-1". Three different amino acids (glycine, aspartic acid and leucine) are found to follow the N-terminal amino acid which is indicative of a non-identity of its subunits. Gossipulin-1 dissociated in 0.1 M HCl, depending on the time of incubation, into 3.87, 2.39 and 1.35 subunits. It had a sedimentation constant of 0.37S in alkaline medium and of 1.24S on 8 M urea. A sedimentation peak of 1.1S was discovered in reduced and alkylated Gossipulin-1.     

Cosuppression of the alpha subunits of beta-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies

The expression of the alpha and alpha' subunits of beta-conglycinin was suppressed by sequence-mediated gene silencing in transgenic soybean seed. The resulting seeds had similar total oil and protein content and ratio compared with the parent line. The decrease in beta-conglycinin protein was apparently compensated by an increased accumulation of glycinin. In addition, proglycinin, the precursor of glycinin, was detected as a prominent polypeptide band in the protein profile of the transgenic seed extract. Electron microscopic analysis and immunocytochemistry of maturing transgenic soybean seeds indicated that the process of storage protein accumulation was altered in the transgenic line. In normal soybeans, the storage proteins are deposited in pre-existing vacuoles by Golgi-derived vesicles. In contrast, in transgenic seed with reduced beta-conglycinin levels, endoplasmic reticulum (ER)-derived vesicles were observed that resembled precursor accumulating-vesicles of pumpkin seeds and the protein bodies accumulated by cereal seeds. Their ER-derived membrane of the novel vesicles did not contain the protein storage vacuole tonoplast-specific protein alpha-TIP, and the sequestered polypeptides did not contain complex glycans, indicating a preGolgi and nonvacuolar nature. Glycinin was identified as a major component of these novel protein bodies and its diversion from normal storage protein trafficking appears to be related to the proglycinin buildup in the transgenic seed. The stable accumulation of proteins in a protein body compartment instead of vacuolar accumulation of proteins may provide an alternative intracellular site to sequester proteins when soybeans are used as protein factories.     

Sunflower seed protein: size and charge heterogeneity in subunits of the globulin fraction

The subunit heterogeneity of the globulin fraction of sunflower seeds was investigated by two dimensional electrophoresis, using isoelectric focusing in the first dimension and sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension. Under non reducing conditions, intermediary subunits B, C and D (molecular weight 54 000, 48 000 and 40 000, respectively) were focused within a pI range 5.4-6.0 but intermediary subunits A (molecular weight 60 000) focused within a pI range 6.3-6.8. Under reducing conditions the electrophoretic patterns show that intermediary subunits consist in large "acidic" and small "basic" subunits linked by disulphide bonds. The large subunits of B species are more acidic and less heterogeneous than the corresponding subunits of the A species. These results confirm that helianthinin had a "legumin-type" structure.     

Structural homology among the major 7s globulin subunits of soybean seed storage proteins

The soybean seed 7S globulin subunits, i.e. α, α′, β and γ-subunits of β-conglycinin, the γ-conglycinin subunit and the HI/HII and LII subunits of basic 7S globulin were purified and the NH₂-terminal amino acid sequences of all these subunits except the γ-subunit of β-conglycinin were determined. Only the NH₂-terminal regions of the α and α′-subunits showed high sequence homology. However, sequencing of tryptic peptides from the seven subunits revealed that internal region sequences were highly homologous among the four subunits of β-conglycinin. In contrast to the β-conglycinin subunits, no sequence homology was found among the other subunits. On the basis of these results, the major 7S globulin fraction is considered more heterogeneous in primary structure than another major globulin fraction, 11S globulin (glycinin), in soybean seeds.     

Closely related families of genes code for the alpha and alpha' subunits of the soybean 7S storage protein complex

Nineteen cloned cDNAs encoding the alpha and alpha'-subunits of the 7S seed storage protein in the soybean, Glycine max, have been isolated from a recombinant cDNA library constructed with mRNA from maturing seeds. In addition, a gene encoding an alpha'-subunit has been isolated from a recombinant Charon 4A phage library containing genomic Glycine max DNA. The cloned DNAs have been divided, on the basis of their endonuclease sites, into two main classes of sequences which differ in approximately 6% of their nucleotides. Whereas the proteins encoded within each DNA class are nearly identical, the proteins encoded by the two different classes of soybean DNAs are distinct and correspond to alpha and alpha'-subunits. Thus, the alpha and alpha'-subunits are coded for by two closely related multigene families. The amino acid differences in the portions of the alpha and alpha'-subunits presented in this paper occur primarily near the carboxyl-terminus. The 3' noncoding nucleotides of the cloned alpha and alpha'-subunit DNAs are more highly conserved than are the coding nucleotides. This conservation suggests that the 3' untranslated sequences of the alpha and alpha'-subunit mRNAs are functional in the expression of the alpha and alpha'-subunit proteins or in the stabilization of the 7S subunit mRNAs.     

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 epsilon-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 (deltaG(diss)) and molar volume change (deltaV(diss)) of dissociation of vicilin subunits. For unmodified vicilin, deltaG(diss) = 18.2 kcal/mol and deltaV(diss) = -102 ml/mol. Glycosylated vicilin derivatives were significantly stabilized against subunit dissociation, with deltaG(diss) of 19.4, 19.2, 20.6, and 22.1 kcal/mol for glucose, galactose, lactose, and galacturonic acid derivatives, respectively. No changes in deltaV(diss) were found for the glucose and galactose derivatives, whereas deltaV(diss) 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.     

Antigenic relationship of legume seed proteins to the 7S seed storage protein of soybean

Extracts enriched for globulin proteins were prepared from the seeds of a large number of legume species and were tested for homology to antisera prepared against the glycosylated 7S seed storage protein of the soybean (Glycine max). Electrophoretic identification and subsequent analysis of proteins precipitated with 7S antisera was useful at relatively short taxonomic distances, particularly within the tribe Phaseoleae, to which G. max belongs. Glycine and most other members of the subtribe Glycininae are unusual within the Phaseoleae in having high molecular weight ($\text{> 70 000}$ dalton) subunit polypeptides. Seeds from other plants representing other subtribes of the Phaseoleae also contained proteins that cross-reacted with the G. max antisera; the molecular weights of these proteins varied from 30 000 to nearly 90 000 daltons. Homology was detected across a wider range of legume tribes within the subfamily Papilionoideae by enzyme-linked immunosorbent assay (ELISA). The results of these experiments suggest both that the 7S proteins of these tribes are evolutionarily related and that at least some features of these apparently rapidly-evolving proteins are under relatively strong selectional constraint.     

Expression of functional recombinant human growth hormone in transgenic soybean seeds

We produced human growth hormone (hGH), a protein that stimulates growth and cell reproduction, in genetically engineered soybean [Glycine max (L.) Merrill] seeds. Utilising the alpha prime (α') subunit of β-conglycinin tissue-specific promoter from soybean and the α-Coixin signal peptide from Coix lacryma-jobi, we obtained transgenic soybean lines that expressed the mature form of hGH in their seeds. Expression levels of bioactive hGH up to 2.9% of the total soluble seed protein content (corresponding to approximately 9?g?kg(-1)) were measured in mature dry soybean seeds. The results of ultrastructural immunocytochemistry assays indicated that the recombinant hGH in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Specific bioassays demonstrated that the hGH expressed in the soybean seeds was fully active. The recombinant hGH protein sequence was confirmed by mass spectrometry characterisation. These results demonstrate that the utilisation of tissue-specific regulatory sequences is an attractive and viable option for achieving high-yield production of recombinant proteins in stable transgenic soybean seeds.     

Limited proteolysis regulates massive degradation of glycinin, storage 11S globulin from soybean seeds: An in vitro model

The time course of glycinin hydrolysis by papain was followed using densitometry of SDS-PAGE patterns, quantification of the residual protein and determination of its molecular mass by gel filtration, and by other appropriate methods. The hydrolysis occurs in two steps. In the first step, a limited proteolysis was observed consisting of a gradual detachment of the α-chain C-terminal sequence region, leading to the formation of glycinin-P, a relatively stable proteolysis product retaining the primordial hexameric structure. Glycinin-P was found to be composed of the intact β-chains covalently bound with the C-terminally truncated α-chains lacking the helix domain, strand J', and the C-terminal disordered region. Glycinin-P is further hydrolyzed in the second step exclusively by a one-by-one mechanism. Comparison of the kinetics of the limited and one-by-one proteolyses analyzed separately indicated that the decrease of protein concentration by 24-25% in the first step occurs almost exclusively due to the decrease of the molecular mass of the residual protein. Thus, the onset of the one-by-one proteolysis is delayed, suggesting a regulatory role of the preceding limited proteolysis in the subsequent massive degradation of glycinin. Probable structural alterations of glycinin generated by this limited proteolysis are discussed.     

Improvement of protein quality in transgenic soybean plants

Glycinin is one of the abundant storage proteins in soybean seeds. A modified Gy1 (A1aB1b) proglycinin gene with a synthetic DNA encoding four continuous methionines (V3-1) was connected between the hpt gene and the modified green fluorescent protein sGFP(S65T) gene, and a resultant plasmid was introduced into soybean by particle bombardment in order to improve nutritional value of its seeds. After the selection with hygromycin, the efficiency of gene introduction was evaluated. More than 60 % of the regenerated plants tolerant to hygromycin yielded the hpt and V3-1 fragment by polymerase chain reaction (PCR) analysis, and the expression of sGFP was detected in about 50 % of putative transgenic soybeans. Southern hybridization confirmed the presence of transgenes in T0 plants and the transgenic soybeans hybridized with the hpt and V3-1 genes were analyzed showed different banding patterns. Most of the transgenic plants were growing, flowering normally and produced seeds. Analysis of seed obtained from transgenic soybean plants expressing hpt and V3-1 genes showed higher accumulation of glycinin compared with non-transgenic plants. In addition, protein expression in transgenic soybean plants was observed by using 2D-electrophoresis.     

Subcellular localization of the 2S globulin narbonin in seeds of Vicia narbonensis

Narbonin is a 2S protein from the globulin fraction of narbon bean (Vicia narbonensis L.) cotyledons. Its amino acid composition and the pattern of its regulated accumulation in developing seeds led to the suggestion that narbonin could be a storage protein. Therefore, it was expected to be present in protein bodies of the storage tissue cells. Comparison of the cDNA-derived amino acid sequence with a directly determined partial N-terminal sequence revealed that the primary translation product of narbonin mRNA lacks a transient N-terminal signal peptide (V.H. Nong et al., 1995, Plant Mol Biol 28: 61–72). Narbonin polypeptides that had been synthesized in a cell-free translation system supplemented with dog pancreas microsomes were not protected against degradation by posttranslationally added proteases (protease protection assay). In accordance with the lack of a signal peptide this indicates that the polypeptide was not cotranslationally sequestered into the microsomes. The protein-body fraction that had been isolated from mature narbon bean cotyledons by a non-aqueous gradient centrifugation procedure was free of narbonin; this was found in the soluble cell fraction. In electron micrographs, narbonin could be localized in the cytoplasm using the immuno gold-labelling technique. Previously, it had already been shown that narbonin is too slowly degraded during narbon bean germination to act as a storage protein. From all these results it has to be concluded that narbonin is a cytoplasmic protein which does not belong to the storage proteins in the restricted sense. Other possible functions are discussed. Received: 18 November 1996 / Accepted: 28 February 1997     

Methionine analogs inhibit production of β-subunit of soybean 7S protein

We have previously reported that exogenous methionine inhibits production of the β-subunit of the 7S storage protein in cultured soybean cotyledons, and that this inhibition involves lack of functional mRNA for the β-subunit. Analogs of methionine were used to study this inhibition. Cycloleucine, norleucine, norvaline and S-ethylcysteine treatments prevented accumulation of the β-subunit. The effects of cycloleucine and norleucine on β-subunit synthesis might have been indirect, since these compounds inhibited growth and caused a 2- to 3-fold increase in free methionine concentration. Norvaline did not affect free methionine concentration, but it did inhibit growth. Treatment with a combination of S-ethylcysteine and aminoethoxyvinylglycine prevented appearance of the β-subunit without inhibiting growth or raising the S-adenosylmethionine concentration. Thus, accumulation of S-adenosylmethionine does not appear to mediate the effect of exogenous methionine on β-subunit production. Treatment with S-ethylcysteine raised free methionine concentration only 34%, so S-ethylcysteine was probably acting directly to inhibit β-subunit production. Measurements of free methionine concentrations in seeds of different sizes, taken from intact plants, suggested that the relatively late appearance of the β-subunit in normal soybean seed development may be due to the presence of high levels of free methionine in very young seeds.     

Contribution of different sources and origins of nitrogen in above- and below-ground structures to the partial nitrogen balance in soybean.

Plant and Soil - Most studies that quantified the biological N fixation (BNF) and partial N balances of soybean have ignored the belowground structures. Our objectives were to evaluate the...