Role of posttranslational cleavage in glycinin assembly.

Glycinin, like other 11S seed storage proteins, undergoes a complex series of posttranslational events between the time proglycinin precursors are synthesized in endoplasmic reticulum and the mature glycinin subunits are deposited in vacuolar protein bodies. According to the current understanding of this process, proglycinin subunits aggregate into trimers in endoplasmic reticulum, and then the trimers move to the vacuolar protein bodies where a protease cleaves them into acidic and basic polypeptide chains. Stable glycinin hexamers, rather than trimers, are isolated from mature seeds. We used a re-assembly assay in this study to demonstrate that proteolytic cleavage of the proglycinin subunits is required for in vitro assembly of glycinin oligomers beyond the trimer stage. The possibility that the cleavage is a regulatory step and that it triggers the deposition of 11S seed storage proteins as insoluble aggregates in vivo is considered.     

The role of proteolysis in the processing and assembly of 11S seed globulins.

11S seed storage proteins are synthesized as precursors that are cleaved post-translationally in storage vacuoles by an asparaginyl endopeptidase. To study the specificity of the reaction catalyzed by this asparaginyl endopeptidase, we prepared a series of octapeptides and mutant legumin B and G4 glycinin subunits. These contained amino acid mutations in the region surrounding the cleavage site. The endopeptidase had an absolute specificity for Asn on the N-terminal side of the severed peptide bond but exhibited little specificity for amino acids on the C-terminal side. The ability of unmodified and modified subunits to assemble into hexamers after post-translational modification was evaluated. Cleavage of subunits in trimers is required for hexamer assembly in vitro. Products from a mutant gene encoding a noncleavable prolegumin subunit (LeBDeltaN281) accumulated as trimers in seed of transgenic tobacco, but products from the unmodified prolegumin B gene accumulated as hexamers. Therefore, the asparaginyl endopeptidase is required for hexamer assembly.     

Processing and assembly in vitro of engineered soybean beta-conglycinin subunits with the asparagine-glycine proteolytic cleavage site of 11S globulins

A short interdomain sequence between the N- and C-terminal domains of beta-conglycinin, the major 7S seed storage protein of soybean, was selected as a target for insertion of amino acid residues specifically cleaved by an asparaginyl endopeptidase that processes globulins into acidic and basic chains. Modified beta-conglycinin subunits containing the proteolytic cleavage site self-assembled into trimers in vitro at an efficiency similar to that of the unmodified subunit. In contrast to the absence of cleavage of the unmodified subunits, however, the modified beta-conglycinin trimers were processed by purified soybean asparaginyl endopeptidase into two polypeptides, each the size expected for the beta-conglycinin N- and C-terminal domains, respectively. The cleavage did not alter the assembly of mutant beta-conglycinins and the cleaved mutant trimers remained stable to further proteolytic attack. To examine the possibility of coassembly between the cleaved 11S and 7S subunits, in vitro processed mutant beta-conglycinin subunits were mixed with native dissociated 11S globulin preparations. Reassembly at a high ionic condition did not induce the 7S subunits to interact with 11S subunits to form hexameric complexes. Thus, cleavage of 7S globulin subunits into acidic and basic domains may not be sufficient for hexamer assembly to occur. Biotechnological implications of the engineered proteins are discussed.     

Purification and reconstitution of the proton-pumping ATPase of fungal and plant plasma membranes

The 11S storage protein (glycinin) of soybean [Glycine max (L.) Merr., cv. Raiden] was studied by polyacrylamide gel electrophoresis and amino acid sequence analysis. It contained the following subunits composed of acidic (A) and basic (B) polypeptides: A1aB2, A1bB1b, A2B1a, and A3B4. However, it lacked polypeptides A4, A5, and B3 which are present in many other cultivars. A new acidic polypeptide called A6 was present in a low amount and was characterized by amino acid sequence analysis. It was homologous to A4, although of a smaller apparent molecular weight. Since Raiden has an average protein content of about 40% and its glycinin fraction can be purified as a 350,000 D complex which is typical of other cultivars, the results imply polymorphism with respect to glycinin subunit composition. Because there is a wide variation in the methionine content of the various subunits, these findings suggest the possibility of genetically manipulating the nutritional quality of soybean seed protein by altering glycinin subunit composition.     

Genomic organization of glycinin genes in soybean

Glycinin is the predominant seed storage protein in most soybean varieties. Previously, five major genes (designated Gy1 to Gy5) encoding glycinin subunits have been described. In this report two new genes are identified and mapped: a glycinin pseudogene, gy6, and a functional gene, Gy7. Messenger RNA for the gy6 pseudogene is not detected in developing seeds. While Gy7 mRNA was present at the midmaturation stage of seed development in the soybean variety Resnik, the steady state amount of this message was at least an order of magnitude less-prevalent than the mRNA encoding each of the other five glycinin subunits. Even though the amino-acid sequence of the glycinin subunit G7 is related to the other five soybean 11S subunits, it does not fit into either the Group-1 (G1, G2, G3) or the Group-2 (G4, G5) glycinin subunit families. The Gy7 gene is tandemly linked 3' to Gy3 on Linkage Group L (chromosome 19) of the public molecular linkage map. By contrast, the gy6 gene occupies a locus downstream from Gy2 on Linkage Group N (chromosome 3) in a region that is related to the position where Gy7 is located on chromosome 19.     

Mobilization of storage proteins in soybean seed (Glycine max L.) during germination and seedling growth

During germination and early growth of the seedling, storage proteins are degraded by proteases. Currently, limited information is available on the degradation of storage proteins in the soybean during germination. In this study, a combined two-dimensional gel electrophoresis and mass spectrometry approach was utilized to determine the proteome profile of soybean seeds (Glycine max L.; Eunhakong). Comparative analysis showed that the temporal profiles of protein expression are dramatically changed during the seed germination and seedling growth. More than 80% of the proteins identified were subunits of glycinin and β-conglycinin, two major storage proteins. Most subunits of these proteins were degraded almost completely at a different rate by 120h, and the degradation products were accumulated or degraded further. Interestingly, the acidic subunits of glycinin were rapidly degraded, but no obvious change in the basic chains. Of the five acidic subunits, the degradation of G2 subunit was not apparently affected by at least 96h but the levels decreased rapidly after that, while no newly appearing intermediate was detected upon the degradation of G4 subunit. On the other hand, the degradation of β-conglycinin during storage protein mobilization appeared to be similar to that of glycinin but at a faster rate. Both α and α' subunits of β-conglycinin largely disappeared by 96h, while the β subunits degraded at the slowest rate. These results suggest that mobilization of subunits of the storage proteins is differentially regulated for seed germination and seedling growth. The present proteomic analysis will facilitate future studies addressing the complex biochemical events taking place during soybean seed germination.     

Glycinin A3B4 mRNA. Cloning and sequencing of double-stranded cDNA complementary to a soybean storage protein

The cDNA clones encoding the precursor form of glycinin A3B4 subunit have been identified from a library of soybean cotyledonary cDNA clones in the plasmid pBR322 by a combination of differential colony hybridizations, and then by immunoprecipitation of hybrid-selected translation product with A3-mono-specific antiserum. A recombinant plasmid, designated pGA3B41425, from one of six clones covering codons for the NH2-terminal region of the subunit was sequenced, and the amino acid sequence was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 516 amino acids. Analysis of this cDNA also showed that it contained 1786 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 46 nucleotides, a signal peptide region corresponding to 24 amino acids, an A3 acidic subunit region corresponding to 320 amino acids followed by a B4 basic subunit region corresponding to 172 amino acids, and a 3'-terminal nontranslated region of 192 nucleotides, which contained two characteristic AAUAAA sequences that ended 110 nucleotides and 26 nucleotides from a 3'-terminal poly(A) segment, respectively. Our results confirm that glycinin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs via disulfide bonds. The inferred amino acid sequence of the mature basic subunit, B4, was compared to that of the basic subunit of pea legumin, Leg Beta, which contained 185 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall 42% of the amino acid positions are identical in both proteins. These results led us to conclude that both storage proteins have a common ancestor.     

A complete cDNA coding for the sequence of glycinin A2B1a subunit precursor

Analysis of the A₂B_1a subunit precursor, one of the A₂-subunit family of glycinin, the main storage protein of soybean, revealed that it is composed of a signal peptide segment (18 amino acids), the A₂ acidic polypeptide (282 amino acids), followed by the B_1a basic polypeptide (185 amino acids). There was overall 63% homology between this subunit complex and pea legumin, which is an analogous protein to glycinin. As this degree of homology is rather higher than that in the A₃B₄ subunit, one of the A₃ subunit family, it seems that the genes encoding the A₂ subunit family are phylogenetically more strongly related to the legumin genes.     

大豆球蛋白研究以及在改良稻米营养品质中的应用

11S大豆球蛋白是大豆贮藏蛋白的重要成分,目前已发现6种有功能的大豆球蛋白,G1、G2、G3、G4、G5和G7,编码这些大豆球蛋白的基因家族分别是Gy1、Gy2、Gy3、Gy4、Gy5和Gy7。本文在简要介绍大豆球蛋白组分、结构及基因家族后,采用生物信息学方法分析比较了不同种类大豆球蛋白基因之间的序列同源性,并对不同大豆球蛋白基因的遗传距离作了分析;重点比较分析不同大豆球蛋白的氨基酸序列和组成,以及不同酸性肽和碱性肽中重要氨基酸的含量,提出了利用大豆球蛋白基因进行水稻营养品质改良研究的新思路和新策略。     

Partial characterization of the acidic and basic polypeptides of glycinin.

The subunits of the 11 S storage protein from soybean cultivar CX635-1-1-1 were purified and characterized. Six polypeptides with acidic isoelectric points and four with basic isoelectric points were isolated from the purified storage protein. The acidic polypeptides had phenylalanine, leucine, isoleucine, and arginine at the NH2 termini, while the basic polypeptides all had glycine at the NH2 termini. Amino acid analysis indicated that certain acidic and basic polypeptides contained 3 to 6 times more methionine than the other polypeptides. Since the low nutritional quality of legume storage proteins is due to a deficiency in methionine, this observation will have significance in efforts to improve soybean quality. The purified polypeptides were further characterized by NH2-terminal sequence analysis. Considerable homology was found between the members of individual families of acidic and basic polypeptides, indicating that the members of each family arose from a common ancestral gene. This study showed that the glycinin polypeptide composition is more complex than previous reports indicated, and for the first time characterized the various polypeptides of the 11 S storage protein by structural analysis.     

Role of the sulfhydryl redox state and disulfide bonds in processing and assembly of 11S seed globulins.

Seed legumins contain two conserved disulfide bonds: an interchain bond (IE) connecting the acidic and basic chains and an intrachain bond (IA) internal to the acidic chain. Mutant subunits were constructed in which these disulfide bonds were disrupted. Oxidized glutathione stimulated the rate of assembly of trimers with unmodified prolegumin subunits. Stimulation was not detected during assembly of IE mutant subunits and was diminished for the IA mutant. Hexamer assembly with trimers of mature unmodified subunits required oxidizing conditions. Trimers composed of mature IE mutants did not form hexamers. Both mutant and non-mutant subunits accumulated in hexamers when the cDNAs were expressed in tobacco. Hexamer assembly in seeds probably involved trimers with a mixture of mutant and non-mutant subunits. Similarly, mixed trimers that were a mixture of mutant and non-mutant subunits assembled into hexamers in vitro. The results demonstrate the importance of disulfide bonds during the assembly of 11S globulins.     

Interaction Involving Disulfide Bridges between Subunits of Soybean Seed Globulin and between Subunits of Soybean and Sesame Seed Globulins

Native subunit proteins of glycinin, the acidic and the basic subunits designated as AS₁₊₂, AS₂₊₃, AS₄, AS₅, and AS₆ and BS, respectively, were isolated by DEAE-Sephadex A-50 column chromatography in the presence of 6 m urea and 0.2 m 2-mercaptoethanol.

Reconstitution of intermediary subunits involving a disulfide bridge from native acidic and basic subunits was investigated. Formation of the intermediary subunit was observed in combinations between BS and each acidic subunit except AS₆. The yields of the reconstituted intermediary subunits differed from one another.

Further, formation of the intermediary complexes was observed when native acidic and basic subunits of soybean glycinin and sesame 13 S globulin, respectively (or reverse combinations), were mixed under reductively denatured condition and subjected to the reconstitution procedure. Considerring the overall evidence, we may conclude that the complexes are probably a hybrid intermediary subunit.     

Genetic mapping of the Gy4 and Gy5 glycinin genes in soybean and the analysis of a variant of Gy4

The predominant storage protein of soybean [Glycine max (L.) Merr.] seed is a globulin called glycinin. Thus far five genes encoding glycinin subunits have been described, and these are denoted by the gene symbols Gy1 to Gy5. The objectives of this study were to map two of these genes, Gy4 and Gy5, and to conduct a genetic analysis of a subunit size-variant from an allele of Gy4. For this purpose a population was formed with an interspecific cross between PI 468916 (G. soja) and A81-356022 (G. max). The two size forms of G4, the subunit from Gy4, segregated codominantly in the mapping population, and were due to a short insertion in the hypervariable region of the mutant protein. The biochemical and molecular characteristics of the two subunits indicate that they are produced from alternate alleles of the same gene. The gene symbols Gy ^a and Gy ^b have been assigned to the normal and variant genes, respectively. When genomic DNA from the two parents was probed with a Gy4 cDNA, RFLPs were identified for both Gy4 and Gy5. Using these genetic markers, the Gy4 and Gy5 glycinin genes were mapped in linkage group O and F on the public soybean genomic map.Joint contribution of North Central Region, USDA-ARS and Journal Paper No. J-14736 of the Iowa Agric. and Home Economics Exp. Stn., Ames, IA 50011; Project 2763. This work was supported, in part, with grants from the Iowa State Biotechnology Program (No. 480-46-09) and the Iowa Soybean Promotion Board to RCS, and the American Soybean Association to NCN     

A cDNA clone encoding a glycinin A1a subunit precursor of soybean.

A cDNA clone covering the whole coding region for a glycinin subunit precursor containing the A1a acidic subunit, one of the A2 family, has been identified from a library of soybean cotyledonary cDNA clones using a mixed oligonucleotide probe. Analysis of the cDNA insert revealed that it contained 1746 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 54 nucleotides, a signal peptide region corresponding to 19 amino acids, an acidic subunit region (A1a) corresponding to 291 amino acids followed by a basic subunit region corresponding to 185 amino acids, and a 3'-terminal nontranslated region of 207 nucleotides. By comparing the predicted protein sequence of this precursor with that of the legumin A precursor of pea, it was found that glycinin A2 subunit family appeared to be more closely related to the legumin than to the A3 subunit family, and that the evolutional rearrangement of glycinin genes has occurred.     

Proteomic and genetic analysis of glycinin subunits of sixteen soybean genotypes.

We investigated proteomic and genomic profiles of glycinin, a family of major storage proteins in 16 different soybean genotypes consisting of four groups including wild soybean (Glycine soja), unimproved cultivated soybean landraces from Asia (G. max), ancestors of N. American soybean (G. max), and modern soybean (G. max) genotypes. We observed considerable variation in all five glycinin subunits, G1, G2 G3, G4 and G5 using proteomics and genetic analysis. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) analysis showed that the wild genotypes had a range of 25-29 glycinin protein spots that included both acidic and basic polypeptides followed by the ancestors with 24-28, modern cultivars with 24-25, and landraces with 17-23 protein spots. Overall, the wild genotypes have a higher number of protein spots when compared to the other three genotypes. Major variation was observed in acidic polypeptides of G3, G4 and G5 compared to G1 and G2, and minor variation was observed in basic polypeptides of all subunits. Our data indicated that there are major variations of glycinin subunits between wild and cultivated genotypes rather than within the same groups. Based on Southern blot DNA analysis, we observed genetic polymorphisms in group I genes (G1, G2, and G3) between and within the four genotype groups, but not in group II genes (G4 and G5). This is the first study reporting the comparative analysis of glycinin in a diverse set of soybean genotypes using combined proteomic and genetic analysis.     

Cloning and structural analysis of DNA encoding an A2B1a subunit of glycinin

The partial DNA sequence of a glycinin gene in a genomic clone and a homologous cDNA clone were determined. They have nearly identical nucleotide sequences and encode the basic polypeptide and part of the acidic polypeptide for an A2B1a glycinin subunit. The protein primary structure deduced from the DNA sequence is in close agreement with the amino acid sequence of the subunit determined chemically and confirms assignment of part of the amino acid sequence in the basic component where we were able to establish an overlap using conventional approaches. The coding part of the basic subunit is interrupted by a 625-base pair A + T-rich intron whose boundaries correlate with the established consensus sequences for the exon-intron junctions. Comparison of the nucleotide sequence of the basic subunit of pea legumin gene with that of the gene for A2B1a subunit reveals 70% homology in coding regions, although there is considerably less in the 3'-flanking regions.     

Engineering two mutants of cDNA-encoding G2 subunit of soybean glycinin capable of self-assembly <Emphasis Type="Italic">in vitro</Emphasis> and rich in methionine

The main goal of this work was to make the cDNA-encoding subunit G2 of soybean glycinin, capable of self-assembly in vitro and rich in methionine residues. Two mutants (pSP65/G4SacG2 and pSP65/G4SacG2HG4) were therefore constructed. The constructed mutants were successfully assembled in vitro into oligomers similar to those occurred in the seed. The successful self-assembly was due to the introduction of Sac fragment of Gy4 (the codons of the first 21 amino acid residues), which reported to be the key element in self-assembly into trimers. The mutant pSP65/G4SacG2HG4 included the acidic chain of Gy4 (HG4), which was previously molecularly modified to have three methionine residues. This mutant will be useful in the efforts to improve the seed quality.     

Identification of Gy4 nulls and development of multiplex PCR-based co-dominant marker for Gy4 and α’ subunit of β-conglycinin in soybean

Alpha prime (α’) subunit of β-conglycinin and Gy4 subunit of glycinin are two important subunits of soybean storage protein which have negative effects on food processing, total amino acid content, and hypersensitivity reactions. It has been possible to reduce or remove some of these problems from soybean by screening or developing mutant lines. The objective of this study was to establish a simple, cheap DNA marker for Gy4 and α’ subunit for use in non-seed destructive, marker-assisted selection (MAS) that can identify these two mutants at the same time in a unique PCR reaction. To achieve this objective, we identified eight of Gy4 mutants from diverse soybean accessions from the USDA Soybean Germplasm Collection and described a multiplex PCR based co-dominant DNA marker for Gy4 subunit of glycinin. Then we crossed one of these Gy4 mutants with Keburi (α’ mutant) for development of double mutant variety and established a multiplex PCR based, co-dominant DNA marker for screening Gy4 and α’ mutants. Thus, using this newly developed marker to identify Gy4 and α’ mutants in breeding programs we could save our time, labor, and resources.     

The amino acid sequence of the A2B1a subunit of glycinin

The amino acid sequences of the acidic and basic components of the A2B1a subunit of glycinin, the major seed reserve protein of the soybean (Glycine max L. Merr.), were determined. They contain 278 and 180 amino acids, respectively, and have molecular weights of 31,600 +/- 100 and 19,900 +/- 100. The molecular weight of the acidic component is considerably less than that estimated by sodium dodecyl sulfate-gel electrophoresis (37,000). Sequence heterogeneity was detected at several positions scattered throughout the primary structures of both components, indicating that the preparation sequenced was composed of several nearly identical polypeptides. These data, in conjunction with a recently determined nucleotide sequence of the 3'-terminal two-thirds of the analogous glycinin subunit gene, illustrate the complexity of the gene family responsible for synthesis of glycinin subunits.