RESUMEN

ß-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean ß-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the α'- and α-subunits of ß-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the ß-conglycinin complexes containing the disulfide-linked α'/α-subunits were complexes of more than 720 kD. The α'- and α-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between α'/α-subunits residing in different ß-conglycinin hexamers, but the binding of P34 to α'- and α-subunits reduces the linkage between ß-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when ß-conglycinin was expressed under nonreducing conditions.

Asunto(s)

Antígenos de Plantas/metabolismo , Cotiledón/metabolismo , Globulinas/metabolismo , Glycine max/metabolismo , Proteínas de Almacenamiento de Semillas/metabolismo , Proteínas de Soja/metabolismo , Antígenos de Plantas/aislamiento & purificación , Western Blotting , Cromatografía en Gel , Disulfuros/metabolismo , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Globulinas/aislamiento & purificación , Complejos Multiproteicos/metabolismo , Concentración Osmolar , Oxidación-Reducción , Unión Proteica , Proteínas de Almacenamiento de Semillas/aislamiento & purificación , Proteínas de Soja/aislamiento & purificación

RESUMEN

Protein disulfide isomerase (PDI) and other PDI family proteins are members of the thioredoxin superfamily and are thought to play important roles in disulfide bond formation and isomerization in the endoplasmic reticulum (ER). The exact functions of PDI family proteins in plants remain unknown. In this study, we cloned two novel PDI family genes from soybean leaf (Glycine max L. Merrill cv. Jack). The cDNAs encode proteins of 520 and 523 amino acids, and have been denoted GmPDIL-3a and GmPDIL-3b, respectively. GmPDIL-3a and GmPDIL-3b are the first plant ER PDI family proteins reported to contain the nonclassic redox center motif CXXS/C, and both proteins are ubiquitously expressed in the plant body. However, recombinant GmPDIL-3a and GmPDIL-3b did not function as oxidoreductases or as molecular chaperones in vitro, although a proportion of each protein formed complexes in both thiol-dependent and thiol-independent ways in the ER. Expression of GmPDIL-3a and GmPDIL-3b in the cotyledon increased during seed maturation when synthesis of storage proteins was initiated. These results suggest that GmPDIL-3a and GmPDIL-3b may play important roles in the maturation of the cotyledon by mechanisms distinct from those of other PDI family proteins.

Asunto(s)

Glycine max/enzimología , Glycine max/genética , Proteína Disulfuro Isomerasas/genética , Dominio Catalítico , Clonación Molecular , Codón/genética , Cartilla de ADN , ADN Complementario/química , ADN Complementario/genética , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Cinética , Hojas de la Planta/enzimología , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/metabolismo , ARN de Planta/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo

RESUMEN

In this study, HepG2 cells were treated with short peptides (7S-peptides) derived from highly purified soybean beta-conglycinin (7S), which was free from lipophilic protein, and the effect of the peptide treatment on lipid metabolism was determined. 7S-peptide treatment suppressed the secretion of apolipoprotein B-100 from HepG2 cells into the medium. The 7S-peptides also suppressed the incorporation of (3)H-glycerol and (14)C-acetate into triacylglyceride but not into major phospholipids, such as phosphatidylcholine and phosphatidylethanolamine. Additionally, the synthesis of cholesterol esters was dramatically decreased for 2 h after the addition of the 7S-peptides, whereas the synthesis of cholesterol remained unchanged by 4 h and increased by 8 h after the addition of the 7S-peptides. The cleaved nuclear form of SREBP-2 increased 8 h after the addition of the 7S peptides, suggesting a decrease in intracellular cholesterol levels. Analysis of changes in mRNA expression after 7S-peptide treatment suggested that the 7S-peptides lower the level of cholesterol in the endoplasmic reticulum, increase the mRNA of genes related to beta-oxidation of fatty acids, and increase the synthesis of cholesterol. From these results, it may be concluded that the peptides derived from 7S altered the lipid metabolism to decrease secretion of apolipoprotein B-100-containing lipoprotein from HepG2 cells.

Asunto(s)

Globulinas/química , Glycine max/química , Metabolismo de los Lípidos/efectos de los fármacos , Péptidos/farmacología , Proteínas de Almacenamiento de Semillas/química , Proteínas de Soja/química , Antígenos de Plantas , Apolipoproteína B-100/análisis , Apolipoproteína B-100/metabolismo , Carcinoma Hepatocelular , Línea Celular Tumoral , Globulinas/metabolismo , Humanos , Lipoproteínas/química , Lipoproteínas/metabolismo , Neoplasias Hepáticas , Péptido Hidrolasas/metabolismo , Proteínas de Almacenamiento de Semillas/metabolismo , Proteínas de Soja/metabolismo , Triglicéridos/biosíntesis

RESUMEN

Protein disulfide isomerase family proteins play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill. cv Jack). The cDNAs encode proteins of 525 and 551 amino acids, named GmPDIL-1 and GmPDIL-2, respectively. Recombinant versions of GmPDIL-1 and GmPDIL-2 expressed in Escherichia coli exhibited oxidative refolding activity for denatured RNaseA. Genomic sequences of both GmPDIL-1 and GmPDIL-2 were cloned and sequenced. The comparison of soybean genomic sequences with those of Arabidopsis, rice and wheat showed impressive conservation of exon-intron structure across plant species. The promoter sequences of GmPDIL-1 apparently contain a cis-acting regulatory element functionally linked to unfolded protein response. GmPDIL-1, but not GmPDIL-2, expression was induced under endoplasmic reticulum-stress conditions. GmPDIL-1 and GmPDIL-2 promoters contain some predicted regulatory motifs for seed-specific expression. Both proteins were ubiquitously expressed in soybean tissues, including cotyledon, and localized to the endoplasmic reticulum. Data from coimmunoprecipitation experiments suggested that GmPDIL-1 and GmPDIL-2 associate with proglycinin, a precursor of the seed storage protein glycinin, and the alpha'-subunit of beta-conglycinin, a seed storage protein found in cotyledon cells under conditions that disrupt the folding of glycinin or beta-conglycinin, suggesting that GmPDIL-1 and GmPDIL-2 are involved in the proper folding or quality control of such storage proteins as molecular chaperones.

Asunto(s)

Globulinas/metabolismo , Glycine max/enzimología , Chaperonas Moleculares/química , Conformación Proteica , Proteína Disulfuro Isomerasas/química , Proteínas de Soja/química , Secuencia de Aminoácidos , Antígenos de Plantas , Clonación Molecular , Retículo Endoplásmico/metabolismo , Globulinas/química , Globulinas/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Almacenamiento de Semillas , Semillas , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Distribución Tisular

RESUMEN

The protein disulfide isomerase is known to play important roles in the folding of nascent polypeptides and in the formation of disulfide bonds in the endoplasmic reticulum (ER). In this study, we cloned a gene of a novel protein disulfide isomerase family from soybean leaf (Glycine max L. Merrill. cv Jack) mRNA. The cDNA encodes a protein called GmPDIM. It is composed of 438 amino acids, and its sequence and domain structure are similar to that of animal P5. Recombinant GmPDIM expressed in Escherichia coli displayed an oxidative refolding activity on denatured RNase A. The genomic sequence of GmPDIM was also cloned and sequenced. Comparison of the soybean sequence with sequences from Arabidopsis thaliana and Oryza sativa showed significant conservation of the exon/intron structure. Consensus sequences within the promoters of the GmPDIM genes contained a cis-acting regulatory element for the unfolded protein response, and other regulatory motifs required for seed-specific expression. We observed that expression of GmPDIM was upregulated under ER-stress conditions, and was expressed ubiquitously in soybean tissues such as the cotyledon. It localized to the lumen of the ER. Data from co-immunoprecipitation experiments suggested that GmPDIM associated non-covalently with proglycinin, a precursor of the seed-storage protein glycinin. In addition, GmPDIM associated with the alpha' subunit of beta-conglycinin, a seed-storage protein in the presence of tunicamycin. These results suggest that GmPDIM may play a role in the folding of storage proteins and functions not only as a thiol-oxidoredactase, but also as molecular chaperone.

Asunto(s)

Glycine max/enzimología , Proteína Disulfuro Isomerasas/genética , Semillas/enzimología , Proteínas de Soja/genética , Animales , Antígenos de Plantas , Western Blotting , Clonación Molecular , Cotiledón/enzimología , Cotiledón/genética , ADN Complementario/química , ADN Complementario/genética , ADN de Plantas/química , ADN de Plantas/genética , Retículo Endoplásmico/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Globulinas/metabolismo , Inmunoprecipitación , Datos de Secuencia Molecular , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Unión Proteica , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/metabolismo , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Almacenamiento de Semillas , Semillas/genética , Análisis de Secuencia de ADN , Proteínas de Soja/química , Proteínas de Soja/metabolismo , Glycine max/genética

RESUMEN

Protein disulfide isomerase family proteins are known to play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill cv. Jack) mRNA by RT-PCR using forward and reverse primers designed from the expressed sequence tag clone sequences. The cDNA encodes a protein of either 364 or 362 amino acids, named GmPDIS-1 or GmPDIS-2, respectively. The nucleotide and amino acid sequence identities of GmPDIS-1 and GmPDIS-2 were 68% and 74%, respectively. Both proteins lack the C-terminal, endoplasmic reticulum-retrieval signal, KDEL. Recombinant proteins of both GmPDIS-1 and GmPDIS-2 were expressed in Escherichia coli as soluble folded proteins that showed both an oxidative refolding activity of denatured ribonuclease A and a chaperone activity. Their domain structures were identified as containing two thioredoxin-like domains, a and a', and an ERp29c domain by peptide mapping with either trypsin or V8 protease. In cotyledon cells, both proteins were shown to distribute to the endoplasmic reticulum and protein storage vacuoles by confocal microscopy. Data from coimmunoprecipitation and crosslinking experiments suggested that GmPDIS-1 associates with proglycinin, a precursor of the seed storage protein glycinin, in the cotyledon. Levels of GmPDIS-1, but not of GmPDIS-2, were increased in cotyledons, where glycinin accumulates during seed development. GmPDIS-1, but not GmPDIS-2, was induced under endoplasmic reticulum-stress conditions.

Asunto(s)

Glycine max/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Proteínas de Soja/metabolismo , Secuencia de Aminoácidos , Western Blotting , Clonación Molecular , Cotiledón/metabolismo , ADN Complementario/química , ADN Complementario/genética , Retículo Endoplásmico/metabolismo , Perfilación de la Expresión Génica , Globulinas/metabolismo , Inmunoprecipitación , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Datos de Secuencia Molecular , Unión Proteica , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Proteínas de Soja/química , Proteínas de Soja/genética , Glycine max/genética , Glycine max/crecimiento & desarrollo