Báo cáo khoa học: A novel plant protein disulfide isomerase family homologous to animal P5 – molecular cloning and characterization as a functional protein for folding of soybean seed-storage proteins docx

A novel plant protein disulfide isomerase family homologous to animal P5 – molecular cloning and characterization as a functional protein for folding of soybean seed-storage proteins Hiroyuki Wadahama1,*, Shinya Kamauchi1,*,†, Yumi Nakamoto2, Keito Nishizawa2, Masao Ishimoto2, Teruo Kawada1 and Reiko Urade1 Graduate School of Agriculture, Kyoto University, Uji, Japan National Agricultural Research Center for Hokkaido Region, Sapporo, Japan Keywords endoplasmic reticulum; protein disulfide isomerase; soybean; storage protein; unfolded protein response Correspondence R Urade, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan Fax: +81 774 38 3758 Tel: +81 774 38 3757 E-mail: urade@kais.kyoto-u.ac.jp †Present address Osaka Bioscience Institute, Suita, Japan Database The nucleotide sequence data for the cDNA of GmPDIM and genomic GmPDIM have been submitted to the DDBJ ⁄ EMBL ⁄ GenBank databases under accession numbers AB189994 and AB295118, respectively *These authors contributed equally to this article (Received 11 October 2007, revised 18 November 2007, accepted 20 November 2007) 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 a¢ subunit of b-conglycinin, a seedstorage 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 doi:10.1111/j.1742-4658.2007.06199.x Secretory, organellar and membrane proteins are synthesized and folded with the assistance of molecular chaperones and other folding factors in the endoplasmic reticulum (ER) In many cases, protein folding in the ER is accompanied by N-glycosylation and the formation of disulfide bonds [1] Formation of disulfide bonds between correct pairs of cysteine residues in a nascent polypeptide chain is thought to be catalyzed Abbreviations DSP, dithiobis[succinimidylpropionate]; ER, endoplasmic reticulum; ERSE, ER stress responsive element; PDI, protein disulfide isomerase FEBS Journal 275 (2008) 399–410 ª 2007 The Authors Journal compilation ª 2007 FEBS 399 Soybean protein disulfide isomerase family H Wadahama et al by the protein disulfide isomerase (PDI) family of proteins [2–4] In humans, 17 genes of the PDI family have been identified [5] The physiological role of each PDI protein and their interactions with each other and with other ER-resident molecular chaperones have been partially elucidated P5, an animal PDI family, was first discovered in Chinese hamster [6] P5 has both thiol-oxidoreductase activity and chaperone activity [7,8] In addition, roles for P5 other than the folding of nascent proteins have been reported in animal cells Zebrafish P5 is involved in the production of midline-derived signals required to establish left ⁄ right asymmetry [9] In human tumor cells, cell-surface P5 was required for shedding of the soluble major histocompatibility complex class I-related ligand, resulting in the promotion of tumor immune evasion [10] In plants, a set of 22 orthologs of known PDI-like proteins was discovered using a genome-wide search of Arabidopsis thaliana and these were separated into 10 phylogenetic groups [11] Among these groups, group V genes show structural similarities to animal P5 However, group V gene products in plant cells have not been identified Large quantities of storage protein are synthesized in the ER during seed development in soybean cotyledon cells [12] Approximately 70% of seed-storage proteins are composed of the two major globulins glycinin and b-conglycinin They are folded and assembled into trimers in the ER, and then transported and deposited in the protein storage vacuoles [13] Glycinin is synthesized as a 60 kDa precursor polypeptide and is proteolytically processed into 40 kDa acidic and 20 kDa basic subunits in the protein storage vacuoles [14–16] A1aB1b, a major glycinin, possesses two intradisulfide bonds between Cys12–Cys45 and Cys88–Cys298 These disulfide bonds are required for assembly into hexamers and for the structural stability of the protein [17–19] Thus, proper folding and disulfide bond formation is important for the effective deposition of glycinin in the vacuoles ER-resident PDI proteins may play a central role in this folding process Previously, we identified two novel PDI proteins belonging to group IV, GmPDIS-1 and GmPDIS-2, and demonstrated that GmPDIS-1 is associated with proglycinin in the ER [20] However, involvement of the other PDI proteins in the folding of storage proteins remains unclear In this study, we isolated cDNA clones and genomic sequences encoding a soybean group V gene of the PDI family The tissue distribution and cellular localization of GmPDIM and changes in its expression during seed development are described In addition, our 400 data suggest that GmPDIM and proglycinin or b-conglycinin associate during the course of the folding process Results cDNA cloning of GmPDIM To clone the soybean ortholog of group V Arabidopsis PDI-like2-2 or PDI-like2-3 [11], a blast search was performed using the nucleotide sequences of these cDNAs from the Institute for Genomic Research Soybean Index The tentative consensus sequence BU926832 was obtained Using primer sets designed from this sequence, we cloned a cDNA from the RNA extracted from young soybean leaves by 3¢-RACE and 5¢-RACE This cDNA encoded GmPDIM, a protein of 438 amino acids (supplementary Fig S1) containing a putative N-terminal secretary signal sequence and a C-terminal tetrapeptide, KDEL, which acts as a signal for retention in the ER [21,22] GmPDIM possesses two tandem thioredoxin-like motifs, each containing a CGHC active site Arginine residues R126 and R255, which are involved in the regulation of the active site redox potential in human PDI [5,23], were conserved In addition, glutamic acid residues E58 and E186, which have been suggested to facilitate ‘the escape’ of the cystein residue of the active site from a mixed disulfide bond with substrate [5,23], were also conserved The amino acid sequence of GmPDIM and orthologs from other plant species were  80% similar, excluding the putative N-terminal signal peptide The amino acid sequence identity between GmPDIM and human P5 was 46% Recombinant GmPDIM was expressed in Escherichia coli as a soluble protein and was purified by affinitycolumn and gel-filtration chromatography (supplementary Fig S2A) Recombinant GmPDIM had a CD spectrum typical of a folded protein (supplementary Fig S3) The domain structure of GmPDIM was predicted to be a linear sequence of three domains in an a–a¢–b from the sequence homology to the conserved domains Therefore, we subjected the recombinant GmPDIM protein to limited proteolysis with either trypsin or V8 protease to determine their domain boundaries The native recombinant protein was digested to give smaller peptide fragments after treatment with either protease The sites of proteolytic cleavage were determined to be Lys150 (K150) and R255 by N-terminal sequencing of the trypsin peptide fragments The N-terminal sequences of other fragments generated by protease digestion were AHHHHH and corresponded to the N-terminal histidine tag of FEBS Journal 275 (2008) 399–410 ª 2007 The Authors Journal compilation ª 2007 FEBS H Wadahama et al Soybean protein disulfide isomerase family Fig Putative domain structure of GmPDIM Schematic representation of cleavage sites in GmPDIM by limited proteolysis The upper line represents recombinant protein The lower boxes indicate the domain boundaries predicted by an NCBI conserved domain search The arrows indicate cleavage sites Black boxes in domain a and a’ represent the CGHC motif SP, signal peptide the recombinant protein We next determined the C-terminal amino acid residues of the peptide fragments by measuring their masses by MALDI-TOF MS Most cleavage sites resided in two narrow regions, overlapping the putative boundary regions in GmPDIM between a and a¢, and a¢ and b, respectively (Fig 1) These results show that GmPDIM has a linear sequence of three domains in an a–a¢–b pattern similar to animal P5 [5] We next determined the activity of recombinant GmPDIM, which catalyzed oxidative refolding of the reduced and denatured RNase A The specific activity of GmPDIM was 45 mmol RNasemin)1Ỉmol)1 (supplementary Fig S2B) Despite the fact that human P5 has molecular chaperone activity [7], no such activity was detected with recombinant GmPDIM, (data not shown) Cloning of genomic sequences of GmPDIM The genomic sequence encoding GmPDIM was cloned and sequenced Alignment and comparison with the cDNA sequence showed that GmPDIM contains nine exons (supplementary Fig S4) Nucleotide sequence of the ORF of the GmPDIM gene was identical to that of the cDNA Comparison of the soybean genomic sequence of GmPDIM with those of A thaliana (AGI number At1g04980 and At2g32920) and Oryza sativa (MOsDb number Os09g27830) identified significant conservation in the exon ⁄ intron structure across these species Moreover, all introns matched degenerate consensus sequence of branch points of plants (YTNAN) upstream of the 3¢ splice site [24] We next analyzed the promoter region of GmPDIM, 2340 bp upstream of the translational initiation codon ATG A search of the database of plant promoters (PLACE: http://www.dna.affrc.go.jp/PLACE/) using the sequences upstream of the coding region of GmPDIM as the query detected an ER stress responsive elements (ERSE; CCAAT-N9-CCACG) [25] and a number of cis-acting regulatory elements involved in the regulation of endosperm specific genes (Table 1) Expression of GmPDIM in soybean tissues We next prepared antiserum against recombinant GmPDIM Anti-GmPDIM serum immunoreacted to recombinant GmPDIM by western immunoblot (Fig 2A, lane 1), and also to two bands from cotyledon cell extract of 50 and 52 kDa (Fig 2A, lane 2) The intensity of these bands decreased when antiGmPDIM serum was pre-incubated with purified recombinant GmPDIM (Fig 2A, lanes 3–5), suggesting that the antibodies specifically immunoreacted with GmPDIM or a protein homologous to GmPDIM Further western immunoblot analyses indicated that GmPDIM is expressed ubiquitously in roots, stems, trifoliolate leaves, flowers and cotyledons (Fig 2B) The approximate quantity of this protein in leaves decreased during leaf expansion Large amounts of seed-storage proteins are synthesized and are translocated to the ER during the maturation stage of embryogenesis Previously, we demonstrated that the synthesis of glycinin was initiated when the seeds achieved a mass of 50 mg and increased gradually until they grew to 300 mg We also demonstrated that the synthesis of b-conglycinin was initiated when the seeds achieved a mass of 40 mg, increased until the seeds grew to 70 mg, and then decreased [20] Under such conditions, the folding machinery comprised of molecular chaperones and other functional proteins must be strengthened in response to the increased de novo synthesis of seedstorage proteins Therefore, we next measured the mRNA and protein levels of GmPDIM using real-time RT-PCR or western immunoblot, respectively The relative level of GmPDIM mRNA was higher in the early stages of seed development and subsequently decreased (Fig 3A) The amount of GmPDIM protein was also higher in the early stages, but decreased until the seed grew to 100 mg Expression of GmPDIM increased in the late stage of seed development (Fig 3B) These results suggest that upregulation of the expression of GmPDIM occurs at a time when the requirement for molecular chaperones is high FEBS Journal 275 (2008) 399–410 ª 2007 The Authors Journal compilation ª 2007 FEBS 401 Soybean protein disulfide isomerase family H Wadahama et al Table Putative regulatory motifs found within the promoter sequences of GmPDIM Motif Consensus sequence ERSE CCAAT-N9-CCACG )300CORE TGTAAAG DPBFcore Dc3 ACACNNG E-box CANNTG GCN4 motif TGAGTCA Prolamine box TGCAAAG RY repeat CATGCA SEF3 motif AACCCA SEF motif RTTTTTR a Distance from ATG Sequencea Function Strand Putative cis-acting element involved in unfolded protein response found in upstream of the promoter from the B-hordein gene of barley and the alpha-gliadin, gamma-gliadin, and low molecular weight glutenin genes of wheat bZIP transcription factors, DPBF-1 and (Dc3 promoter-binding factor-1 and 2) binding core sequence; Found in the carrot (D.c.) Dc3 gene promoter; Dc3 expression is normally embryo-specific, and also can be induced by ABA E-box of napA storage-protein gene of Brassica napus Sequence is also known as RRE (R response element) Conserved in many storage-protein gene promoters ) )117 ) ) )1073 )1474 TGTAAAG TGTAAAG + ) + )95 )1100 )1470 ACACacG ACACttG ACACaaG + + + + ) ) ) ) ) )140 )1100 )1596 )1632 )140 )1100 )1596 )1632 )1874 CAaaTG CAagTG CAaaTG CAaaTG CAaaTG CActTG CAttTG CAgtTG TGAGTCA + )23 TGCAAAG + )270 CATGCA + )1509 AACCCA + + + + ) ) ) ) )299 )419 )476 )1031 )372 )1608 )1656 )2298 gTTTTTa gTTTTTa aTTTTTa gTTTTTa aTTTTTa gTTTTTa aTTTTTg gTTTTTa cis-acting element required for endosperm-specific expression cis-acting element involved in quantitative regulation of the GluB-1 gene RY repeat found in RY ⁄ G box (the complex containing the two RY repeats and the G-box) of napA gene in Brassica napus; Required for seed specific expression Soybean consensus sequence found in the 5’-upstream region of b-conglycinin gene Soybean consensus sequence found in the 5’-upstream region of b-conglycinin gene CCAAT CCAAT-catatattt-aCACG Conserved bases of the motifs are in large letters Upregulation of GmPDIM by ER stress Many ER-resident chaperones are upregulated by the accumulation of unfolded protein in the ER (i.e ER stress) [26–29] Because the consensus sequences to ERSE were found within the promoter region of GmPDIM, we next tested whether expression of GmPDIM responded to ER stress When ER stress was induced by treatment with tunicamycin or 402 l-azetidine-2-carboxylic acid in soybean cotyledons, GmPDIM mRNA increased (Fig 4A,B) Upregulation of mRNA of GmPDIM was detected by DNA array analysis with a genechip (Affymetrix, Santa Clara, CA, USA) designed from soybean expression sequence tags (data not shown) In addition, protein levels of GmPDIM, BiP and calreticulin were also increased in the cotyledons treated with tunicamycin (Fig 4C) FEBS Journal 275 (2008) 399–410 ª 2007 The Authors Journal compilation ª 2007 FEBS H Wadahama et al A Soybean protein disulfide isomerase family A B B Fig Expression of GmPDIM in soybean tissues (A) Purified recombinant GmPDIM (20 ng) (lane 1) and proteins extracted from the cotyledon (30 lg) (lanes 2–5) were analyzed by western immunoblot with anti-GmPDIM serum (1 lL) treated without (lanes 1, 2) or with 16 lg (lane 3), 80 lg (lane 4) or 400 lg (lane 5) purified recombinant GmPDIM (B) Thirty micrograms of protein extracted from the cotyledon (80 mg bean) (lane 1), root (lane 2), stem (lane 3), cm leaf (lane 4), cm leaf (lane 5), cm leaf (lane 6) and flower (lane 7) were analyzed by western immunoblot with anti-GmPDIM serum GmPDIM is an ER luminal protein GmPDIM has an N-terminal signal sequence for targeting it to the ER, and a C-terminal ER-retention signal sequence KDEL We performed a magnesium-shift assay to confirm the localization of GmPDIM in the rough ER Microsomes were prepared from the cotyledons and centrifuged through a sucrose gradient in the presence of magnesium or EDTA The buoyant density of rough ER is decreased by dissociation of ribosomes in the presence of EDTA Fractions were collected from the sucrose gradient and were analyzed by western immunoblot The peak of GmPDIM at a density of 1.21 gỈmL)1 in the presence of magnesium was shifted to fractions of lighter sucrose (1.16 gỈmL)1) in the presence of EDTA (Fig 5A), indicating that GmPDIM localized in the rough ER Next, microsomes were purified from cells and treated with proteinase K in the absence or presence of Triton X-100 GmPDIM Fig Expression of GmPDIM in soybean cotyledons during maturation (A) GmPDIM mRNA was quantified by real time RT-PCR Each value was standardized by dividing the value by that for actin mRNA Values are calculated as a percentage of the highest value obtained during maturation Data represent the mean ± SD for four experiments (B) Proteins (25 lg) extracted from cotyledons were analyzed by western immunoblot with anti-GmPDIM serum was resistant to protease treatment in the absence of detergent (Fig 5B, lane 3), but when the microsomal membranes first were disrupted by Triton X-100, GmPDIM was degraded (Fig 5B, lane 4) These results indicate that GmPDIM is an ER luminal protein Association of GmPDIM with proglycinin and b-conglycinin a’ in the cotyledon GmPDIM has oxidative folding activity in vitro and localizes to the ER lumen of the cotyledon, suggesting that it may function on folding of glycinin [17] Because nascent polypeptides and molecular chaperones transiently associate with each other in the ER, we next attempted to detect an interaction between GmPDIM and proglycinin, which is translocated into the lumen of the ER for folding Because a transient association between a chaperone and nascent polypeptide is generally unstable, immunoprecipitation experiments were carried out after treatment with the protein cross-linker dithiobis[succinimidylpropionate] (DSP) GmPDIM was detected in the immunoprecipitate with FEBS Journal 275 (2008) 399–410 ª 2007 The Authors Journal compilation ª 2007 FEBS 403 Soybean protein disulfide isomerase family A H Wadahama et al B A B C Fig Responses of the GmPDIM gene to ER stress induced by reagent treatment Cotyledons from 137–142 or 210–263 mg beans were divided into two halves and incubated in the absence or presence of tunicamycin (TM) for 24 h (A) or L-azetidine-2-carboxylic acid for 18 h (B) GmPDIM mRNA was quantified by real time RTPCR Each value was standardized by dividing the value by that for actin mRNA Fold expression change was calculated as the ratio of mRNA in the samples treated with the stress reagent to that in the untreated sample Data represent the mean ± SD for three experiments BiP* and CRT* (calreticulin) are from Wadahama et al [20] (C) Proteins (15 lg) extracted from cotyledons treated without (lanes 1, and 5) or with (lanes 2, and 6) tunicamycin for 24 h were analyzed by western immunoblot with anti-GmPDIM serum (lanes and 2), anti-BiP serum (lanes and 4) and anti-calreticulin serum (lanes and 6) anti-GmPDIM serum by western immunoblot (Fig 6A) The efficiency of immunoprecipitation of GmPDIM was not influenced by cross-linking with DSP We next tried to detect an association between GmPDIM with proglycinin In order to detect trace amounts of nascent proglycinin, cotyledons were metabolically labeled with [35S]-methionine and [35S]-cysteine After labeling, microsomes were prepared from the cotyledons in the presence of DSP, were solubilized, and were subjected to immunoprecipitation with anti-GmPDIM serum or non-immunized serum Immunoprecipitates were treated with dithiothreitol to reduce the disulfide bonds formed by cross-linking with DSP, and were then subjected to a second immunoprecipitation using anti-glycinin acidic subunit serum No precipitation of proglycinin with nonimmunized serum, was confirmed (Fig 6B, lanes and 2) Proglycinin was detected in the immunoprecipi404 Fig Localization of GmPDIM in the ER lumen (A) Microsomes were isolated from cotyledons (100 mg beans), and microsomes were fractionated on isopyknic linear sucrose gradients in the presence of MgCl2 or EDTA Proteins from each gradient fraction were analyzed by western immunoblot with anti-GmPDIM serum The top of the gradient is on the left Density (gỈmL)1) is indicated on the top (B) Microsomes were treated without (lanes and 2) or with (lanes and 4) proteinase K, in the absence (lanes and 3) or presence (lanes and 4) of Triton X-100 Microsomal proteins (10 lg) were analyzed by western immunoblot with anti-GmPDIM serum A B Fig Co-immunoprecipitation of GmPDIM and proglycinin (A) Confirmation of immunoprecipitation of GmPDIM with anti-GmPDIM serum Microsomes were isolated from cotyledons (150 mg beans) and treated with (+) or without ()) DSP Proteins were extracted and immunoprecipitated with anti-GmPDIM serum Microsomes (lane 1) and the immunoprecipitants (lanes and 3) were analyzed by western immunoblot with anti-GmPDIM serum (B) Co-immunoprecipitation experiments Cotyledons were pretreated without (lanes 1–4) or with (lanes and 6) dithiothreitol and labeled with Pro-mix L-[35S] in vitro labeling mix for h After labeling, microsomes were isolated and treated with (+) or without ()) DSP The extracts from the microsomes were subjected to immunoprecipitation with non-immuninized serum (lanes and 2) or antiGmPDIM serum (lanes 3–6) The precipitants were subjected to a second immunoprecipitation with anti-glycinin acidic subunit serum The final precipitants were subjected to SDS ⁄ PAGE and analyzed by fluorography The positions of proglycinins (pro 11S) are indicated on the right tate using anti-GmPDIM serum (Fig 6B, lane 4) These results suggest that GmPDIM may associate with proglycinin in the ER FEBS Journal 275 (2008) 399–410 ª 2007 The Authors Journal compilation ª 2007 FEBS H Wadahama et al Because co-immunoprecipitation between proglycinin and GmPDIM was dependent on cross-linking by DSP, it is possible that GmPDIM associates non-covalently with proglycinin in the ER To test this, similar experiments were performed using cotyledon cells treated with dithiothreitol Dithiothreitol is a membranepermeable reducing agent that inhibits disulfide bond formation in the ER Therefore, it was expected that unfolded proglycinin would increase in the ER in the presence of dithiothreitol A small amount of proglycinin was detected in immunoprecipitates using cotyledon cells that were not treated with DSP (Fig 6B, lane 5), whereas a large amount of proglycinin was detected in immunoprecipitates from cotyledon cells treated with DSP (Fig 6B, lane 6) The PDI family of proteins play roles not only as thiol-oxidereductases, but also as molecular chaperones in the ER [7,8,30] To test whether GmPDIM also functions as a molecular chaperone in the folding of b-conglycinin, we examined if GmPDIM associates with the b-conglycinin a¢ subunit No precipitation of the b-conglycinin a¢ subunit with non-immunized serum was confirmed (Fig 7, lanes and 2) The b-conglycinin a¢ subunit was barely detected in the immunoprecipitate with anti-GmPDIM serum (Fig 7, lanes and 4) We next performed the immunoprecipitation experiment using cotyledon cells treated with tunicamycin Tunicamycin may increase the amount of Fig Co-immunoprecipitation of GmPDIM and the b-conglycinin a’ subunit Cotyledons were pretreated without (lanes 1–4) or with (lanes and 6) and labeled with Pro-mix L-[35S] in vitro labeling mix for h After labeling, microsomes were isolated and treated with (+) or without ()) DSP The extracts from the microsomes were subjected to immunoprecipitation with non-immuninized serum (lanes and 2) or anti-GmPDIM serum (lanes 3–6) The precipitants were subjected to a second immunoprecipitation with anti-b-conglycinin a’ subunit serum The final precipitants were subjected to SDS ⁄ PAGE and analyzed by fluorography The position of the b-conglycinin a’ subunit (7S-a’) is indicated on the right Soybean protein disulfide isomerase family unfolded b-conglycinin a¢ subunit in the ER, because the folding efficiency of glycoproteins like b-conglycinin is reduced by inhibition of N-glycosylation [1] A small amount of b-conglycinin a¢ subunit was detected in the immunoprecipitate with anti-GmPDIM serum from cotyledon cells untreated with DSP (Fig 7, lane 5), but larger amounts were detected when the cells were treated with DSP (Fig 7, lane 6) These results suggest that GmPDIM may associate with an unglycosylated form of the b-conglycinin a¢ subunit in the ER in the presence of tunicamycin Discussion In this study, we cloned the cDNA of GmPDIM and characterized it as a member of the PDI family of proteins GmPDIM has two tandem redox-active thioredoxin-like domains, a and a’, and a redox-inactive thioredoxin-like domain, b The amino acid sequence of GmPDIM is highly similar to that of the animal thiol-oxidoreductase P5 [7] The recombinant form of GmPDIM was active as it could refold RNase A that had been reduced and denatured The specific activity of GmPDIM was very similar to those of GmPDIS-1 and GmPDIS-2 (other soybean PDI family proteins) [20] However, the specific activities of GmPDIM, GmPDIS-1 and GmPDIS-2 were relatively low and corresponded to 10% of bovine PDI activity [20] Because the amino acid sequences of the two thioredoxin domains and a region essential for oxidative refolding activity were conserved in GmPDIM, GmPDIS-1 and GmPDIS-2, their low activities may be due to their low affinities for unfolded RNase A Specific antiserum against recombinant GmPDIM reacted with two bands in the soybean cell lysate, and both migrated to similar positions on the SDS ⁄ PAGE gel It is unclear whether both proteins were products from a single GmPDIM gene or from two separate genes, as gene duplications are common in higher plants In A thaliana, two group V genes (AtPDIL2-2 and AtPDIL2-3) were identified (supplementary Fig S1) Therefore, it is possible that one of the bands is GmPDIM and the other is a homolog However, it cannot be excluded that the second band seen on immunoblotting is due to modification of GmPDIM Expression of GmPDIM mRNA was upregulated by ER stress Likewise, the DNA microarray analysis demonstrated that expression of AtPDIL2-2 and AtPDIL23 were also upregulated by ER stress [26,27] The ERSE consensus sequence was identified in the promoter regions of both GmPDIM and AtPDIL2-2; this cisacting regulatory element is frequently found in genes responsive to ER stress [26–28] In addition, the FEBS Journal 275 (2008) 399–410 ª 2007 The Authors Journal compilation ª 2007 FEBS 405 Soybean protein disulfide isomerase family H Wadahama et al A thaliana transcription factor, AtbZIP60, activates transcription from ERSE [31] Therefore, GmPDIM and AtPDIL2-2 may be unfolded protein responsive genes regulated by AtbZIP60 and may play an important role in maintaining homeostasis of the ER under ER stress Consensus sequences regulating seed-specific expression were found in the promoter region of GmPDIM For example, RY repeat, which was reported to function for seed-specific transcription of b-conglycinin, was found [32] However, mRNA expression patterns of GmPDIM and b-conglycinin [12] were different, suggesting that expression of these genes in the cotyledon is regulated in a different manner It is not known how the level of GmPDIM mRNA is controlled in the cotyledon cells The protein levels of GmPDIM increased dramatically during seed maturation, suggesting that it may also play an important role in this process The regulation of GmPDIM protein levels during this stage was a post-transcriptional event rather than a transcriptional one It is obscure how the protein levels of GmPDIM are controlled PDI family proteins are thought to catalyze the formation of disulfide bonds on nascent polypeptide chains in the ER Therefore, GmPDIM was assumed to relate to proglycinin folding that accompanies the formation of disulfide bonds in the ER of cotyledon cells Because the interaction between these two proteins was detected only in microsomes treated with DSP, it is likely that the majority of GmPDIM associates non-covalently with proglycinin Interaction between GmPDIM and proglycinin was also detected in the presence of dithiothreitol, which inhibits disulfide bond formation in the ER and may cause accumulation of unfolded proglycinin Because the active center (CGHC) of GmPDIM is reduced in the presence of dithiothreitol, it cannot form disulfide bonds with the cysteine residues in proglycinin Therefore, it is possible that GmPDIM associates non-covalently with proglycinin in the presence of dithiothreitol This also suggests that GmPDIM may function as a molecular chaperone for proglycinin However, because the chaperone activity of GmPDIM for rhodanase was not detected (data not shown), it is likely that GmPDIM recognizes specific protein elements other than hydrophobic structures exposed in unfolded proteins No association of GmPDIM with the b-conglycinin a¢ subunit was detected under normal conditions, but a positive interaction was detected in the presence of tunicamycin Tunicamycin inhibits N-glycosylation and may cause accumulation of the unfolded b-conglycinin a¢ subunit in the ER GmPDIM may play a role as a molecular chaperone for the b-conglycinin a¢ subunit, because the mature form possesses no disulfide bonds 406 [33,34] However, it is not clear if GmPDIM can interact with both the glycosylated and non-glycosylated forms of the b-conglycinin a¢ subunit, or only with the nonglycosylated form Alternatively, it is possible that GmPDIM catalyzes disulfide bond formation during folding, because five cysteine residues are present in the pro-sequence of the b-conglycinin a¢ subunit, which are subsequently removed in the post-ER compartment The results obtained using the anti-GmPDIM serum must be interpreted cautiously, because association between both GmPDIM and a homolog of GmPDIM with proglycinin or the b-conglycinin a¢ subunit may be detected, because anti-GmPDIM serum immunoreacted to two similar cotyledon proteins (Fig 2A) Future experiments with antibodies specific to individual proteins will clarify this result Molecular chaperones in the ER are believed to collaborate with each other in different ways so that they adapt to each substrate protein Exhaustive study of the interaction between the PDI family and other molecular chaperones, including their recognition sites on substrate polypeptides, is necessary to clarify the folding mechanism of each protein Experimental procedures Plants Soybean (Glycine max L Merrill cv Jack) seeds were planted in 5-L pots and grown in a controlled environmental chamber at 25 °C under 16 : h day ⁄ night cycles Roots were collected from plants 10 days after seeding Flowers, leaves, and stems were collected from plants 45 days after seeding All samples were immediately frozen and stored in liquid nitrogen until use DNA cloning of GmPDIM The cloning of GmPDIM cDNA was performed by 3¢- and 5¢-RACE Soybean trifoliolate center leaves were frozen under liquid nitrogen and then ground into a fine powder with a micropestle SK-100 (Tokken, Inc., Chiba, Japan) Total RNA was isolated using the RNeasy Plant Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol Messenger RNA was isolated from total RNA with the PolyATtractÒ mRNA Isolation System (Promega, Madison, WI) The 3¢-RACE method was performed using the SMARTTM RACE cDNA Amplification kit (Clontech, Mountain View, CA) according to the manufacturer’s protocol using the primer 5¢-TCCTCACCCGTG CTTCAACTCACTCC-3¢ The 5¢-RACE method was performed using the primer 5¢-CTGTTGGCTGAATGCT CATTGATAGGG-3¢, which was designed based on the FEBS Journal 275 (2008) 399–410 ª 2007 The Authors Journal compilation ª 2007 FEBS H Wadahama et al sequence obtained by 3¢-RACE The amplified DNA fragment was subcloned into the pT7Blue vector (TaKaRa Bio Inc., Shiga, Japan) The inserts in the plasmid vectors were sequenced using the fluorescence dideoxy chain termination method and an ABI PRISMÒ 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) Cloning of genomic sequences encoding GmPDIM Genomic sequence encoding GmPDIM was isolated from the transformation-competent artificial chromosome (TAC; pYLTAC7) library of soybean variety ‘Misuzudaizu’ by the three-dimensional screening system [35,36] Screening was performed by PCR using the primer set 5¢-CAATTGA TGCTGATGCTCATCCGTC-3¢ and 5¢-CATGGCCCAG TTTAACCTTCCCTT-3¢ Construction of His-tagged expression plasmid Expression plasmid encoding His-tagged GmPDIM without the putative signal peptide was constructed as follows The DNA fragment was amplified from GmPDIM cDNA by PCR using the primers 5¢-GACGACGACAAGATGC ACGCACTCTATGGAGC-3¢ and 5¢-GAGGAGAAGC CCGGTTCATAGCTCATCCTTGCTTGAAG-3¢ The amplified DNA fragment was subcloned into the ligationindependent cloning site of the pET46Ek ⁄ LIC vector (EMD Biosciences, San Diego, CA, USA) The recombinant protein has the His-tag linked to the N-terminus Soybean protein disulfide isomerase family (2 lg) (Sigma-Aldrich) at 25 °C for 30 or 120 min, respectively The cleavage sites of the resulting peptides were mapped as described previously [20] Oxidative refolding assay with reduced RNase A PDI activity was assayed by measuring RNase activity produced through the regeneration of the active form from reduced and denatured RNase A Reduced and denatured RNase A was prepared as described previously by Creighton [38] Each reaction mixture contained 200 mm [4-(2hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (pH 7.5), 150 mm NaCl, mm CaCl2, 0.5 mm glutathione disulfide, mm glutathione, mgỈmL)1 reduced RNase A and 0.25 mgỈmL)1 recombinant GmPDIM and was incubated at 25 °C An aliquot (16 lL) of the reaction mixture was removed and RNase A activity was measured spectrophotometrically at 284 nm with cytidine 2¢,3¢-cyclic monophosphate as the substrate [39] Reactivation of reduced RNase A in the absence of recombinant protein was subtracted from reactivation in the presence of GmPDIM Antibodies Anti-GmPDIM serum was prepared using recombinant GmPDIM by Operon Biotechnologies, K.K (Tokyo, Japan) Preparation of antibody specific to BiP, calreticulin, the glycinin acidic subunit and the b-conglycinin a¢ subunit has been described previously [20] Western immunoblot analysis Expression and purification of recombinant GmPDIM BL21(DE3) cells were transformed with the His-tagged expression vector described above Expression of recombinant protein was induced by the addition of 0.4 mm isopropyl thio b-d-galactoside at 30 °C for h Cells from L of culture were collected by centrifugation, disrupted by sonication in mL of 20 mm Tris ⁄ HCl (pH 8.0) containing mm imidazole and 0.5 m NaCl Affinity-column chromatography using His-Bind resin and gel-filtration chromatography were carried out as described previously [20] The concentration of purified recombinant GmPDIM was determined by absorbance values at 280 nm using the molar extinction coefficient calculated by the modified method of Gill and von Hippel [37] An extinction coefficient of 53 400 m)1Ỉcm)1 was used for recombinant GmPDIM Limited proteolysis of GmPDIM Purified recombinant GmPDIM (50 lg) was digested in100 mm Tris ⁄ HCl (pH 0) with either trypsin (1 lg) (Sigma-Aldrich Co., St Louis, MO, USA) or V8 protease Soybean tissues that had been frozen under liquid nitrogen were ground into fine powders with a micropestle SK-100 Proteins were extracted by boiling for in SDS ⁄ PAGE buffer [40] containing a 1% cocktail of protease inhibitors (Sigma-Aldrich) Protein concentration in the sample was measured with a protein assay kit (RC DC protein assay, Bio-Rad, Hercules, CA, USA) Proteins were subjected to SDS ⁄ PAGE [40] and blotted onto a poly(vinylidene difluoride) membrane Blots were immunostained with specific antibodies as described in the text, and with horseradish peroxidase-conjugated IgG antiserum (Promega) as secondary Blots were developed with the Western Lightning Chemiluminescence Reagent (Perkin Elmer Life Sciences, Boston, MA, USA) Real time RT-PCR analysis Measurement of mRNA was performed as described previously [20] Briefly, 250 lgỈmL)1 tunicamycin or 50 lm l-azetidine-2-carboxylic acid (Sigma-Aldrich) was administered to the inner surface of the divided half of the cotyledon and incubated at 25 °C Total RNA was isolated from FEBS Journal 275 (2008) 399–410 ª 2007 The Authors Journal compilation ª 2007 FEBS 407 Soybean protein disulfide isomerase family H Wadahama et al plant tissues using an RNeasy Plant Mini kit Quantification of mRNA was performed by real-time RT-PCR with a Thermal Cycler DiceÔ Real Time System (TaKaRa Bio Inc.) The forward primer 5¢-CGGAACCAAAACATGC TAACATTTTC[FAM]G-3¢ (Invitrogen, Carlsbad, CA, USA) and the reverse primer 5¢-CGTTACAGGCAACTTGTTTCTCA-3¢ were used for detection of GmPDIM mRNA ER fractionation Slices of cotyledons were homogenized by 20 strokes of a Dounce homogenizer in ice-cold buffer containing 100 mm Tris ⁄ HCl (pH 7.8), 10 mm KCl containing 12% (w ⁄ v) sucrose and either mm MgCl2 or mm EDTA The homogenate was centrifuged for 10 at 1000 g at °C Approximately 600 lL of supernatant was loaded onto a 12 mL linear 21–56% (w ⁄ w) sucrose gradient made in the same buffer After centrifugation for h at 154 400 g and °C, mL fractions were collected from the bottom of the gradient and assayed by western immunoblot using the anti-GmPDIM serum The sucrose concentration of each fraction was measured with a refractometer NAR-1T (ATAGO CO., LTD, Tokyo, Japan) Proteinase K treatment of microsomes Microsomes were prepared from cotyledons as described previously [20], and were treated with 0.5 lgỈmL)1 proteinase K in the presence or absence of 1% Triton X-100 for at °C Proteins were precipitated with 10% trichloroacetic acid for 30 at °C, and were analyzed by western immunoblot with anti-GmPDIM serum somes were prepared as described previously [20] Proteins were extracted from the microsomes with 50 mm Tris ⁄ HCl buffer (pH 7.5) containing 150 mm NaCl, and pre-cleared with protein A-conjugated Sepharose beads (50% slurry) (Sigma-Aldrich) Initial immunoprecipitation was performed at °C for 16 h with non-immmunized serum or affinity-purified anti-GmPDIM sera The immunoprecipitate was dissolved in 2% SDS and 0.4 m dithiothreitol The second immunoprecipitation was performed with anti-glycinin acidic subunit serum or anti-b-conglycinin a¢ subunit serum at °C for 16 h The antigen–antibody complexes were subjected to SDS ⁄ PAGE, and radiolabeled proteins were detected by fluorography with ENLIGHTING (NEN Life Science Products, Inc., Boston, MA, USA) Part of the immunoprecipitant obtained in the first immunoprecipitation using anti- GmPDIM serum was subjected to SDS ⁄ PAGE and analyzed by western immunoblot using anti-GmPDIM and the One-StepTM Complete IP-Western kit (GenScript Co., Piscataway, NJ, USA) Acknowledgements We thank Ms Masatoshi Izumo for identifying the C-terminal amino acid of the peptide produced by protease digestion We thank Ms Kensuke Iwasaki and Ms Akie Ko for assaying the oxidative refolding activity This study was supported by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences and a Grant-in-Aid for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (18658055) References Immunoprecipitation experiments Six pairs of cotyledons were isolated, divided into two halves and labeled flat-side up in a Petri dish at 25 °C for h with a mixture of 1.48 MBq per mL Pro-mix L-[35S] in vitro labeling mix (37 TBqỈmmol)1) (GE Healthcare, Little Chalfont, UK) and mL FN Lite [41] For treatment of cotyledons under ER-stress conditions, a cotyledon was pretreated with or without 250 lgỈmL)1 tunicamycin or mm dithiothreitol at 25 °C for h and labeled in the presence of the stress reagent The cotyledons were rinsed with FN Lite containing 10 mm cold methionine and cysteine three times and with 20 mm sodium pyrophosphate buffer (pH 7.5) containing 0.3 m mannitol (buffer A), after which slices from the flat side of each cotyledon were cut The slices were homogenized by 20 strokes of a Dounce homogenizer at °C in mL buffer A with or without mgỈmL)1 DSP The homogenate was placed on ice for h The cross-linking reaction was terminated by the addition of mm glycine for 30 on ice The micro- 408 Helenius A & Aeb M (2004) Roles of N-linked glycans in the endoplasmic reticulum Annu Rev Biochem 73, 1019–1049 Freedman RB, Hirst TR & Tuite MF (1994) Protein disulphide isomerase: building bridges in protein folding Trends Biochem Sci 19, 331–336 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Merrill] embryogenic cultures: the role of sucrose and total nitrogen content on proliferation In Vitro Cell Dev Biol Plant 34, 8–13 Supplementary material The following supplementary material is available online: Fig S1 Multiple amino acid sequence alignment of GmPDIM and orthologs from other species A multiple alignment was generated using clustal w AtPDIL2-2, Arabidopsis PDI-like2-2 (At1g04980); AtPDIL2-3, Arabidopsis PDI-like2-3 (At2g32920); Os PDIL2-3, rice PDI-like 2-3 (Os09g27830); ZmPDIL2-3, maize PDI-like 2-3 (AY739290); HsP5, human P5 (D49489) Numbers refer to the amino acid number, asterisks indicate amino acid matches, and dashes represent gaps between the sequences Putative signal sequence (underlined), active site CGHC motifs 410 (shaded in black), conserved arginine residues (shaded in gray) and conserved glutamic acid residues (boxes) are indicated Fig S2 Expression and oxidative refolding activity of recombinant GmPDIM (A) Recombinant GmPDIM in Escherichia coli (lane 1) was purified by Histag column chromatography (lane 2), followed by gel filtration chromatography (lane 3) Proteins in each sample were separated by 10% SDS ⁄ PAGE and stained with Coomassie Brilliant Blue (B) Oxidative refolding activity of recombinant GmPDIM Activity was assayed by the measurement of RNase A activity restored through the regeneration of the active form of reduced RNase A Each value represents the mean ± error of two experiments Fig S3 Far-UV CD spectrum of recombinant GmPDIM The spectrum was measured with a spectropolarimeter J-720 (JASCO Corporation, Tokyo, Japan) in a mm path length cell at 25 °C The proteins were dissolved in 100 mm potassium phosphate buffer, pH 7.0 Fig S4 Comparison of intron ⁄ exon structures of GmPDIM with homologous genes from different plant species Open boxes indicate exons and the solid black boxes denote introns Numbers represent exon and intron size (bp) The position of two putative thioredoxin-like active site sequences (CGHC) and the ER retention signal (KDEL or KDEL-related sequence) are also indicated This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 399–410 ª 2007 The Authors Journal compilation ª 2007 FEBS ... TGTAAAG TGTAAAG + ) + )95 )1100 )1470 ACACacG ACACttG ACACaaG + + + + ) ) ) ) ) )140 )1100 )1596 )1632 )140 )1100 )1596 )1632 )1874 CAaaTG CAagTG CAaaTG CAaaTG CAaaTG CActTG CAttTG CAgtTG TGAGTCA... The Authors Journal compilation ª 2007 FEBS 407 Soybean protein disulfide isomerase family H Wadahama et al plant tissues using an RNeasy Plant Mini kit Quantification of mRNA was performed by real-time... peptide was constructed as follows The DNA fragment was amplified from GmPDIM cDNA by PCR using the primers 5¢-GACGACGACAAGATGC ACGCACTCTATGGAGC-3¢ and 5¢-GAGGAGAAGC CCGGTTCATAGCTCATCCTTGCTTGAAG-3¢