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
1 Graduate School of Agriculture, Kyoto University, Uji, Japan
2 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:
†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.

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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

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H. Wadahama et al.

Soybean protein disulfide isomerase family

Fig. 1. 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: 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.

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Table 1. 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 4 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 2 (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).

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A

Soybean protein disulfide isomerase family

A

B

B

Fig. 2. 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), 3 cm leaf (lane 4), 6 cm leaf (lane 5), 9 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. 3. 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

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A

H. Wadahama et al.

B

A

B

C

Fig. 4. 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, 3 and 5) or with (lanes 2, 4 and 6) tunicamycin for 24 h
were analyzed by western immunoblot with anti-GmPDIM serum
(lanes 1 and 2), anti-BiP serum (lanes 3 and 4) and anti-calreticulin
serum (lanes 5 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 1
and 2). Proglycinin was detected in the immunoprecipi404

Fig. 5. 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 1 and 2) or
with (lanes 3 and 4) proteinase K, in the absence (lanes 1 and 3)
or presence (lanes 2 and 4) of Triton X-100. Microsomal proteins
(10 lg) were analyzed by western immunoblot with anti-GmPDIM
serum.

A

B

Fig. 6. 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 2 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 5 and 6) dithiothreitol and labeled
with Pro-mix L-[35S] in vitro labeling mix for 1 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 1 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.

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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 1 and 2). The
b-conglycinin a¢ subunit was barely detected in the
immunoprecipitate with anti-GmPDIM serum (Fig. 7,
lanes 3 and 4). We next performed the immunoprecipitation experiment using cotyledon cells treated with
tunicamycin. Tunicamycin may increase the amount of

Fig. 7. Co-immunoprecipitation of GmPDIM and the b-conglycinin

a’ subunit. Cotyledons were pretreated without (lanes 1–4) or with
(lanes 5 and 6) and labeled with Pro-mix L-[35S] in vitro labeling mix
for 1 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 1 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

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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 : 8 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 PolyATtractỊ 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, 2 mm CaCl2, 0.5 mm glutathione disulfide,
2 mm glutathione, 1 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 4 h. Cells from 1 L of
culture were collected by centrifugation, disrupted by sonication in 8 mL of 20 mm Tris ⁄ HCl (pH 8.0) containing
5 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 8 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 5 min 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

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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 5 mm MgCl2 or 5 mm EDTA. The
homogenate was centrifuged for 10 min at 1000 g at 4 °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 2 h at 154 400 g and
4 °C, 1 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
5 min at 4 °C. Proteins were precipitated with 10% trichloroacetic acid for 30 min at 4 °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 4 °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 4 °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
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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 1 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
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