Molecular cloning and characterization of soybean
protein disulfide isomerase family proteins with nonclassic
active center motifs
Kensuke Iwasaki
1
, Shinya Kamauchi
1,
*, Hiroyuki Wadahama
1
, Masao Ishimoto
2
, Teruo Kawada
1
and Reiko Urade
1
1 Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Japan
2 National Agricultural Research Center for Hokkaido Region, Sapporo, Japan
Introduction
Secretory, organelle and membrane proteins are synthe-
sized and folded with the assistance of molecular chap-
erones and other folding factors in the endoplasmic
reticulum (ER). In many cases, the process of protein
folding is accompanied by N-glycosylation and the for-
mation of disulfide bonds [1]. Disulfide bonds are
essential for structural stabilization and for regulation
of the functions of many secretory and plasma mem-
brane proteins [2,3]. The formation and isomerization
of disulfide bonds are catalyzed by protein disulfide
isomerase (PDI) and other PDI family proteins located
in the ER [4,5]. PDI has two thioredoxin domains
containing the redox active site CGHC (a and a¢) and

DIL-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-inde-
pendent 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.
Structured digital abstract
l
MINT-7137566: Bip (uniprotkb:Q587K1), GmPDIL-3b (genbank_nucleotide_g:51848586)
and GmPDIL-3a (genbank_nucleotide_g:
51848584) colocalize (MI:0403)bycosedimentation
through density gradients (
MI:0029)
Abbreviations
ER, endoplasmic reticulum; PDI, protein disulfide isomerase; PDILT, testis-specific protein disulfide isomerase-like protein; PVDF,
poly(vinylidene difluoride).
4130 FEBS Journal 276 (2009) 4130–4141 ª 2009 The Authors Journal compilation ª 2009 FEBS
members contain one or more thioredoxin domains [7].
PDI family proteins containing the redox active center
transfer the disulfide bond between the two cysteine
residues of their active site to the substrate protein [8].
Recently, it has been shown that PDI family proteins
containing nonclassic redox motifs, such as yeast
Eug1p and mammalian testis-specific PDI-like protein
(PDILT) and ERp44, may function in protein folding,

important roles in folding and in formation and
rearrangement of disulfide bonds in the storage
proteins.
Group III PDI family proteins have not been stud-
ied. Putative amino acid sequences obtained from
Arabidopsis genome sequence predict the typical PDI
domain structure a–b–b¢–a¢, but that both the
a-domain and a¢-domain contain nonclassic CXXS ⁄ C
motifs as opposed to the more traditional CGHC
sequence. In this study, we describe soybean group III
PDI family ER proteins, namely GmPDIL-3a and
GmPDIL-3b, and identify nonclassic redox center
CXXS ⁄ C motifs in each. Characterization of GmP-
DIL-3a and GmPDIL-3b and changes in their
expression during seed development are described. In
addition, our data suggest that GmPDL-3a and
GmPDIL-3b form protein complexes in both thiol-
dependent and thiol-independent ways in the ER.
Results
cDNA cloning of GmPDIL-3a and GmPDIL-3b
In order to clone the soybean orthologs of Arabidopsis
PDI-like1-5 and PDI-like1-6 (group III PDIs) [13], we
first obtained their nucleotide sequences from the Insti-
tute for Genomic Research Soybean Index and used
them in blast searches. We identified the tentative
consensus sequence TC183516, and primer sets were
designed on the basis of this sequence. Two cDNAs
were cloned using RNA extracted from young soybean
leaves by 5¢-RACE and 3¢-RACE using these primers.
Genomic GmPDIL-3b was cloned and sequenced,

ing only the AUG1 mutation (lane 3) or wild-type
mRNA (lane 2), suggesting that initiation events can
also begin at AUG2, but are unproductive because of
the stop codon located just upstream of AUG3. On
the other hand, AUG1 in GmPDIL-3a mRNA may
K. Iwasaki et al. Novel plant PDI family proteins
FEBS Journal 276 (2009) 4130–4141 ª 2009 The Authors Journal compilation ª 2009 FEBS 4131
not be used as efficiently for translation initiation,
and therefore may not interfere with translation from
AUG3. For GmPDIL-3b, a 59 kDa polypeptide was
generated from wild-type mRNA, and from mRNA
that contained AGG in place of both AUG1 and
AUG2 (Fig. 1C, lanes 2–5), whereas it was not trans-
lated from mutant mRNA in which both AUG3 and
AUG4 were changed to AGG (lane 6). The amino
acid sequence identity shared between GmPDIL-3a
and GmPDIL-3b, excluding the signal peptides, was
92%. The structure of GmPDIL-3a and GmPDIL-3b
was predicted to contain the four domains a–b–b¢–a¢
(Fig. 1D). GmPDIL-3a and GmPDIL-3b have two
predicted thioredoxin domains between amino acids
65–164 and 403–481, and 68–167 and 406–508,
respectively, corresponding to the a-domain and
a¢-domain of PDI [7]. Notably, both GmPDIL-3a and
GmPDIL-3b lack the two classic PDI redox-active
CGHC motifs within the a-domain and a¢-domain.
Instead, they both contain the sequence CPRS in the
a-domain and CMNC or CINC in the a¢-domain.
GmPDIL-3a and GmPDIL-3b contain a C-terminal
KDEL sequence that probably functions in ER reten-

formed without (lane 1) or with (lane 2) 1 lg
of wild-type GmPDIL-3a mRNA or mutant
GmPDIL-3a mRNA, of which the first (lane
3), second (lane 4), first and second (lane 5)
or third AUG (lane 6) was replaced with
AGG. Products were separated by
SDS ⁄ PAGE and detected by fluorography.
(C) In vitro translation of GmPDIL-3b. Trans-
lation reactions were performed without
(lane 1) or with (lane 2) 1 lg of wild-type
GmPDIL-3b mRNA, or with mutant GmP-
DIL-3b mRNA, of which the first (lane 3),
second (lane 4), first and second (lane 5)
or third and fourth AUGs (lane 6) were
replaced with AGG. (D) Putative domain
structure of GmPDIL-3a and GmPDIL-3b.
The boxes indicate the domain boundaries
predicted by an NCBI conserved domain
search. Black boxes in domain-a and
domain-a¢ represent the CPRS and CXXC
motifs. A closed circle with a bar represents
an N-glycosylation consensus site. SP,
signal peptide.
Novel plant PDI family proteins K. Iwasaki et al.
4132 FEBS Journal 276 (2009) 4130–4141 ª 2009 The Authors Journal compilation ª 2009 FEBS
As shown in Fig. 2A,B, neither protein was able to
catalyze the oxidation of thiol residues on the synthetic
peptide and the oxidative refolding of reduced and
denatured RNase A. In addition, neither protein
reduced the disulfide bond in insulin (Fig. 2C). As it

3a (open circles), GmPDIL-3b (solid circles) and bovine PDI (solid squares) were assayed using the synthetic peptide as a substrate as
described in Experimental procedures. (B) Oxidative refolding activity of recombinant GmPDIL-3a (3a), GmPDIL-3b (3b), GmPDIL-1 (L-1),
L-1 plus 3a or 3b, GmPDIL-2 (L-2), or L-2 plus 3a or 3b. Activity was assayed by measuring the RNase activity produced through the
regeneration of the active form of reduced RNase A. Data represent the mean ± standard deviation for three experiments. (C) Thiol
reductase activities of recombinant GmPDIL-3a (open circles), GmPDIL-3b (solid circles) and bovine PDI (solid squares) were assayed
using insulin as a substrate. (D) Chaperone activities of recombinant GmPDIL-3a, GmPDIL-3b and GmPDIL-2 were assayed by measuring
the aggregation of rhodanese in the absence (open triangles) or presence of GmPDIL-3a (open circles), GmPDIL-3b (solid circles), or
GmPDIL-2 (solid squares).
K. Iwasaki et al. Novel plant PDI family proteins
FEBS Journal 276 (2009) 4130–4141 ª 2009 The Authors Journal compilation ª 2009 FEBS 4133
Expression of GmPDIL-3a and GmPDIL-3b in
soybean tissue
We next prepared antiserum directed against recombi-
nant GmPDIL-3a and a synthetic peptide containing
sequences found in GmPDIL-3b, but not in GmPDIL-
3a. Anti-GmPDIL-3a serum recognized both recombi-
nant GmPDIL-3a and GmPDIL-3b (Fig. 3A, lanes 1
and 2), whereas anti-GmPDIL-3b serum reacted exclu-
sively with recombinant GmPDIL-3b (Fig. 3A, lanes 4
and 5). Anti-GmPDIL-3a serum reacted with both a
55 kDa and a 59 kDa band in western blot analysis
of cotyledon proteins (Fig. 3A, lane 3). Both the 55
and 59 kDa bands were N-glycosylated, as digestion
experiments using glycosidase F resulted in the
bands shifting to 53 and 57 kDa, respectively (Fig. 3B).
The cotyledon proteins that were deglycosylated
with glycosidase F and detected with the serum were
characterized by two-dimensional gel electrophoresis
and western blot analysis. Two spots of 53 and
57 kDa, with isoelectric points of 5.3 and 5.1, respec-

blot using anti-GmPDIL-3a serum. (C) Cotyledon proteins were
treated with glycosidase F, separated by two-dimensional electro-
phoresis, and analyzed by western blot using anti-GmPDIL-3a
serum (upper panel) or anti-GmPDIL-3b serum (lower panel). pI, iso-
electric point. (D) Thirty micrograms of protein extracted from the
cotyledon (80 mg bean) (lane 1), root (lane 2), stem (lane 3), leaf
(lane 4) and flower (lane 5) were analyzed by western blot using
anti-GmPDIL-3a serum.
A
B
Fig. 4. Localization of GmPDIL-3a and GmPDIL-3b in the ER
lumen. (A) Microsomes were isolated from cotyledons (100 mg
bean), and were fractionated on isopyknic sucrose gradients in the
presence of MgCl
2
or EDTA. Proteins from each fraction were ana-
lyzed by western blot using anti-GmPDIL-3a serum or anti-BiP
serum. The top of the gradient is on the left, and density (gÆmL
)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 blot using anti-GmPDIL-
3a serum.
Novel plant PDI family proteins K. Iwasaki et al.
4134 FEBS Journal 276 (2009) 4130–4141 ª 2009 The Authors Journal compilation ª 2009 FEBS
ER, and a C-terminal ER retention sequence (KDEL).
To confirm localization of GmPDIL-3a and GmPDIL-
3b to the ER, microsomes were prepared from cotyle-

synthesized and translocated into the ER lumen during
the maturation stage of embryogenesis. Therefore, we
next measured the mRNA and protein levels of GmP-
DIL-3a and GmPDIL-3b by real-time RT-PCR and
western blotting, respectively, during different stages of
development. The amounts of pro-b-conglycinin and
proglycinin are considered to be nearly equivalent to
the synthesis levels of both b-conglycinin and glycinin,
as pro-b-conglycinin and proglycinin are transient pro-
tein forms that are present in the ER prior to process-
ing in the protein storage vacuoles. The synthesis of
proglycinin and pro-b-conglycinin was initiated when
the seeds achieved a mass of 50 mg (Fig. 5A, lanes 2
and 4). The amount of GmPDIL-3a and GmPDIL-3b
proteins increased until the seeds grew from 40 to
80 mg (Fig. 5A, lane 1). Thereafter, the level remained
constant. This event correlated with the amount of
GmPDIL-3a mRNA, although the amount of GmP-
DIL-3b mRNA was not consistent with the amount of
GmPDIL-3b protein expression (Fig. 5B).
Expression of many ER-resident proteins can be
upregurated by ER stress in plant cells [26–28]. There-
fore, we next measured the amounts of GmPDIL-3a
and GmPDIL-3b mRNA in cotyledon cells under
stress by treatment with tunicamycin or dithiothreitol.
The amount of neither mRNA was affected by either
treatment, whereas the mRNA of BiP, which is a
representative unfolded protein response gene, was
dramatically upregulated (data not shown). These data
suggest that expression of neither GmPDIL-3a nor

GmPDIL-3a or GmPDIL-3b with molecular sizes
larger than those of monomeric GmPDIL-3a or
GmPDIL-3b were detected in the region of 130–
300 kDa (Fig. 6A). A proportion of GmPDIL-3a or
GmPDIL-3b in these complexes was detected as mixed
disulfides. When western blots were performed in the
presence of N-ethylmaleimide to trap any disulfide-
bound intermediates under nonreducing conditions,
trace amounts of GmPDIL-3a and GmPDIL-3b mole-
cules were found to be engaged in intermolecular,
disulfide-linked complexes of approximately 130 kDa
(Fig. 6B, lane 3). As these mixed disulfide bonds disap-
peared under reducing conditions (Fig. 6B, lane 1), it
is likely that GmPDIL-3a or GmPDIL-3b interacts
with proteins in the ER through a redox-dependent
mechanism. When nonreducing experiments were per-
formed after crosslinking treatment of associated pro-
teins with dithiobis(succinimidyl propionate), the
130 kDa complexes decreased in abundance, whereas
complexes ranging in size from 200 kDa to greater
than 250 kDa appeared (Fig. 6B, lane 4). This could
suggest that a proportion of the 130 kDa disulfide-
linked complexes associated noncovalently with other
proteins. Partner proteins for GmPDIL-3a or GmP-
DIL-3b in these complexes remain to be identified.
Discussion
In this report, we characterized new members of the
plant PDI family, which we now refer to as GmP-
DIL-3a and GmPDIL-3b. The conserved exon struc-
ture of the GmPDIL-3a and GmPDIL-3b genomic

A
B
Fig. 6. GmPDIL-3a or GmPDIL-3b form protein complexes in a
thiol-dependent or thiol-independent manner in the ER. (A) Cotyle-
don proteins (100 mg bean) were extracted with digitonin and ana-
lyzed by two-dimensional electrophoresis on blue native (BN) PAGE
and SDS ⁄ PAGE and western blot using anti-GmPDIL-3a serum. (B)
Cotyledon proteins (100 mg bean) treated with (lanes 2 and 4) or
without (lanes 1 and 3) dithiobis(succinimidyl propionate) (DSP)
were lysed in the presence of N-ethylmaleimide and analyzed by
10% reducing (R) or nonreducing (NR) SDS ⁄ PAGE and western blot
using anti-GmPDIL-3a serum.
Novel plant PDI family proteins K. Iwasaki et al.
4136 FEBS Journal 276 (2009) 4130–4141 ª 2009 The Authors Journal compilation ª 2009 FEBS
their a¢-domain. Additionally, GmPDIL-3a and GmP-
DIL-3b showed no reductase activity. Replacement of
the second and third amino acids in classic redox-
active CGHC motifs with methionine or isoleucine
and asparagine in GmPDIL-3a and GmPDIL-3b may
be the cause of the lack of such enzymatic activities.
Alternatively, the lack of other amino acids, such as
arginine, which is important for the regulation of the
active site redox potential in human PDI [8,33], may
cause the lack of enzymatic activity. Mammalian
PDILT, which has the same domain structure as
PDI, but lacks oxidoreductase activity, has been dem-
onstrated to have chaperone activity in vitro [34]. As
PDILT forms a complex with the calnexin homolog
calmegin in vitro, this protein is thought to function
as a redox-inactive chaperone for glycoprotein folding

and GmPDIL-3b will be required to establish their
physiological function.
Little is known about the coordinated function of
ER chaperones in the plant. Previously, we observed
that at least four types of PDI family proteins (GmP-
DIL-1, GmPDIL-2, GmPDIM, and GmPDIS) were
expressed ubiquitously in the plant body [16–18]. Thus,
it may be difficult to substitute other PDI family pro-
teins for GmPDIL-3a or Gm-PDIL-3b, as they proba-
bly have unique functions in the plant. The details of
how PDI family proteins contribute to ER function
and protein folding are beginning to emerge, and,
importantly, knowledge concerning GmPDIL-3a and
GmPDIL-3b can now be applied to the understanding
of how divergent PDI family proteins contribute to
quality control in the ER, and how this process influ-
ences vital plant function.
Experimental procedures
Plants
Soybean seeds (Glycine max L. Merrill cv. Jack) were
planted in 5 L pots and grown in a controlled environment
chamber at 25 °C under 16 h day ⁄ 8 h 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.
Cloning of GmPDIL-3a and GmPDIL-3b
Cloning of the cDNAs for GmPDIL-3a and GmPDIL-3b
was performed by 3¢-RACE and 5¢-RACE. Soybean trifolio-
late center leaves were frozen under liquid nitrogen and then

was performed using the reaction mixture of the first PCR
and a reverse primer (Table S1). Wild-type and mutagenic
DNA fragments were subcloned at the SpeI restriction site
into pT7Blue (TaKaRa Bio Inc.) and sequenced. Plasmids
were linearized by digestion with KpnI, and were tran-
scribed in vitro using a RiboMax Large Scale RNA produc-
tion systems kit (Promega Corporation). In vitro translation
reactions were performed in in a total volume of 25 lL
containing 1 lg of mRNA, 555 kBq of L-[
35
S] in vitro cell
labeling mix (37 TBqÆmmol
)1
; GE Healthcare BioSciences
Corporation, Piscataway, NJ, USA), 80 lm cysteine ⁄ methi-
onine-free amino acid mixture, 0.8 units of RNasin ribo-
nuclease inhibitor, 120 mm potassium acetate, and 12.5 lL
of wheat germ extract (Promega Corporation) at 25 °C
for 90 min. Proteins were separated by 10% SDS ⁄ PAGE,
and were detected by fluorography with ENLIGHTNING
(Perkin Elmer Life Sciences, Boston, MA, USA).
Construction of expression plasmids
Expression plasmids encoding mature GmPDIL-3a (Thr24–
Leu520) and GmPDIL-3b (Ser27–Leu523), excluding the
putative signal peptides, were constructed as follows. DNA
fragments were amplified from GmPDIL-3a and GmPDIL-
3b cDNAs by PCR using the primers 5¢-GACGACGACA
AGATGGAGGTTAAGGATGAGTTG-3¢ and 5¢-GAG
GAGAAGCCCGGTCTATAACTCATCTTTGAGTAC-3¢
for GmPDIL-3a, and 5¢ -GACGACGACAAGATGGAGG

regeneration of the active form from reduced and denatured
RNase A in the presence of 0.5 lm recombinant GmPDIL-
3a, GmPDIL-3b, GmPDIL-1, or GmPDIL-2 [41,42]. Thiol
reductase activity was measured as previously described,
where the glutathione-dependent reduction of insulin was
measured by Morjana and Gilbert [43]. Briefly, 50 lgof
bovine PDI (TaKaRa Bio Inc.), recombinant GmPDIL-3a or
recombinant GmPDIL-3b was incubated in 1 mL of 0.2 m
sodium phosphate buffer (pH 7.5) containing 5 mm EDTA,
3.7 mm reduced glutathione, 0.12 mm NADPH, 16 U of glu-
tathione reductase (Sigma-Aldrich Inc., St Louis, MO, USA)
and 30 lm insulin (Sigma-Aldrich Inc.) at 25 °C, and absor-
bance was monitored at 340 nm. Oxidase activity was
assayed using a synthetic peptide, NH
2
-NRCSQGSCWN-
COOH, as described by Alanen et al. [44]. Briefly, 0.5 lm
bovine PDI, recombinant GmPDIL-3a or recombinant
GmPDIL-3b was incubated in 0.2 m sodium phosphate ⁄
citrate buffer (pH 6.5), 2 mm reduced glutathione, 0.5 mm
oxidized glutathione and 5 lm synthetic peptide at 25 °C,
and fluorescence was monitored at 350 nm with excitation at
280 nm on a Hitachi F-3000 fluorescence spectrophotometer
(Hitachi Ltd, Tokyo, Japan).
Chaperone activity assays
Chaperone activity was assayed as described previously
[45]. Briefly, aggregation of 0.4 lm rhodanese (Sigma-
Aldrich Inc.) during refolding was measured spectrophoto-
metrically at 320 nm (25 °C) in the absence or presence
of 1.2 lm recombinant GmPDIL-3a, GmPDIL-3b, and

(PVDF) membranes. For two-dimensional separation by
isoelectric focusing and SDS ⁄ PAGE, SDS was removed
from the samples with the 2D clean-up kit (GE Healthcare
UK Ltd). Proteins (100 lg) were applied to 7 cm Ready-
Strip IPG Strips (Bio-Rad Laboratories), and isoelectric
focusing was performed using a Protean IEF Cell (Bio-Rad
Laboratories). The strips were then subjected to SDS ⁄
PAGE, and proteins on the gel were transferred to PVDF
membranes. For two-dimensional electrophoresis of blue
native PAGE [30] and SDS ⁄ PAGE, slices of cotyledons
were homogenized with 20 strokes of a Dounce homo-
genizer in ice-cold buffer containing 50 mm Bis-Tris (pH
7.2), 50 mm NaCl containing 10% (w ⁄ v) glycerol, 0.001%
ponceau S, and 1% digitonin. After standing at 4 °C for
1 h, the homogenate was centrifuged for 30 min at
14 000 g. Five per cent Coomassie Brilliant Blue G-250
solution was added to the supernatant to a final concentra-
tion of 0.25%, and the supernatant was subjected to
3–12% polyacrylamide gradient gel electrophoresis accord-
ing to the manufacturer’s protocol for the Native PAGE
Novex Bis-Tris Gel System (Invitrogen Corporation, Carls-
bad, CA, USA). Blue native PAGE gels were then sub-
jected to SDS ⁄ PAGE, and proteins on the gel were
transferred to PVDF membranes. Membranes were incu-
bated with primary antibody, followed by a horseradish
peroxidase-conjugated IgG secondary antibody (Promega
Corporation), and were developed using Western Lightning
Chemiluminescence Reagent (Perkin Elmer Life Science) as
previously described [18].
Real-time RT-PCR

genates were centrifuged for 10 min at 1000 g at 4 °C. Next,
600 lL of the supernatant was loaded onto a 12 mL linear
21–56% (w ⁄ w) sucrose gradient prepared in the same buffer.
Samples were centrifuged at 154 400 g for 2 h at 4 °C, and
1 mL fractions were collected and assayed by western blot.
Acknowledgements
We thank Dr M. Kito for critical reading of the man-
uscript and warm encouragement. This work 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).
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Supporting information
The following supplementary material is available:
Fig. S1. Comparison of intron–exon structures of
GmPDIL-3a, GmPDIL-3b and orthologs of other
plants.
Fig. S2. Multiple amino acid sequence alignment of
GmPDIL-3a, GmPDIL-3b and orthologs of other
plants.
Fig. S3. Expression and CD analysis of recombinant
GmPDIL-3a and GmPDIL-3b.
Table S1. List of primers for ATG-mutagenesis of
GmPDIL-3a and GmPDIL-3b.
This supplementary material can be found in the
online version of this article.
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