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All Three Subunits of Soybean #-Conglycinin Are Potential Food Allergens
Hari B. Krishnan, Won-Seok Kim, Sungchan Jang, and Monty S. Kerley
J. Agric. Food Chem., 2009, 57 (3), 938-943• DOI: 10.1021/jf802451g • Publication Date (Web): 12 January 2009
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All Three Subunits of Soybean ?-Conglycinin Are
Potential Food Allergens
HARI B. KRISHNAN,*,†,§WON-SEOK KIM,§SUNGCHAN JANG,§AND
MONTY S. KERLEY#
Plant Genetics Research Unit, Agricultural Research Service, U.S. Department of Agriculture, and
Divisions of Plant Sciences and Animal Sciences, University of Missouri, Columbia, Missouri 65211
Soybeans are recognized as one of the “big 8” food allergens. IgE antibodies from soybean-sensitive
patients recognize more than 15 soybean proteins. Among these proteins only the R-subunit of
?-conglycinin, but not the highly homologous R′- and ?-subunits, has been shown to be a major
allergenic protein. The objective of this study was to examine if the R′- and ?-subunits of ?-conglycinin
can also serve as potential allergens. Immunoblot analysis using sera collected from soybean-allergic
patients revealed the presence of IgE antibodies that recognized several soy proteins including 72,
70, 52, 34, and 21 kDa proteins. Matrix-assisted laser desorption ionization time-of-flight mass
spectrometry (MALDI-TOF) analysis of trypsin-digested 72, 70, and 52 kDa proteins indicated that
these proteins were the R′-, R-, and ?-subunits of ?-conglycinin, respectively. Additionally, purified
R′-, R-, and ?-subunits of ?-conglycinin were recognized by IgE antibodies present in the soybean-
allergic patients. The IgE reactivity to the ?-subunit of ?-conglycinin was not abolished when this
glycoprotein was either deglycosylated using glycosidases or expressed as a recombinant protein in
Escherichia coli. The results suggest that in addition to the previously recognized R-subunit of
?-conglycinin, the R′- and ?-subunits of ?-conglycinin also are potential food allergens.
KEYWORDS: Allergen; ?-conglycinin; IgE reactivity; glycoprotein; soybeans
Soybeans are the major source of protein and vegetable oil
in the world. The United States is the leading producer and
exporter of soybean. It is estimated that the farm value of
soybean produced in 2006/2007 was $20.4 billion, which is
second only to corn (http://www.ers.usda.gov/Briefing/Soy-
beansOilCrops/). Soybean meal is extensively used for livestock
because it contains high-quality protein with an amino acid
composition complementary to corn protein. Furthermore, it is
relatively inexpensive compared to other protein sources used
for livestock. In addition to their extensive use in the animal
industry, humans also are increasingly consuming soybeans and
soy products. The use of soybeans in human food is widespread
in the Orient and Southeast Asia. For several centuries, ancient
populations have recognized the multiple health benefits of
soybean. Epidemiological studies have shown populations that
consume soybeans and soy products have lower incidences of
cancer, heart diseases, and other chronic illness (1, 2). The use
of soybeans in United States has steadily increased recently
primarily due to these well-documented health benefits.
Soybean has been recognized by regulatory authorities as one
of the “big 8” food allergens (3, 4). Soybean allergy is most
common among children nourished with soybean-based infant
formula (5). However, the incidence of soybean allergies, while
lower in adults, is still estimated at about 0.5% of the U.S.
population (6, 7). Allergic symptoms to soybean include skin,
gastrointestinal, and respiratory reactions and in some cases
anaphylaxis (6, 7). Several soybean proteins have been identified
as allergens in the molecular mass range from 7 to 71 kDa (8).
Prominent among them are the Gly m Bd 30 K, Gly m Bd 28
K, Gly m Bd 60 K, and G1 and G2 glycinin proteins (9-13).
Of the 33 identified IgE-binding allergenic proteins in soybeans,
only a limited number of proteins are responsible for a majority
of adverse reactions to soybeans (8).
Soybean 7S storage protein, the ?-conglycinin, has been
identified as one of the most allergenic proteins (14, 15). Ogawa
et al. (12) showed about 25% of soybean-sensitive Japanese
patients with atopic dermatitis developed IgE antibodies against
the R-subunit of ?-conglycinin. ?-Conglycinin is composed of
three subunits, namely, R′ (76 kDa), R (72 kDa), and ? (52
kDa) (16). These subunits share extensive amino acid sequence
homology (17). Despite the close similarity among these
subunits, the IgE antibodies from soybean-sensitive patients
failed to cross-react against the R′- and ?-subunits (18). Here,
* Address correspondence to this author at 108 Curtis Hall, USDA-
ARS, University of Missouri Columbia, MO 65211 [telephone
(573) 882-8151; fax (573) 884-7850; e-mail Hari.Krishnan@
†U.S. Department of Agriculture.
§Division of Plant Sciences.
#Division of Animal Sciences.
J. Agric. Food Chem. 2009, 57, 938–943
10.1021/jf802451g CCC: $40.75
2009 American Chemical Society
Published on Web 01/12/2009
it was demonstrated that only the R-subunit of ?-conglycinin,
but not the other two subunits, could elicit IgE antibodies in
soybean sensitive-patients with atopic dermatitis. In this study,
we employed immunoblot analysis to detect soy proteins that
elicited IgE antibodies in patients who were allergic to soybeans.
Sera from soybean-allergic patients contained IgE antibodies
that strongly reacted against several proteins including 72, 70,
52, 34, and 21 kDa proteins. The 72, 70, and 52 kDa proteins
Our results demonstrate that all three subunits of ?-conglycinin
can elicit IgE antibody in soybean-sensitive patients.
MATERIALS AND METHODS
Human Sera. Sera from five adult soybean-allergic patients with
high soybean-specific IgE levels were obtained from PlasmaLab
International (Everett, WA). Sera from two soy-sensitive patients that
contained IgE antibodies against soy proteins were a generous gift from
Dr. Michael Zeece at the University of Nebraska. Sera from individuals
with no known history of soybean allergic reactions were used as
Seed Protein Extraction and SDS-PAGE. Dry soybean cultivar
Williams 82 seeds were ground to a fine powder by mortar and pestle.
Ground seed powder (10 mg) was extracted with 1.0 mL of a solution
containing 125 mM Tris-HCl buffer, pH 6.8, 4% sodium dodecyl sulfate
(w/v), 20% glycerol (v/v), 50 µL of 2-mercaptoethanol, and 0.03 mM
bromophenol blue. Samples were heated in a boiling water bath for 5
min and clarified by centrifugation (5000g, 15 min). The supernatant
was transferred to a clean tube, and 10 µL was loaded onto a 13.5%
SDS-PAGE gel (19) using the Hoefer SE260 minigel electrophoresis
apparatus (GE Healthcare, Piscataway, NJ). After separation, the gels
were stained with 0.1% Coomassie Blue R-250.
Two-Dimensional PAGE Analysis. The procedure employed to
isolate soybean seed proteins and their fractionation by 2D PAGE
analysis has been described earlier (20). Briefly, isoelectric focusing
was performed using 13 cm IPG strips (pH 4-7) in the IPGphor System
(GE Healthcare). Following this, the proteins were separated with a
13.5% SDS-PAGE (19). The gels were fixed overnight in a solution
of 50% ethanol (v/v) and 3% phosphoric acid. After a distilled water
wash, the gels were prestained for 1 h in 34% methanol, 17%
ammonium sulfate, and 3% phosphoric acid and then stained in 0.066%
Coomassie Blue G250 (w/v).
Immunoblot Analysis. Proteins separated by SDS-PAGE were
electrophoretically transferred to a Protran nitrocellulose membrane
(Schleicher & Schuell Inc., Keene, NH). Membranes were blocked with
5% milk in Tris-buffered saline (TBS, pH 7.3) for 2 h and then
incubated in 1:500 dilution of plasma from individual adult soybean-
allergic patients (PlasmaLab International) overnight at room temper-
ature with gentle rocking. In some cases, sera from soybean-allergic
patients reacting against the 72, 70, and 52 kDa proteins were pooled
and used for immunoblot analyses. After three washings with TBS
containing 0.05% Tween-20 (TBST, 10 min/each), the membrane was
incubated for 2 h in a 1:5000 dilution of goat anti-human IgE-horseradish
peroxidase conjugate secondary antibody (Biosource, Camarillo, CA).
Following this the membranes were washed three times for 10 min
with TBST and one time with TBS. Immunoreactive polypeptides were
detected with the Super Signal West Pico enhanced chemiluminescent
substrate (Pierce Biotechnology, Rockford, IL) according to the
Matrix-Assisted Laser Desorption Ionization Time-of-Flight
Mass Spectrometry (MALDI-TOF-MS) Analysis. Protein spots
selected for mass spectrographic analysis were excised from the gels,
washed in distilled water, and destained in a 50% solution of acetonitrile
(v/v) containing 25 mM ammonium bicarbonate. Gel spots were
subjected to digestion with 20 µL (10 µg/mL) modified porcine trypsin
in 25 mM ammonium bicarbonate (Promega, Madison, WI). Resulting
peptides were analyzed with a Voyager DE-STR MALDI-TOF mass
spectrometer (Applied Biosystems, Framingham, MA). The peptides
were cocrystallized with R-cyano-4-hydroxycinnamic acid matrix. A
337 nm nitrogen laser operating at 20 Hz was used in sample ionization.
Trypsin autolysis peaks of charge mass ratios 842.51 and 2211.10 served
as internal standards. For MALDI-TOF-MS data to qualify as a positive
identification, a protein’s molecular weight search (MOWSE) score had
to equal or exceed the minimum significant score of 64.
Partial Purification and Enzymatic Deglycosylation of ?-Cong-
lycinin. The 7S ?-conglycinin globulin fraction from soybean seeds
was partially purified essentially as described by Nagano et al. (21)
and separated using a 10% preparative SDS-PAGE. Following elec-
trophoresis, the gel was briefly stained with Coomassie Blue. The 52
kDa ?-subunit of ?-conglycinin was eluted from the gel as described
(22) and deglycosylated utilizing the GlycoProfile II enzymatic N-
deglycosylation kit (Sigma, St. Louis, MO). Ten micrograms of gel-
purified ?-conglycinin was deglycosylated following the denaturing
protocol suggested. Deglycosylation was achieved with the use of
PNGase F, which removes all Asn-linked oligosaccharides, and a
combination of NANase II and O-glycosidase DS that releases all Ser/
Thr-linked Gal (?1,3)GalNAc(R1) and all sialic acid substituted
Gal(?1,3)LGalNAc(R1) from glycoproteins. The efficiency of the
deglycosylation was verified by a shift in mobility of the protein using
Expression of Recombinant ?-Subunit of ?-Conglycinin in
Escherchia coli. The coding region of the ?-subunit of ?-conglycinin
lacking the signal peptide was obtained following RT-PCR amplification
of the total seed RNA using gene specific primer pairs. The N- and
C-terminal specific primers were 5′-CATATGTTAAAGGTGAGAGAG-
GATGAGAATAAC-3′ and 5′-CTCGAGTCAGTAGAGAGCACCTAA-
GATTGAAG-3′, which included NdeI and XhoI restriction sites,
respectively, to facilitate cloning. The PCR product was purified from
an agarose gel, digested with NdeI and XhoI (Takara Mirus Bio, Inc.,
Madison, WI) and ligated into the NdeI/XhoI site of E. coli expression
vector pET 28(a)+ (Calbiochem-Novabiochem, San Diego, CA) using
the ExTaq ligase kit (Takara Mirus Bio, Inc.). The resultant plasmid,
pBBCON, was introduced into ER2566 E. coli strain (New England
Biolabs, Beverly, MA) and grown in 5 mL of Luria broth medium in
the presence of 100 µg/mL kanamycin at 37 °C. This culture was used
to inoculate 100 mL of Luria broth containing 100 µg/mL kanamycin
and grown at 37 °C. When the culture reached an optical density of
0.5 (A600nm), isopropyl-?-D-thiogalactopyranoside (IPTG) was added to
a final concentration of 1 mM, and growth was allowed to continue
overnight at 37 °C. Recombinant ?-subunit of ?-conglycinin was
purified under denaturing conditions following the manufacturer’s
suggested protocol (Calbiochem-Novabiochem). Protein concentration
was determined spectrophotometrically utilizing the DC Standard
Protein Assay Kit (Bio-Rad Laboratories, Richmond, CA).
Different Soybean Seed Proteins Elicit IgE Antibodies in
Soybean-Sensitive Patients. Western blot analysis was per-
formed to identify soy proteins that are recognized specifically
by IgE antibodies from patients who are sensitive to soybeans.
Sera from seven adult soybean-allergic patients were examined
in this analysis. As shown in Figure 1, the sera from these
individuals showed cross-reaction against several soy proteins.
Prominent among them were 72, 70, 52, 34, and 21 kDa
proteins. Of the seven sera tested, three of them showed cross-
reactivity to the 72, 70, and 52 kDa protein. Sera from two
patients showed very specific reaction to a 21 kDa protein. The
72, 70, and 52 kDa proteins were located at the same positions
in the acrylamide gels that correspond to the R′-, R-, and
?-subunits of ?-conglycinin (Figure 1). A previous study has
shown that the 70 kDa R-subunit of ?-conglycinin is a major
allergenic protein (18). The R′- (72 kDa) and ?-subunits (52
kDa), which show extensive sequence homology to the R-sub-
unit of ?-conglycinin, were not reported as allergenic proteins.
However, in our study both the 72 and 52 kDa proteins were
recognized by sera from soybean-sensitive patients, leading to
the hypothesis that all three subunits of ?-conglycinin may be
Soybean ?-Conglycinin Food AllergensJ. Agric. Food Chem., Vol. 57, No. 3, 2009
All Three Components of 7S Globulin Fractions Are
Recognized by IgE Antibodies. To examine if the 72 and 52
kDa proteins correspond to the R′- and ?-subunits of ?-cong-
lycinin, 2D gel electrophoresis was performed (Figure 2). The
three subunits of ?-conglycinin were well resolved by this
procedure. The R′- and R-subunits separated into distinct spots
with isoelectric points of 5.2 and 4.9, respectively (Figure 2A).
The ?-subunit was resolved into four distinct spots having
isoelectric points ranging from 5.6 to 6.0 (Figure 2A). Proteins
resolved by 2D gels were transferred to a nitrocellulose
membrane and incubated with pooled sera from individuals
reacting against the 72, 70, and 52 kDa soybean proteins.
Western blot analysis showed strong reaction against the two
spots corresponding to the ?-subunit and a weaker reaction
against the R′- and R-subunits (Figure 2B). From an identical
2D gel, protein spots showing cross-reaction were excised from
the gels and subjected to MALDI-TOF-MS analysis. A com-
parative search of peptides of known protein listed in the
National Center for Biotechnology Information nonredundant
database with peptides generated by trypsin digestion showed
significant homology to the R′-, R-, and ?-subunits of ?-con-
glycinin (Table 1).
Additional confirmation that the 72, 70, and 52 kDa proteins
are the three subunits of ?-conglycinin was obtained by Western
blot analysis using gel-purified components of ?-conglycinin.
First, we obtained a partially purified 7S ?-conglycinin globulin
fraction from soybean seeds essentially as described (21). This
fraction was further fractionated on SDS-PAGE, and the three
subunits of ?-conglycinin were purified from the gels (Figure
3A). The gel-purified subunits were tested by SDS-PAGE and
immunoblot analyses with pooled sera from patients containing
IgE antibodies against 72, 70, and 52 kDa soybean proteins.
This analysis clearly demonstrated that the IgE antibodies
reacted against the R′-, R-, and ?-subunits of ?-conglycinin
Removal of Oligosaccharides from the ?-Subunit of
?-Conglycinin Does Not Prevent IgE Binding. It is well-
known that the carbohydrate moieties of a glycoprotein may
be involved in IgE reactivity (23). Because the ?-conglycinin
proteins are glycoproteins, we examined the IgE reactivity
against these proteins directly against the N-linked glycans. Gel-
purified ?-subunit of ?-conglycinin was subjected to deglyco-
sylation by incubating the protein sample with glycosidases
PNGase F, NANase II, and O-glycosidase DS. Treatment with
glycosidases resulted in faster migration of the ?-subunit of
?-conglycinin in comparison to unglycosylated protein (data not
shown). IgE antibodies from patients sensitive to soy proteins
recognized both the unglycosylated and deglycosylated forms
of the ?-subunit of ?-conglycinin. Because it may be possible
that under our experimental conditions enzymatic deglycosy-
lation may be incomplete, we expressed an unglycosylated form
of the ?-subunit of ?-conglycinin as a recombinant protein in
E. coli (Figure 4A). Because E. coli does not possess the same
type of cellular machinery used for glycosylation in plants, the
?-subunit of ?-conglycinin expressed in E. coli will not be
glycosylated. Immunoblot analysis clearly showed that the IgE
antibodies from patients sensitive to soy proteins were able to
recognize the unglycosylated form of the ?-subunit of ?-con-
glycinin (Figure 4B).
Several soybean proteins have been recognized as allergens
primarily on the basis of their reactivity to IgE antibodies from
soybean-sensitive patients (24, 25). Some of these proteins can
bring about food allergies, whereas others can elicit respiratory
allergies. Three soybean seed coat allergens, Gly m 1A, Gly m
1B, and Gly m 2, are responsible for asthma outbreaks in
Barcelona, Spain (26, 27). Soybean 11S, 7S, and 2S globulin
Figure 1. SDS-PAGE and reactivity analysis of soybean seed proteins
probed with individual sera from soybean-sensitive patients. Lanes: M,
molecular weight markers; 1, soybean total seed proteins stained with
CoomassieBlue; 2-5, correspondingimmunoblot probedwithserafrom
individuals sensitive to soybean. IgE-binding proteins were detected by
Figure 2. Two-dimensional gel electrophoresis of soybean allergenic
proteins. (A) Soybean seed proteins were first separated by isoelectric
focusing using a pHgradient from4 to 7 and then by SDS-PAGE on a
12% gel. Proteins were transferred to a nitrocellulose membrane and
reacting against the 72, 70, and 52 kDa soybean proteins, and bound
and analyzed by MALDI-TOF-MS.
J. Agric. Food Chem., Vol. 57, No. 3, 2009Krishnan et al.
fractions have also been identified as food allergens. Kunitz
trypsin inhibitor is the prominent allergen in the 2S fraction
(14). The 11S glycinins, the most abundant storage protein of
soybean, are synthesized as precursor proteins and posttrans-
lationally processed into 40 kDa acidic and 20 kDa basic
subunits. Both the acidic (28, 29) and basic (10) subunits of
glycinins have been identified as major allergens. IgE immu-
noblot and amino acid sequence analysis confirmed that the basic
glycinin subunits from all five members of the glycinin gene
family are allergens (10). A previous study also reported positive
tests of IgE antibodies to the acidic subunit (40 kDa) but not to
the basic subunit (20 kDa) glycinin (29). In the current study
we did not detect IgE binding to proteins corresponding to the
40 and 20 kDa glycinin subunits. This may be due to differences
among serum sources employed in this investigation. Another
possibility is that soybean protein that is allergenic to a certain
group of individuals may not necessarily elicit the same
antigenic response in other groups of people.
The 7S globulin fraction of soybean, the ?-conglycinin,
consists of R′-, R-, and ?-subunits of approximately 72, 70, and
52 kDa, respectively, and shares extensive sequence homology
(17). Despite this homology, it was reported that only the 70
kDa R-subunit was recognized by IgE antibodies from soybean-
sensitive patients with atopic dermatitis (18). Cleavage of the
peptide bonds with CNBr and chymotrypsin and subsequent
N-terminal amino acid sequence determination of the peptides
indicated that IgE-binding sites was located between amino acid
residues 232 and 383 of the R-subunit of ?-conglycinin (18). A
comparison of the IgE-binding peptide region of R-subunit to
the corresponding regions of the R′- and ?-subunits reveals some
amino acid differences in this region. These differences were
speculated to be one possible reason for the absence of the IgE
binding to R′- and ?-subunits of ?-conglycinin (18). In contrast
Table 1. Identification of Proteins Reactive to Human SerumIgE as Subunits of ?-Conglycinin by MALDI-TOF-MS
R′-subunit of ?-conglycinin
R-subunit of ?-conglycinin
?-subunit of ?-conglycinin
256 4948358 (KV)REDENNPFYLR
aParentheses denote additional residues found on additional matched peptide.
(A) Purified subunits of ?-conglycinin were separated on a 10% SDS-
PAGEandstainedwithCoomassieBlue. (B) Proteins showninpanel A
were transferred to a nitrocellulose membrane and probed with pooled
sera fromindividuals reacting against the 72, 70, and 52 kDa soybean
anti-human IgE horseradish peroxidase conjugate.
Soybean ?-Conglycinin Food Allergens J. Agric. Food Chem., Vol. 57, No. 3, 2009
to the earlier study (18), the results presented in this investigation
clearly demonstrate that all three subunits of ?-conglycinin are
potential food allergens. The different conclusions on the
apparent allergenicity of the three subunits of ?-conglycinin may
be attributed to differences among serum sources and if the
source is from an adult or infant. Our conclusion that all three
subunits of ?-conglycinin are potential allergens is further
supported by recent studies conducted in rats. In this study, in
which rats were fed purified ?-conglycinin, it was concluded
that that dietary soybean ?-conglycinin has negative effects on
growth and immune function in rats (30). Furthermore, it was
demonstrated that the recombinant R′-subunit of soybean
?-conglycinin possesses an intrinsic immune-stimulating capac-
ity and can induce allergic reaction in Brown Norway rats (31).
These results in combination with our current study using
immunoblot analyses indicate that all three subunits of ?-con-
glycinin can be potential allergens. However, in vitro immu-
noblotting studies with commercially obtained sera may some-
times lead to ambiguous results, and therefore the potential
biological significance of IgE binding to soybean ?-conglycinin
for human soy-based allergies in vivo warrants further
We thank Nathan Oehrle for critically reading the manuscript.
Names are necessary to report factually on available data:
however, the USDA neither guarantees nor warrants the standard
of product, and the use of the name by USDA implies no
approval of the product to the exclusion of others that may be
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proteins were detected by chemiluminescence using anti-human IgE
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Received for review August 6, 2008. Revised manuscript received
November 13, 2008. Accepted December 4, 2008.
Soybean ?-Conglycinin Food Allergens J. Agric. Food Chem., Vol. 57, No. 3, 2009