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Theor Appl Genet (2010) 121:1527–1539
DOI 10.1007/s00122-010-1408-4
123
ORIGINAL PAPER
Presence of celiac disease epitopes in modern and old hexaploid
wheat varieties: wheat breeding may have contributed
to increased prevalence of celiac disease
Hetty C. van den Broeck · Hein C. de Jong · Elma M. J. Salentijn ·
Liesbeth Dekking · Dirk Bosch · Rob J. Hamer · Ludovicus J. W. J. Gilissen ·
Ingrid M. van der Meer · Marinus J. M. Smulders
Received: 13 April 2010 / Accepted: 25 June 2010 / Published online: 28 July 2010
© The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract Gluten proteins from wheat can induce celiac
disease (CD) in genetically susceptible individuals. SpeciWc
gluten peptides can be presented by antigen presenting cells
to gluten-sensitive T-cell lymphocytes leading to CD. Dur-
ing the last decades, a signiWcant increase has been
observed in the prevalence of CD. This may partly be
attributed to an increase in awareness and to improved
diagnostic techniques, but increased wheat and gluten con-
sumption is also considered a major cause. To analyze
whether wheat breeding contributed to the increase of the
prevalence of CD, we have compared the genetic diversity
of gluten proteins for the presence of two CD epitopes
(Glia-9 and Glia-20) in 36 modern European wheat
varieties and in 50 landraces representing the wheat varie-
ties grown up to around a century ago. Glia-9 is a major
(immunodominant) epitope that is recognized by the major-
ity of CD patients. The minor Glia-20 was included as a
technical reference. Overall, the presence of the Glia-9
epitope was higher in the modern varieties, whereas the
presence of the Glia-20 epitope was lower, as compared to
the landraces. This suggests that modern wheat breeding
practices may have led to an increased exposure to CD epi-
topes. On the other hand, some modern varieties and land-
races have been identiWed that have relatively low contents
of both epitopes. Such selected lines may serve as a start to
breed wheat for the introduction of ‘low CD toxic’ as a new
breeding trait. Large-scale culture and consumption of such
varieties would considerably aid in decreasing the preva-
lence of CD.
Introduction
Wheat is the third most produced cereal in the world after
maize and rice (http://faostat.fao.org/site/567/default.aspx#
ancor). It is a very important food crop for the daily intake
of proteins, vitamins, minerals and Wbers in a growing part
of the world population (Cummins and Roberts-Thomson
2009). Wheat consumption, and especially the intake of its
gluten, is, however, also a major cause of the development
of celiac disease (CD). CD is an inXammatory disorder of
the small intestine resulting in a wide variety of chronically
symptoms (diarrhea, bowel pain, headache, growth retarda-
tion, osteoporosis, infertility, lymphoma, etc.) in about 1%
of the wheat consuming world population. The prevalence
and the risk of death in undiagnosed CD have increased
dramatically during the last 50 years in the United States
(Rubio-Tapia et al. 2009). Lohi et al. (2007) described a
Communicated by J. Snape.
H. C. van den Broeck (&) · E. M. J. Salentijn · D. Bosch ·
L. J. W. J. Gilissen · I. M. van der Meer · M. J. M. Smulders
Plant Research International, Wageningen UR,
P.O. Box 16, 6700 AA Wageningen, The Netherlands
e-mail: hetty.busink@wur.nl
H. C. de Jong
Limagrain Nederland B.V., P.O. Box 1,
4410 AA Rilland, The Netherlands
L. Dekking
Leiden University Medical Center,
P.O. Box 9600, 2300 RC Leiden, The Netherlands
R. J. Hamer
Laboratory of Food Chemistry, Wageningen UR,
P.O. Box 8129, 6700 EV Wageningen, The Netherlands
Present Address:
L. Dekking
Department of Immunology, Dynomics BV,
Erasmus Medical Centre, P.O. Box 82,
1400 AB Bussum, The Netherlands
1528 Theor Appl Genet (2010) 121:1527–1539
123
doubling of the prevalence of CD in Finland in the last two
decades, which deWnitely could not be ascribed to improved
detection only. In Asia, the prevalence of CD is increasing
because of a change toward Western-style diets (Cummins
and Roberts-Thomson 2009). Changes in life style (e.g., the
increasing exclusion of breast feeding) and the time and
amount of the Wrst introduction of wheat containing prod-
ucts in early life can be considered major environmental
factors causing this increase (Ventura et al. 1999; Ivarsson
et al. 2000; Fasano 2006). Wheat consumption as wheat
Xour and wheat-based products per capita is high in Europe
and the Middle East and increasing in Asia (Rubio-Tapia
et al. 2009) and is again increasing in the United States
(http://www.ers.usda.gov/AmberWaves/september08/find-
ings/wheatflour.htm). In addition, wheat gluten is increas-
ingly applied as an additive in a wide and growing variety
of processed foods and in other products, including medi-
cines (Day et al. 2006; Hlywiak 2008; Maltin et al. 2009;
Atchison et al. 2010).
Bread wheat (Triticum aestivum) is an allohexaploid
species resulting from natural hybridization between a tet-
raploid T. turgidum (dicoccum) carrying the AB-genome
and a wild diploid species Aegilops tauschii carrying the
D-genome (Gupta et al. 2008, and references therein).
Especially, the introduction of the D-genome improved the
bread-making properties (Payne et al. 1981a; Payne 1987;
Gupta and MacRitchie 1994; Branlard et al. 2001). Over
100 years ago, breeders started to systematically cross and
select bread wheat for higher yields, adaption to climate
changes, better bread-making characteristics, and improved
disease resistance. Little information is available about the
breeding history of landraces on these aspects (Zeven
2000). Breeding has resulted in many thousands of diVerent
wheat varieties that are stored in genetic resource centers
and breeding company stocks. Modern wheat breeding,
focusing on the increase of yield, initially narrowed down
the genetic base of its germplasm (Leinová et al. 2007).
However, genetic diversity has increased again in the set of
varieties released since the 1990s because breeders started
to use wild relatives and synthetic wheats for introgression
of, among others, disease resistances (Van de Wouw et al.
2010). The net eVect was neither a decrease nor an increase
of genetic diversity, as measured by neutral genetic mark-
ers, over the last century. However, this does not exclude
the possibility that diversity in some traits may have been
reduced or increased. Notably, for prolamins, landraces can
contain many diVerent chemotypes in a single population
(Damania et al. 1983), but modern varieties have only a
small number of diVerent gene combinations for some of
the prolamins (Payne et al. 1981b). This raises the impor-
tant question about possible breeding-induced diVerences
in the presence of T-cell stimulatory epitopes in modern
varieties compared to landraces and older varieties.
In wheat, gluten proteins comprised gliadins and glute-
nins, which are present in approximately equal amounts and
form 80% of the total storage protein content in the wheat
kernel, next to albumins (12%) and globulins (8%). The
gliadins form a large protein family in which /-, -, and
-gliadins can be distinguished (Woychik et al. 1961),
whereas the glutenins can be subdivided into low-molecu-
lar weight glutenin subunits (LMW-GS) and high-molecu-
lar weight glutenin subunits (HMW-GS) (Shewry and
Tatham 1999). The high proline and glutamine content
makes gluten proteins resistant to complete proteolytic
digestion (Hausch et al. 2002; Shan et al. 2002, 2004). Glu-
ten peptides resulting from partial digestion of all gluten
protein groups (/-, -, -gliadins, LMW-GS, and HMW-
GS) may contain T-cell stimulatory epitopes (Koning 2008;
Stepniak et al. 2008), but the epitopes from the -gliadins
are considered to have by far the highest clinical relevance
with regard to both the adaptive immune response and the
innate immune response that lead to the development of CD
(Sjöström et al. 1998; Arentz-Hansen et al. 2000a, b, 2002;
Anderson et al. 2000; Janatuinen et al. 2002; Vader et al.
2002; Maiuri et al. 2003; Molberg et al. 2003; Schuppan
et al. 2003; Qiao et al. 2005; Marti et al. 2005; Camarca
et al. 2009).
In the present paper, we use two monoclonal antibodies
(mAbs) that were raised against the Glia-9 and Glia-20
epitopes (Spaenij-Dekking et al. 2004, 2005; Mitea et al.
2008b) for comparison of the presence of T-cell stimulatory
epitopes in gluten protein extracts from diVerent wheat
landraces and modern varieties. The Glia-9 epitope is
especially known as a major immunodominant epitope that
can be recognized by the majority of CD patients (Vader
et al. 2002; Camarca et al. 2009). The Glia-9 epitope
sequence (I) is part of the proteolytic-resistant 33-mer in
-gliadins that has a strong T-cell stimulatory eVect (Shan
et al. 2002; Shan et al. 2005). The Glia-20 epitope, which
is used in this study as a technical reference, is a minor epi-
tope that is recognized by a minority of patients. For these
epitopes, proper mAbs are available. Unfortunately, not all
epitopes can be studied properly with existing antibodies,
and the consequence of the shorter epitope recognition site
of the mAbs compared to the T cell recognition site might
be over-staining, which would result in overestimation of
the toxicity.
Recent research using protein extracts from a limited
selection of wheat varieties demonstrated a large variation
in immune responses, as measured as epitope-speciWc T-cell
responses or in mAb binding studies (Molberg et al. 2005;
Spaenij-Dekking et al. 2005). In the present study, we set
out to compare the occurrence of T-cell stimulatory gluten
epitopes of modern European varieties with landraces to
determine to what extent breeding might have changed the
presence of T-cell stimulatory epitopes in wheat. The
Theor Appl Genet (2010) 121:1527–1539 1529
123
results will be discussed in view of the question to what
extent wheat breeding can contribute to the prevalence of
CD.
Materials and methods
Search for the occurrence of sequences recognized
by mAbs and T-cell epitopes
The frequency of known T-cell stimulatory epitope
sequences in deduced -gliadin proteins was analyzed
using the expressed sequence tag (EST) sequences obtained
from hexaploid varieties Lavett and Baldus as described by
Salentijn et al. (2009). The deduced -gliadin sequences
were analyzed for the diVerent minimal recognition
sequences of mAbs and T-cells for Glia-9 (QPFPQPQ and
PFPQPQLPY, respectively) and Glia-20 (RPQQPYP and
FRPQQPYPQ, respectively) (Spaenij-Dekking et al. 2004,
2005; Mitea et al. 2008a, b). No mismatches were allowed.
Grain samples
A set of 36 modern hexaploid wheat varieties, available for
the European market, were obtained from Limagrain,
Lelystad, The Netherlands. The varieties were selected for
maximum Glu-3 and Gli-1 diversity, in the genetic back-
ground of the most frequent composition of HMW-GS alle-
les (Glu-A1: 0 or 1; Glu-B1: 7 + 9; Glu-D1: 5 + 10;
Table 1), based on allozyme patterns that are routinely
produced for wheat varieties. The set of landraces
(Table 2) was obtained from the Centre for Genetic
Resources (CGN), the Netherlands (http://www.cgn.wur.nl/
uk/), except for accession RICP01C0203330, which was
obtained from the Research Institute of Crop Production
(RICP), Czech Republic. The accessions were selected
based on collection period and diversity of geographic
origin.
Gluten protein extraction
For extraction of gluten proteins from wheat, we used the
method as described by Van den Broeck et al. (2009a),
which combines three sequentially obtained extracts to
extract nearly all gliadins and glutenins. Extraction of the
residue left after the third extract with 25 mM Tris/HCl (pH
8.0) containing 2% SDS and with 25 mM Tris/HCl (pH
8.0) containing 2% SDS and 1% DTT showed that only
some HMW-GS and probably some omega-gliadins/D-type
LMW-GS were still present in the sample. The gluten
extracts analyzed in this study, therefore, contained most of
the gluten proteins present.
Wheat grains were ground in an analytical mill (A 11
Basic, IKA-Werke) and sieved through mesh (0.5 mm).
Gluten proteins were extracted from 50 mg wheat Xour by
addition of 0.5 ml of 50% aqueous iso-propanol with con-
tinuous mixing (MS1 Minishaker, IKA Works, Inc.) at
1,000 rpm for 30 min at room temperature, followed by
centrifugation at 10,000 rpm for 10 min at room tempera-
ture. The residue was re-extracted twice with 50% aqueous
iso-propanol/1% DTT/50 mM Tris–HCl, pH 7.5, for
30 min at 60°C with mixing every 5–10 min followed by
centrifugation at 10,000 rpm for 10 min at room tempera-
ture. After addition of each next extraction solution, the res-
idue was resuspended by shaking in a Fastprep® FP220A
Instrument for 10 s at speed 6.5 m/s followed by soniWca-
tion for 10 min in an ultrasonic bath (Branson 3510, Branson
Ultrasonics Corporation). The three obtained superna-
tants were combined and considered the gluten protein
extract. The protein content was quantiWed using Biorad
Protein Assay (Bio-Rad Laboratories), based on the
Bradford dye-binding procedure, according to manufacturer’s
instruction.
SDS-PAGE and immunoblotting
Equal amounts of gluten proteins were loaded and were
separated on SDS-PAGE gels (10%) (Laemmli 1970)
using a Hoefer SE 260 mighty small II system (GE
Healthcare). Proteins were blotted onto nitrocellulose
(0.2 m, Bio-Rad Laboratories), omitting methanol from
the blotting buVer, using a Mini Trans-Blot Cell (Bio-
Rad Laboratories) at 100 V for 1 h. Blots were incubated
as described by Cordewener et al. (1995) using mAbs
speciWc for T-cell stimulatory epitopes Glia-9 (Mitea
et al. 2008b), Glia-20 (Mitea et al. 2008a, b), Glt-156
(LMW-1 and LMW-2) (Spaenij-Dekking et al. 2005;
Mitea et al. 2008b), and HMW-glt (Spaenij-Dekking
et al. 2004; Mitea et al. 2008b). Antibody binding to the
blots was visualized by staining for alkaline phosphatase-
conjugated secondary antibody, using Nitro Blue tetrazo-
lium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate
(BCIP) (Sigma). The gluten protein extract of ‘Toronto’
was used on each separate immunoblot as an ‘inter-gel’
control.
Blots were scanned using a Bio-Rad GS-710 Calibrated
Imaging Densitometer (Bio-Rad Laboratories) and saved as
TIFF images. Pixel intensities were calculated per lane
using Quantity One software (Bio-Rad Laboratories). Rela-
tive intensities diVered speciWcally per mAb used, but were
normalized to values obtained for the ‘inter-gel’ control.
Intensities per lane were categorized into three classes: low
(+), medium (++), and high (+++) according to linear sub-
division of the maximum intensities.
1530 Theor Appl Genet (2010) 121:1527–1539
123
Two-dimensional gel electrophoresis (2-DE)
For 2-DE, gluten proteins were separated in the Wrst
dimension by IEF. Immobiline Drystrips pH 3–10 of 7 cm
(GE Healthcare) were rehydrated overnight with 5 g pro-
tein in rehydration buVer (6 M urea, 2 M thio-urea, 2%
CHAPS, 20 mM DTT) complemented with 0.5% IPG
buVer pH 3–10 (GE Healthcare) to reach a Wnal volume of
125 l, according to manufacturer’s instructions. The
rehydrated strips were focused on an IPGphor (GE Health-
care) using the following conditions: 300 V during 30 min,
gradient to 1,000 V in 30 min, gradient to 5,000 V in 1 h
20 min, step and hold at 5,000 V until 6,500 Vh. Prior to
second dimension, strips were equilibrated for 15 min in
equilibration buVer (6 M urea, 30% glycerol, 2% SDS,
50 mM Tris–HCl, pH 8.8) containing 1% (w/v) DTT, fol-
lowed by 15 min in 5 ml equilibration buVer containing
2.5% (w/v) iodoacetamide. Separation in the second
dimension was performed using SDS-PAGE gels (10%)
and the SE 260 mighty small II system (GE Healthcare).
Gels were used for immunoblotting or stained with Page-
Blue™ (Fermentas).
Table 1 Details of modern
wheat varieties Variety Type Quality
group
Glu-1Glu-3Gli-1Year
of release
Country
of origin
1 Kornett W A 0, 7 + 9, 5 + 10 f, g, c f, f, b 1997 Germany
2 Eta S B 1, 7 + 9, 5 + 10 f, f, c f, g, b 1986 Poland
3 Thasos S E 1, 7 + 9, 5 + 10 f, f, c a, f, g 1994 Germany
4 Triso S E 1, 7 + 9, 5 + 10 f, c, c f, f, b 1996 Germany
5 Star S A 0, 7 + 9, 5 + 10 f, c, c a, f, g 1986 Germany
6 Ramiro W A 1, 7 + 9, 5 + 10 f, c, a f, b, g 1969 Germany
7 Bovictus W B 0, 7 + 9, 5 + 10 e, j, c b, l, b 1993 Germany
8 Combi S E 0, 7 + 9, 5 + 10 e, g, c f, f, l 1990 Germany
9 Zentos W E 0, 7 + 9, 5 + 10 e, g, c a, f, g 1989 Germany
10 Naxos S A 1, 7 + 9, 5 + 10 e, g, c a, c, a 1992 Germany
11 Astron W A 0, 7 + 9, 5 + 10 d, g, c o, f, b 1989 Germany
12 Belisar W A 0, 7 + 9, 5 + 10 d, g, c o, e, a 1995 Germany
13 Glockner W E 0, 7 + 9, 5 + 10 d, g, c l, b, b 1993 Germany
14 Sperber W A 0, 7 + 9, 5 + 10 d, g, c a, h, d 1982 Germany
15 Rektor W E 1, 7 + 9, 5 + 10 d, g, c a, f, a 1980 Germany
16 Tinos S E 1, 7 + 9, 5 + 10 d, f, c a, f, g 1993 Germany
17 Trakos W B 0, 7 + 9, 5 + 10 d, c, c b, f, b 1997 Germany
18 Toronto W A 0, 7 + 9, 5 + 10 a, j, c b, l, b 1990 Germany
19 Tambor W A 0, 7 + 9, 5 + 10 a, h, c b, f, b 1993 Germany
20 Aristos W A 0, 7 + 9, 5 + 10 a, g, c f, f, d 1997 Germany
21 Pegassos W A 1, 7 + 9, 5 + 10 a, g, c f, f, a 1996 Germany
22 Bussard W E 1, 7 + 9, 5 + 10 a, g, c f, c, b 1990 Germany
23 Mikon W B 1, 7 + 9, 5 + 10 a, g, c f, b, l 1988 Germany
24 Winni W B 1, 7 + 9, 5 + 10 a, g, c b, f, d 1998 Germany
25 Klaros S A 1, 7 + 9, 5 + 10 a, g, c a, f, b 1995 Germany
26 Quattro S A 1, 7 + 9, 5 + 10 a, g, c a, f, a 1995 Germany
27 Batis W A 1, 7 + 9, 5 + 10 a, g, c a, e, l 1994 Germany
28 Aron W E 0, 7 + 9, 5 + 10 a, g, c a, e, i 1992 Germany
29 Munk S A 0, 7 + 9, 5 + 10 a, g, c a, e, g 1993 Germany
30 Ambras W A 1, 7 + 9, 5 + 10 a, g, c a, b, k 1990 Germany
31 Urban W E 0, 7 + 9, 5 + 10 a, g, c a, b, i 1980 Germany
32 Ralle S E 1, 7 + 9, 5 + 10 a, c, c a, f, b 1984 Germany
33 Bold W B 0, 6 + 8, 5 + 10 d, c, c o, s, i 1996 Germany
34 Borenos W E 0, 7 + 9, 2 + 12 a, d, a f, h, g 1987 Germany
35 Dakota W B 1, 7 + 8, 2 + 12 e, j, a m, l, a 1996 Germany
36 Cadenza S B 0, 14 + 15, 5 + 10 a, h, c a, d, b 1992 UK
Allele identiWcation for Glu-1,
Glu-3, and Gli-1 loci is
according to Jackson et al.
(1996). W is winter wheat.
S is spring wheat. Quality group
according to the German classi-
Wcation (German Federal OYce
for Plant Varieties): E is Elite
wheat, A is Quality wheat,
and B is Bread wheat
Theor Appl Genet (2010) 121:1527–1539 1531
123
Table 2 Details of landrace accessions
Passport details are from CGN, Wageningen, The Netherlands. A is advanced cultivar; L is landrace/traditional cultivar; I is intermediate; S is
spring wheat; W is winter wheat
Accession no. Accession name T. aestivum
subspecies
Population
type
Type Collection
date
Origin
1 RICP01C0203330 Arcade aestivum AS Belgium
2 CGN19307 Minaret aestivum A S 1982 The Netherlands
3 CGN04210 Weissahr Rotkorn Binkel compactum L S 1951 Italy
4 CGN08510 Hilgendorf aestivum A S 1947 New Zealand
5 CGN08315 Rouge de la Gruyere compactum LS Switzerland
6 CGN12393 Sappo aestivum A S 1969 Sweden
7 CGN19285 Baldus aestivum A S 1990 The Netherlands
8 CGN08025 Sinde aestivum L S 1972/1973 Ethiopia
9 CGN10640 – compactum L S 1974 Pakistan
10 CGN05620 – aestivum L W Afghanistan
11 CGN05359 Alty Agac Mestnaja aestivum LW Armenia
12 CGN12271 Kamtschatka Mestnaja compactum L S 1964 Armenia
13 CGN05414 Chyrda Bugda Mestnaja aestivum L W Azerbaijan
14 CGN06118 Gomborka aestivum L S 1959 Azerbaijan
15 CGN08330 – compactum ? W Azerbaijan
16 CGN08306 – spelta L S Azerbaijan
17 CGN08309 – spelta L S Azerbaijan
18 CGN05500 – aestivum L W 1955 Iran
19 CGN06440 Tritaeg 1 aestivum L S 1955 Iran
20 CGN08274 Weisser Kolbenspelz spelta LS Germany
21 CGN12269 Iran 404c spelta L S Iran
22 CGN12270 Iran 416a spelta L S Iran
23 CGN04065 Bagdad aestivum L S 1902 Iraq
24 CGN06150 Humera aestivum L S Iraq
25 CGN06162 Iraqua aestivum L S 1969 Iraq
26 CGN08327 Irak compactum ? S 1964 Iraq
27 CGN06183 – aestivum L S 1969 Israel
28 CGN04191 – compactum L S 1967 Israel
29 CGN09728 – aestivum L S 1981 Pakistan
30 CGN12071 – aestivum L S 1976 Pakistan
31 CGN10639 – compactum L S 1974 Pakistan
32 CGN11461 BraunerSpelzAusSchettlenz spelta LW Germany
33 CGN04041 Ak 702 aestivum L S 1963 Turkey
34 CGN04267 Ak Guzluk aestivum L W 1948 Turkey
35 CGN05392 Bolu aestivum L W 1948 Turkey
36 CGN10653 Turk 169 aestivum LI Turkey
37 CGN04345 Galickaja aestivum L W USSR
38 CGN05541 Krymka aestivum L W USSR
39 CGN06033 Artemovka/Lutescens 1418 aestivum A S 1945 USSR
40 CGN04042 Akagomughi/Akakomugi aestivum L S 1960 Japan
41 CGN04275 Alton, Ghirka winter aestivum L W 1900 USSR
42 CGN05465 Gelderse Ris aestivum L W <1900 The Netherlands
43 CGN04381 Lammas Red aestivum LW UK
44 CGN04080 Chiddam aestivum L S 1863 France
45 CGN04164 Noe aestivum L S 1880 France
46 CGN04087 Chul Bidaj; Idaho Hard aestivum L S 1902 USSR
47 CGN06361 Red Fife aestivum L S 1900 USA
48 CGN04236 Pyrothrix 28 aestivum A S 1973 USSR
49 CGN05640 Rode Dikkop aestivum L W <1901 The Netherlands
50 CGN11465 Spelt van Hoosterhof spelta LW Belgium
1532 Theor Appl Genet (2010) 121:1527–1539
123
Tandem mass spectrometric analysis and data analysis
Protein bands were excised from SDS-PAGE gel and
digested with chymotrypsin (Boehringer Mannheim).
Digested peptide mixtures were separated and analyzed by
electrospray tandem mass spectrometry (LTQ-Orbitrap,
Thermo Fisher ScientiWc) as described by Van Esse et al.
(2008). Proteins were identiWed by automated database
searching (OMSSA, http://pubchem.ncbi.nlm.nih.gov/omssa/)
against the T. aestivum protein sequence database.
Results
Search for frequency of epitope sequences
Monoclonal Ab staining, which is much easier than T-cell
testing, is currently the only way to perform a comparative
study such as carried out here for a large number of varie-
ties and landraces. It is currently also the only tool to quan-
tify for CD epitopes in breeding programs. One worry
could be that, as a consequence of the shorter epitope rec-
ognition site of the mAb, staining with the mAbs will not
reveal all T-cell epitopes present, while other sites may be
stained that do not represent complete epitopes.
To evaluate if there is an under- or overestimation of CD
epitopes when using mAbs, we analyzed the frequency of
minimal recognition sequences of mAbs and T-cells for
Glia-9 and Glia-20 in deduced -gliadin proteins encoded
by EST sequences obtained from hexaploid varieties Lavett
and Baldus. The numbers of protein sequences that contain
the various sequences recognized by Glia-9 and Glia-20
mAbs and T-cells are shown in Table 3. Sequences
obtained from varieties Lavett and Baldus (Salentijn et al.
2009) contained the sequences recognized by either Glia-9
(mAb and T-cell) or Glia-20 (mAb and T-cell) or both.
Five sequences obtained from the D-genome of ‘Baldus’
contained the T-cell epitope sequence for Glia-9 but only
three contained the Glia-9 mAb sequence. On the other
hand, a single sequence from ‘Lavett’ (B-genome) and two
sequences from ‘Baldus’ (B-genome) were obtained that
contained the Glia-9 mAb sequence but not the corre-
sponding T-cell sequence. The fact that -gliadins encoded
by the B-genome do not contain sequences recognized by
the Glia-9 and Glia-20 T-cells was consistent with the
results of Van Herpen et al. (2006).
As we calculated, over 92% of the found mAb epitopes
were recognized by the corresponding T-cells (Table 3),
which means that there is a small overestimation of the CD
epitopes in a cultivar using mAb staining compared to T-cell
testing.
To analyze whether -gliadin epitopes are present in
other protein than only -gliadins, the NCBI protein data-
base (http://www.ncbi.nlm.nih.gov/sites/entrez?db=protein)
was screened for sequences that could be recognized by the
Glia-9 and Glia-20 mAbs and T-cells. The search showed
that -gliadins and -gliadins/D-type LMW-GS exist that
contain the Glia-9 mAb recognition sequence, although no
corresponding T-cell recognition sequence was present in
these -gliadins and -gliadins/D-type LMW-GS. For the
wheat lines from which the NCBI protein database sequences
have been derived, this would lead to overestimation of
Glia-9 epitopes using antibody staining. No -gliadins and
-gliadins/D-type LMW-GS were found that contain
sequences recognized by the Glia-20 mAb or T-cells.
SDS-PAGE and immunoblotting
The gluten protein content in the grains of 36 modern wheat
varieties was on average 24.4 §3.6 g/mg Xour. In grains
from landrace accessions, the average gluten protein content
was 30.9 §3.4 g/mg Xour. The diVerence in protein con-
tent is most likely caused by the diVerent environments in
which the varieties were grown. A higher amount of starch in
combination with a stable total amount of protein leads to a
reduction of the gluten protein content per mg of Xour.
The wheat varieties and landraces were analyzed by
immunoblotting using mAbs against T-cell stimulatory epi-
topes Glia-9 and Glia-20 to detect the number of proteins
present containing these epitope sequences. Equal amounts
of protein were loaded on the gels.
Modern varieties
Most modern varieties showed a number of the same
(or very comparable) staining patterns for both mAbs
Table 3 Search results for Glia-9 and Glia-20 epitope sequences
present in deduced -gliadin sequences from hexaploid varieties Lavett
and Baldus (Salentijn et al. 2009)
The number in each cell represents the presence of the recognized
sequences by mAb or T-cell. The symbol ‘–’ in a cell means that the
sequence was not present
Variety Genome Glia-9 Glia-20 Glia-9+
Glia-20
mAb T-cell mAb T-cell mAb T-cell
Lavett A (n=4)442222
B (n=1)1–––––
D (n=7)665555
Baldus A (n=2)221111
B (n=7)2–––––
D (n=5)355535
Total
(n=26)
18 17 14 13 11 13
Theor Appl Genet (2010) 121:1527–1539 1533
123
against Glia-9 and Glia-20 epitopes (Fig. 1a, b), but dis-
tinct patterns were obtained for ‘Kornett’ (Table 1, no. 1),
‘Bovictus’ (Table 1, no. 7), ‘Trakos’ (Table 2, no. 17),
‘Toronto’ (Table 1, no. 18), ‘Tambor’ (Table 1, no. 19),
‘Bold’ (Table 1, no. 33), ‘Dakota’ (Table 1, no. 35), and
‘Cadenza’ (Table 1, no. 36).
Some distinct blocks of bands were visible that are iden-
tical in several cultivars. Using the Glia-20 mAb, two
closely linked proteins assigned as -gliadins/D-type
LMW-GS were stained, which we will refer to as ‘block-1’
proteins (Fig. 1b). The molecular masses of these two
linked proteins were »55 kDa. This block occurred in 26
modern wheat varieties. Ten varieties had two proteins of
»60 kDa, probably linked -gliadins/D-type LMW-GS,
which can also be stained with the Glia-9 mAb (Fig. 1a).
The lower one of these two bands from variety Bovictus
(Table 1, no. 7) was characterized by LC–MS/MS and
showed 54.6% protein coverage with a D-type LMW-GS
from T. aestivum (CAR82265) and 29.6% protein coverage
with an -gliadin from T. aestivum (AAG17702) (results
not shown). Within those two sequences, no recognition
sequence is present for the Glia-20 mAb, with which the
protein bands gave a clear signal. However, among the
peptide sequences that were identiWed by LC–MS/MS,
sequences were obtained that carry the Glia-20 mAb and
T-cell epitope sequence. Until now, only a few -gliadin
and D-type LMW-GS proteins have been sequenced
because they are diYcult to clone due to the presence of
large repetitive domains (Hassani et al. 2008). Molecular
masses of 1B- and 1D-encoded -gliadins are often overes-
timated by SDS-PAGE compared to mass spectrometry
(DuPont et al. 2000). These -gliadins are encoded by two
closely linked genes on the Gli-D1 locus (Kasarda et al.
1983). From studies on ‘Chinese Spring’ deletion lines
(Van den Broeck et al. 2009b), we know that these proteins
are absent in plants in which parts of the short arm of chro-
mosome 1D were deleted. In contrast, no -gliadins react-
ing with mAb Glia-9 and Glia-20 were removed in lines
having deletions of the short arms of chromosome 1A or
1B. Our epitope staining data support earlier results (Ensari
et al. 1998; Denery-Papini et al. 1999; Camarca et al. 2009)
showing that -gliadins may contain epitopes that are
involved in gluten-sensitive response of CD patients.
When stained by the Glia-20 mAb, the three varieties
Bovictus (Table 1, no. 7), Toronto (Table 1, no. 18), and
Dakota (Table 1, no. 35) showed another block of tenta-
tively linked gluten proteins between 42 and 50 kDa
(Fig. 1b, referred to as ‘block 2’) that were also stained by
Fig. 1 Analyses of g luten protein extracts from 36 modern wheat vari-
eties and hexaploid landrace accessions. Immunoblots modern varie-
ties using amAb Glia-9, bmAb Glia-20. Immunoblots landraces
using cmAb Glia-9, dmAb Glia-20. Boxes indicate ‘block-1’ and
‘block-2’ proteins
1534 Theor Appl Genet (2010) 121:1527–1539
123
the Glia-9 mAb. This indicates that these proteins con-
tained both epitope sequences. The ‘block-2’ proteins are
also detected in immunoblot analyses of CIMMYT syn-
thetic hexaploid wheat lines (results not shown). The pro-
teins are not visible in the tetraploid parent lines and occur
independently of the ‘block-1’ gluten proteins. Hence, they
are most likely encoded by chromosome 1D.
The epitope sequence search revealed that deduced
-gliadin sequences contained both sequences recognized
by the Glia-9 mAb and the Glia-20 mAb (Table 3). The fact
that -gliadin sequences can contain sequences recognized
by both mAbs is conWrmed by the results in Fig. 2, which
shows gluten proteins containing either the sequence recog-
nized by the Glia-9 mAb, the Glia-20 mAb or both.
Overall, most of the -gliadins contain the Glia-9 epitope
and only few, depending on the variety, contain just the
Glia-20 epitope.
Other mAbs that were used in screening were raised
against epitopes from HMW-glutenin and LMW-GS
(Glt-156). Immunoblotting showed that all HMW-GS present
in our modern varieties (a limited set: Glu-A1: 0 or 1;
Glu-B1: 7 + 9; Glu-D1: 5 + 10; Table 1) stained with the
mAb against the HMW-Glt epitope (not shown). This was
conWrmed by a NCBI database search in which all full-size
HMW-GS were shown to contain the HMW epitope. The
T-cell epitope from LMW-GS (Glt-156) is covered by two
mAbs. Both mAbs were used in screening (data not
shown). The mAb covering the N-terminal part as well as
the antibody against the C-terminal part of the T-cell epi-
tope resulted in one to three bands appearing on immuno-
blots of which mostly one, and sometimes two, were
overlapping. Both epitopes are not considered major epi-
topes.
The existing mAbs against Glia-1, unfortunately, only
recognize two amino acids of the T-cell epitope (Mitea
et al. 2008b) and were therefore not used for screening
for the Glia-1 epitope, which is the most important of
the -gliadin epitopes (Salentijn et al., in prep).
2-DE
When gluten proteins from ‘Bovictus’ (Table 1, no 7) and
‘Sperber’ (Table 1, no. 14) were separated in more detail by
2-DE, a single band on an immunoblot often yielded more
than one protein spot (Fig. 3). Because of the complexity of
protein bands, care has to be taken in assigning protein
bands directly as alleles to varieties, based on one-dimen-
sional SDS-PAGE protein patterns alone.
Landraces
The results for the landraces showed much more diverse
protein patterns for proteins that bind Glia-9 or Glia-20
mAb (Fig. 4a, b). The two closely linked -gliadins/D-type
LMW-GS were also present in the landraces (boxed pro-
teins in Fig. 1c, d). In some landraces, both the higher and
lower molecular mass -gliadins/D-type LMW-GS were
present. This may be caused by genetic heterogeneity
within the accessions that were obtained from a Genebank,
since several kernels were combined for protein extraction.
The ‘block-2’ gluten proteins were not found in any of the
landraces we have tested.
Fig. 2 Images represent immunoblot results for modern wheat varie-
ties Bovictus, Combi, Zentos, Glockner, Toronto, Tambor, Winni, and
Bold. aRed channel shows the results with mAb Glia-9. bGreen
channel for results with mAb Glia-20. cOverlay of both images in
(a)and (b). In yellow, identical gluten protein bands are shown
a
b
c
kDa
Bovictus
Combi
Zentos
Glockner
Toronto
Tambor
Winni
Bold
97.4
66.2
45.0
31.0
97.4
66.2
45.0
31.0
97.4
66.2
45.0
31.0
Theor Appl Genet (2010) 121:1527–1539 1535
123
Fig. 3 2-DE analysis of the
gluten protein extracts from
modern wheat varieties Bovictus
and Sperber. a2-DE gel of
‘Bovictus’ stained with
PageBlue. bImmunoblot of
‘Bovictus’ using mAb Glia-9.
cImmunoblot of ‘Bovictus’
using mAb Glia-20.
dImmunoblot of ‘Sperber’
using mAb Glia-20. Lanes at
the left in (b), (c), and (d)are
one-dimensional immunoblots.
Boxes indicate ‘block-1’ and
‘block-2’ proteins
b
dc
a
Block 1
Block 2
310
Fig. 4 Immunoblots from Fig. 1 were scanned and the relative intensities are shown for mAbs Glia-9 and Glia-20. aGlia-9 in modern wheat
varieties, bGlia-20 in modern wheat varieties, cGlia-9 in wheat landraces, and dGlia-20 in wheat landraces
Modern varieties mAb Glia-α9
1
2
3
4
5
6789
10
11
1213
14
15
1617
18
19
20
21
22
2324
25
2627 28 29
30
31
32
33
34
35
36
0
760
1520
2280
relative intensities
Landraces mAb Glia-α9
1
2
3
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
39
40
41
42
43
44
45
46
47
48
49
50
38
4
0
760
1520
2280
relative intensities
Modern varieties mAb Glia-α20
1
2
345
6
7
8
9
10
11
12
13
14 15
16 17
19
20
21
22
23 24
25 26
27
28 29
30
31
32
33
34
35
36
18
0
55
110
165
relative intensities (x10
2
)
Landraces mAb Glia-α20
1
2
356
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
4
0
55
110
165
relative intensities (x10
2
)
a
b
c
d
1536 Theor Appl Genet (2010) 121:1527–1539
123
Immunoblot intensities
Immunoblots were scanned from Figs. 1a, b, c, and d and
the pixel intensity of each lane was calculated. Our results
indicate that -gliadins/D-type LMW-GS may contain
T-cell stimulatory epitopes Glia-9 and Glia-20. For this
reason, these bands have been included in the pixel intensity
measurements of the variety and landrace gel lanes to quan-
tify the overall intensity as presented in Fig. 4. In addition,
also signals caused by response of -gliadins to the Glia-9
mAb have been included in the intensity measurements.
Although we did not Wnd an intact epitope in our database
analysis, the set of -gliadin sequences present in the NCBI
database might be too limited to exclude the possibility of
the presence of the Glia-9 T-cell epitope. We sorted the
intensities for both mAb stainings in three classes: low (+),
medium (++), or high (+++) (Fig. 4). Clear diVerences in
the immunoblot staining of Glia-9 and Glia-20 epitopes
were found among and between varieties and landraces as
groups. The calculation of relative intensities is used to
measure the level of CD epitopes in the diVerent wheat sam-
ples. It is, at the moment, the most accurate way to compare
the presence and levels of CD epitopes in wheat, as it takes
into account the fact that there are diVerences in relative
expression levels and that some gluten proteins may contain
multiple copies of an epitope. Furthermore, it does not rely
on the daunting task of fully separating all gluten protein
variants into individual spots on a 2-DE gel.
From the modern varieties, Cadenza (Table 1, no. 36)
showed the lowest response to both mAbs. Among the
landraces, CGN08327 (Table 2, no. 26) showed the lowest
response to both mAbs. Other landraces showing low
response to the Glia-9 mAb (but medium to the Glia-20
mAb) are ‘Minaret’ (CGN19307, Table 2, no. 2, advanced
cultivar), ‘Weissahr Rotkorn Binkel’ (CGN04210, Table 2,
no. 3), ‘Rouge de la Gruyere’ (CGN08315, Table 2,
no. 5), CGN12071 (Table 2, no. 30), and ‘Pyrothrix 28’
(CGN04236, Table 2, no. 48, advanced cultivar).
No systematic diVerences were observed between spring
and winter wheat varieties based on the relative intensities for
both mAbs. Among the landraces, the accessions classiWed
as subspecies compactum and spelta did not diVer systemati-
cally from the others. In addition, landraces could not be
grouped according to their country or region of origin. This
may be partly due to because of the fact that the recorded
country is the country of the Wrst Genebank collection, which
often may not be the country where it originated from.
Discussion
Looking back over the last Wve decades, several trends
are apparent in wheat consumption: an increase in wheat
consumption per capita (Rubio-Tapia et al. 2009) (http://www.
ers.usda.gov/AmberWaves/september08/findings/wheatflou r.
htm), an increase in CD-related T-cell stimulatory epitopes
in wheat (as for the major epitope Glia-9, this paper), an
increase in the use of gluten in food processing (Day et al.
2006; Atchison et al. 2010), and an increase in the con-
sumption of processed foods. To some extent this can be
attributed to an increase in awareness and improved diag-
nostic techniques. Given the relation between incidence of
CD and exposure to cereals, it cannot be ruled out that an
increased content of T-cell stimulatory epitopes has also
contributed to this increased prevalence.
A diet based on wheat varieties reduced in T-cell stimu-
latory epitopes may help in the prevention of CD, as it has
been observed that the amount and duration to gluten expo-
sure are associated with the initiation of CD (Ventura et al.
1999; Ivarsson et al. 2000; Fasano 2006). Wheat gluten
proteins determine the elasticity and viscosity of the dough
(Shewry and Tatham 1997; Branlard et al. 2001), but they
also showed to be highly useful in food processing. Gluten
is widely applied in the production of soups, sauces, meat
products, potato chips, candies, ice creams, and even in
medicines, vitamin supplements, etc. Wheat varieties with
very low amounts of T-cell stimulatory epitopes may be
tolerated by many CD patients (Janatuinen et al. 2002;
Vader et al. 2003). The reduction of the amount of major
T-cell stimulatory epitopes in food will especially beneWt
children, in which the onset of CD may be delayed or even
prevented, and in non-diagnosed CD patients (the vast
majority of all CD patients) to strongly reduce their symp-
toms. This means that breeding for wheat with considerably
reduced T-cell stimulatory epitopes is to be considered as a
serious option.
This study explored diVerences in the presence of T-cell
stimulatory gluten epitopes between modern wheat varie-
ties and landraces, aiming at breeding strategies for the
reduction of T-cell stimulatory epitopes in gluten-contain-
ing food products. Immunoblotting was used to compare 36
modern wheat varieties and 50 landraces. Monoclonal Abs
speciWc for the T-cell stimulatory epitopes Glia-9 and
Glia-20 were used. We found that the diversity in banding
patterns was lower in the modern varieties, clearly indicat-
ing a reduced genetic diversity. This implies that non-diag-
nosed CD patients nowadays may encounter a less diverse
set of gluten proteins than several decades ago. The set of
landraces included accessions from all over the world and
the modern varieties are all European varieties. This does
not aVect the comparison as the decrease in diversity of
-gliadins in varieties is the same when only landraces of
European origin are considered.
So, can wheat breeding successfully be used to lower
exposure to T-cell stimulatory epitopes? In the set of wheat
varieties studied, we found a number of candidates that
Theor Appl Genet (2010) 121:1527–1539 1537
123
exhibit a low content of the epitopes measured. Among the
36 modern varieties, only one variety was identiWed with a
low response against the Glia-9 mAb (the immunodomi-
nant epitope), compared to 15 out of 50 landraces, whereas
the frequencies of high responders to this antibody were
almost the same. The opposite was found regarding the
Glia-20 mAb, which showed a signiWcantly higher overall
antibody response in the landraces. Considering the epitope
impact on CD patients of the major immunodominant Glia-9
epitope, it is concluded from these data that in general the
toxicity of modern wheat varieties has increased.
Landraces will require further breeding to increase their
agronomical and food-technological value. On the other
hand, with a worldwide occurrence of 1% of CD patients
and a high frequency (85–90%) of undiagnosed individuals,
some agronomic drawbacks may be acceptable.
Monoclonal Ab staining is a less time-consuming and
much easier method to screen a large number of varieties
for the presence of T-cell stimulatory epitopes than dealing
with T-cell clones, but requires speciWc mAbs. These are
available for a subset of all epitopes that have been identi-
Wed in wheat. As Glia-9 is a major T-cell epitope from
-gliadins which is recognized by most patients (Vader et al.
2002; Camarca et al. 2009), we have used it here as a proxy
for CD toxicity of all -gliadin. The -gliadins also contain
various T-cell epitopes that are recognized by groups of
patients, although by fewer patients than the -gliadins.
Unfortunately, there are no speciWc mAbs against -gliadin
epitopes that recognize any of these T-cell stimulatory epi-
topes reliably. New methods are currently being developed,
using high throughput sequencing of transcripts and proteo-
mics of gluten proteins. Hopefully, it will become possible
to qualitatively and quantitatively assess the presence of all
CD T-cell stimulatory epitopes in wheat varieties. To
prove, however, that wheat varieties can be considered safe
for consumption by CD patients, we will need T-cell testing
using T-cells from a large number of patients and, ulti-
mately, a trial in which CD patients will be challenged by
hopefully non-CD-stimulatory wheat varieties.
Conclusion
It stands to reason that reduction of T-cell stimulatory epi-
topes in wheat may directly contribute to increasing the
quality of life of many individuals. Further selection of
varieties and landraces low in T-cell stimulatory -gliadin
epitopes and other major epitopes (e.g. from -gliadins) can
be considered a responsibility of wheat breeding companies
together with research organizations and government. Start-
ing from such selections of ‘low in CD epitopes’ wheat,
‘low in CD epitopes gluten’ may become an important new
trait in wheat breeding. The inclusion in breeding programs
of varieties from diVerent origins will assure the mainte-
nance of a broad genetic diversity. Such selection strategies
should also include the use of tetraploid (durum) and dip-
loid wheat species. Further application of advanced breed-
ing technologies, including re-synthesizing of hexaploids
and speciWc gene silencing, will additionally be helpful.
Acknowledgments The authors would like to thank Oscar Vorst
(Plant Research International) and Noor Bas (Centre for Genetic
Resources, The Netherlands (CGN) for assisting in making the selec-
tion of modern wheat varieties and the landrace accessions. Thanks to
Sjef Boeren (Wageningen University) for support with LC–MS/MS
analysis. This research was funded by the Celiac Disease Consortium,
an Innovative Cluster approved by the Netherlands Genomics Initia-
tive and partially funded by the Dutch Government (BSIK03009), and
by the Dutch Ministry of Agriculture, Nature, and Food Quality of The
Netherlands through the DLO program ‘Plant and Animal for Human
Health’ (project KB-05-001-019-PRI).
Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution Noncommercial License which permits any
noncommercial use, distribution, and reproduction in any medium,
provided the original author(s) and source are credited.
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