The Scientific World Journal
Volume 2012, Article ID 837416, 8 pages
AreAncientDurumWheats LessToxicto Celiac Patients?
AStudy ofα-GliadinfromGraziellaRa and Kamut
M. StellaColomba andArmando Gregorini
Dipartimento di Scienze della Terra, della Vita e dell’Ambiente (DiSTeVA), Universit` a di Urbino “Carlo Bo”,
Via Maggetti 22, 61029 Urbino, Italy
Correspondence should be addressed to M. Stella Colomba, firstname.lastname@example.org
Received 26 October 2011; Accepted 12 January 2012
Academic Editor: John Sidney
Copyright © 2012 M. S. Colomba and A. Gregorini. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
In the present paper, the controversial hypothesis suggesting ancient grains might show lower immunogenic properties and
therefore the possibility to introduce them in the diet of wheat-sensitive people, including celiac patients, was investigated.
The immunogenic potential of the ancient durum wheats Graziella Ra and Kamut was studied by comparison to the durum
accessions Cappelli, Flaminio, Grazia and Svevo. Experiments were carried out with two monoclonal antibodies (mAbs) raised
against α-gliadin peptides p31–49 and p56–75 (the latter containing the overlapping DQ2-Glia-α1 and DQ2-Glia-α2 epitopes),
toxic for celiac patients. For all accessions, a few α-gliadin alleles were also cloned, sequenced and translated into aminoacid
sequences. Several aminoacid substitutions or deletions were detected in p31–49, DQ2-Glia-α1 and DQ2-Glia-α2 epitopes,
nevertheless, ELISA constantly showed antibody-antigen positive reactions which led us to suggest that mAbs binding was
not apparently affected by polymorphisms. Moreover, a few substitutions were also observed in DQ2-Glia-α3 and DQ8-Glia-
α1 epitopes. Although some DQ2-Glia-α1 and DQ2-Glia-α2 variants evidenced herein were previously reported to have a
diminished or abolished T cell stimulatory capacity, present results cannot confirm that ancient durum wheats would be
less CD-toxic. In conclusion, we strongly advice celiac patients from consuming ancient wheats including Graziella Ra or
Celiac disease (CD) is an intestinal chronic disorder caused
by an intolerance to wheat gluten proteins (gliadins and
glutenins) mainly resulting in small-intestinal mucosal
injuries and nutrient malabsorption in susceptible individ-
uals . In recent years it has become clear that CD is far
more common than previously thought. Several serological
screening studies from Europe, South America, Australasia,
and the USA have shown that approximately 0.5–2% of these
populations suffers from CD. Nevertheless, most affected
spectrum of clinical presentations . Although they are all
characterized by a certain degree of villous atrophy, from a
clinical point of view, it is usual to consider three different
forms of CD: major or classic CD (characterized by the
presence of symptoms of frank malabsorption, i.e., diarrhea,
steatorrhea, and/or weight loss); minor subclinical CD (with
no evident symptoms of malabsorption, but rather with
minor transient or extraintestinal symptoms); and Silent CD
positive to serological tests such as antigliadin, antiendomys-
ial, and antitransglutaminase, showing evidence of histologi-
cal change in the small intestine). Hence, screening for silent
or subclinical CD has become of paramount importance,
especially considering that a proper diagnosis might prevent
and/or reduce the severity of symptoms and ultimately fa-
vour a remission of associated diseases.
CD is a multifactorial disorder including both genetic
and environmental factors whose relative weight is not yet
fully understood. Differences in concordance rates in mono-
zygotic (86%) and dizygotic (20%) twins strongly suggest a
relevant influence of genetic factors, of which HLA (Human
Leukocyte Antigen) is estimated to contribute for 40–50%
2The Scientific World Journal
to disease development [3, 4]. In particular, while roughly
95% of CD patients carries HLA-DQ2 (DQA1∗0501/DQB1∗
0201), most individuals that are not HLA-DQ2 positive
express HLA-DQ8 (DQA1∗0301/DQB1∗0302). Both HLA-
DQ2 and HLA-DQ8 have typical peptide-binding motifs
characterized by a preference for hydrophobic and negatively
mostly from gliadins digestion [5, 6], although the celiac
toxicity of glutenins becoming increasingly appreciated .
According to mobility in lactic acid PAGE (A-PAGE),
gliadins can be subdivided into four subfractions: α/β-
gliadins, γ-gliadins, and ω-gliadins, whereas the glutenins
consist of low and high molecular weight (LMW and HMW)
glutenins, the latter being particularly important for the
baking quality of dough. Gliadins have several unique fea-
tures that contribute to their immunogenic properties.
They are extremely rich in proline (P) and glutamine (Q)
and, consequently, highly resistant to proteolytic degra-
dation within the gastrointestinal tract, since gastric and
pancreatic enzymes lack postproline-cleaving activities .
Additionally, the high-glutamine content makes gliadins a
good substrate for tissue transglutaminase (tTG), an enzyme
constitutively expressed in the lamina propria playing a role
in tissue repair. Under physiological conditions, tTG can also
convert (during the deamidation process) glutamine into the
negatively charged glutamic acid (E), leading to enhanced
immunogenicity of the resulting modified peptides, which
can preferentially bind to HLA-DQ2 or HLA-DQ8 [9, 10].
Deamidation is most likely a crucial event in the gener-
ation of a full-blown gluten-specific T-cell response and
cell stimulatory capacity have been identified in all gliadin
fractions and in low and high molecular weight glutenins
[11, 12]. In particular, as far as concerns α-gliadins, four
peptides (with a minimal 9-mer epitope core), DQ2-Glia-
α1 [P(F/Y)PQPQLPY], DQ2-Glia-α2 (PQPQLPYPQ), DQ2-
Glia-α3 (previously reported as Glia-α20, FRPQQPYPQ),
and DQ8-Glia-α1 (QGSFQPSQQN) are known to provoke a
activation, there is also activity of the innate immune system,
by gliadin peptides, particularly α-gliadin 31–49 (toxic core
cells  but which cause in vitro [15, 16] and in vivo celiac
The only effective treatment available for CD patients is
a strict exclusion of gluten from the diet. Detrimental conse-
quences of gluten and/or analogous proteins (present in rye,
that noncompliance to a gluten-free diet is associated
with increased risk of anemia, infertility, osteoporosis, and
intestinal lymphoma . On the other hand, complying
with a gluten-free diet is difficult and affects the patients’
quality of life, but a strict diet is critical to reduce morbidity
and mortality. New treatment strategies are thus actively
pursued. Most of these treatments aim to put the celiac
patients on a normal diet and add a drug designed to abolish
the T-cell stimulatory capacity of gluten. An alternative
possibility would be to improve the celiac diet with food
items made from baking-quality wheat that do not contain
harmful gluten proteins. In this regard, the existence of
thousands Triticum accessions has raised the question of
whether they are equally toxic for CD patients and promoted
attempts to generate (by selective breeding or genetic mod-
ifications) wheat (and other cereals) with absent or reduced
immunogenicity [18, 19]. Moreover, ancient wheats (most
frequently not subjected to major genetic improvements)
have been speculated, although without any scientific or
clinical evidence, to potentially reduce or absent toxicity and
therefore to be better suited to be introduced into the diets
of people suffering from intolerances or allergies, including
To test such a controversial hypothesis, the CD-immu-
nogenic properties of α-gliadins from two ancient wheats,
Graziella Ra; an historic accession of durum wheat showing
medium-high nutritional properties , and Kamut, con-
sidered an ancient relative of durum wheat, have been inves-
tigated by a comparative analysis with four durum wheats,
including Cappelli (the very first selected Italian strain,
therefore considered a traditional wheat) and three modern
accessions, Flaminio, Grazia, and Svevo. In particular, as
a further step of a previous research aimed at evaluating
the genetic diversity of these accessions , in the present
paper we studied α-gliadin peptides p31–49 (LGQQQPFPP-
QQPYPQPQPF) and p56–75 (LQLQPFPQPQLPYPQPQL-
PY) by two complementary approaches based on standard
proteomic and genomic techniques. ELISA was carried out
using two specific monoclonal antibodies (mAbs), PN3 and
CDC-5, raised against synthetic peptides equivalent to the
aminoacids 31–49 and 56–75, respectively. A-gliadin genes
from all the accessions were also cloned and sequenced to
detect polymorphisms of the two toxic motifs and search
for a possible relationship between sequence variability and
monoclonal antibody/epitope binding reaction. Addition-
on DQ2-Glia-α3 and DQ8-Glia-α1 epitope variants.
2.1. Wheat Accessions and Gliadin Extraction. Graziella Ra
is a type of durum wheat characterized by low yield (15–
20 quintals per hectare), medium-long cycle, tall size (about
120cm), and a phenotype very similar to Kamut’s with large
ears and long aristas [20, 21]. It was brought to Italy at the
end of ‘70s, forgotten for a long time and “rediscovered”,
due to its fine pasta-making qualities, a few years ago when
it appeared on the market as Graziella Ra. Kamut is a
registered trademark of Kamut International, Ltd., used in
marketing products made with the homonymous grain (cv.
QK-77), the correct subspecies of which is still in dispute.
In fact, according to Stallknecht et al. , Kamut has been
classified, from time to time, as T. turgidum polonicum, T.
turgidum turanicum, or T. turgidum durum; more recently,
hybrid between T. durum and T. polonicum. Although its
taxonomy is quite contentious, it is generally considered
an ancient relative of durum subspecies. Cappelli is an
The Scientific World Journal3
Italian traditional strain of durum wheat which deserves
a privileged place among the varieties of old established
durum wheat for being the very first selected variety. Svevo,
Grazia, and Flaminio are modern durum cultivars, with a
great commercial importance, employed for pasta or bread
making. All wheats were provided by Alce Nero Cooperative
(Isola del Piano, PU, Italy) with the exception of Kamut,
kindly supplied by Molini del Conero (Osimo, AN, Italy).
For each accession, wheat kernels were ground in an
electric grinder to produce a homogenous sample powder.
Subsequently, gliadins were extracted following standard
protocols: briefly, 1g of powder was transferred to 10mL
of 60% (v/v) ethanol for 30min, shaking vigorously. After
the supernatant was recovered and stored frozen (−20◦C).
Whole gliadin from Sigma-Aldrich (G3375) was employed
2.2. Enzyme-Linked Immunosorbent Assay (ELISA). For each
accession, total gliadin was assessed by a commercial two
immunological step sandwich assay type, the Gliadin ELISA
tions. Subsequently, α-gliadin was determined by indirect
two-steps ELISA with PN3  and CDC-5  mAbs
following standard procedures . Briefly, 96-microwells
plates were coated with samples (diluted 1:3,000) and stan-
dards (Sigma gliadin at concentrations ranging from 0ng/
mL to 1,000ng/mL) overnight at 4◦C in the dark, incubated
with murine mAbs (PN3, diluted 1:1,000 or CDC-5, diluted
(IgG, H + L) conjugated to horseradish peroxidase (Pierce)
diluted 1:5,000, for one hour. The substrate, tetramethyl-
benzidine (TMB), was added to plates and after 15 minutes
at RT in the dark, absorbance was determined at 450nm.
Results were read off a semilogarithmic calibration curve.
Statistical analysis was performed by the one-way ANOVA
test, followed by the Bonferroni post hoc test.
2.3. DNA Extraction, Amplification, Cloning, and Sequencing.
Fifteen seeds of each cultivar were germinated in the dark
for two days. The seedlings were grown in daylight for seven
days. The leaf tissues, sampled at the four-leaf stage from ten
different plants per accession, were immediately frozen in
liquid nitrogen and ground in a mortar with a pestle. Fifty
mg of powder was used for DNA extraction following the
cetyltrimethylammonium bromide (CTAB) buffer protocol
 with slight modifications.
Forward (5?-ATGAAGACCTTTCTCATCC-3?) and re-
verse (5?-YYAGTTRGTACCGAAGATGCC-3?) primers to
amplify α-gliadin genes from genomic DNA were designed
on the conserved sequences at the 5?and 3?ends of the
coding region of a few α-gliadin gene complete sequences
retrieved from the GenBank database. PCR amplification
was carried out using a high-fidelity Pfu DNA Polymerase
(Promega) as follows: 95◦C for 2min; 95◦C for 1min,
60◦C for 30sec, 72◦C for 2min (30 cycles); 72◦C for 5min.
An aliquot (1μL) of the PCR product was inserted into
a pCR 4-TOPO vector by the TA-cloning system and
Cappelli Flaminio Grazia Graziella Ra KamutSvevo
α-gliadin by PN3
α-gliadin by CDC-5
Figure 1: Total gliadin and α-gliadin determination by indirect
ELISA. Total gliadin was assessed by a commercial kit (Immuno-
tech); α-gliadin was evaluated using anti-p31–49 (PN3) and anti-
p56–75 (CDC-5) mAbs. Results were read off a semilogarithmic
calibration curve, constructed as the dependence of measured
absorbance values (vertical axis—linear scale) of corresponding
calibrators (standard Sigma—gliadin), range 0–1,000ng/mL (hor-
izontal axis-logarithmic scale); values are reported as mean ± SD
from three independent experiments.
transformation was performed on Escherichia coli TOP10
cells following the manufacturer’s instructions (Invitrogen).
The selected transformants were analysed for presence
and correct orientation of the insert by PCR, grown in LB
medium overnight and purified by the Wizard Plus SV
minipreps kit (Promega). Sequencing of plasmid inserts
was done by using automated DNA sequencers at Eurofins
MWG Operon (Germany). Sequences were visualized with
BioEdit Sequence Alignment Editor version 22.214.171.124 ,
aligned with the ClustalW option included in this software
and double checked by eye. Deduced aminoacid sequences
were obtained and analysed by BioEdit-dedicated options.
3.1. ELISA. Kamut (K) (41.40 ± 0.10g/Kg) and Graziella
Ra (Gll) (40.43 ± 0.87g/Kg) had the greater amounts of
total (α/β, γ and ω) gliadin, followed by Cappelli (C)
(30.32 ± 1.06g/Kg), Flaminio (F) (26.80 ± 1.30g/Kg), Svevo
(S) (23.46 ± 4.67g/Kg), and Grazia (Gr) (23.04 ± 3.12g/Kg)
(Figure 1). One-way ANOVA showed means to be signifi-
cantly different (P < 0.001), whereas the Bonferroni post
hoc test determined that Graziella Ra and Kamut were sig-
nificantly different from Flaminio (P < 0.05), Grazia, and
Svevo (P < 0.01) (Table 1).
For α-gliadin assayed using the PN3 (C, 4.09±0.41g/Kg;
F, 3.85 ± 0.02g/Kg; Gr, 3.66 ± 0.05g/Kg; Gll, 5.61 ±
0.19g/Kg; K, 5.80 ± 0.07g/Kg; S, 3.33 ± 0.39g/Kg) and
CDC-5 (C, 3.07 ± 0.25g/Kg; F, 2.88 ± 0.03g/Kg; Gr, 2.79 ±
0.20g/Kg; Gll, 4.25 ± 0.16g/Kg; K, 4.41 ± 0.30g/Kg; S,
2.84 ± 0.12g/Kg) mAbs (Figure 1) one-way ANOVA showed
differences among accessions to be statistically significant
(P < 0.001); results of Bonferroni correction are reported
in Table 1. All experiments gave similar outcomes for both
4The Scientific World Journal
Table 1: Results of Bonferroni multiple comparison post hoc test.
Statistical significance of means
differences for total gliadin
Statistical significance of means
differences for α-gliadin by PN3
Statistical significance of means
differences for α-gliadin by
Cappelli versus Flaminio
Cappelli versus Grazia
Cappelli versus Graziella
Cappelli versus Kamut
Cappelli versus Svevo
Flaminio versus Grazia
Flaminio versus Graziella
Flaminio versus Kamut
Flaminio versus Svevo
Grazia versus Graziella
Grazia versus Kamut
Grazia versus Svevo
Graziella versus Kamut
Graziella versus Svevo
Kamut versus Svevo
Bonferroni method (one of the most common used post hoc test) was employed to compute the P values when comparing pairwise differences of mean values
for total gliadin (assessed by a commercial ELISA assay) and α-gliadin (assayed using PN3 and CDC-5 mAbs) amounts. ns, NOT SIGNIFICANT;∗P < 0.05;
∗∗P < 0.01;∗∗∗P < 0.001.
mAbs showing an antibody-antigen-positive reaction in
every single case. Hence, according to mABs reactivity, all
grains under study, including the two ancient wheats, have
high potential immunogenicity and toxicity.
3.2. A-Gliadin Gene Sequences. A-gliadin genes from all the
accessions were cloned and sequenced. As already known
and in line with the relatively high-copy number of α-gliadin
alleles reported for diploid wheat ancestors (T. speltoides and
T. monococcum) and hexaploid (T. aestivum) species ,
in this study as well, several alleles, identified as short (S)
or long (L), were characterized differing from each other,
at least in this case, mostly in the length of few CAA-rich
were between 894bp and 963bp. Every accession showed at
of α-gliadin alleles containing one or more internal stop
codons (C→T substitution was the most common single-
base change observed) were encountered. In line with other
studies, we refer to them as pseudogenes, although we
cannot predict from the genomic data whether a subset
is being expressed . Such findings agree with previous
observations that at least 50% of the α-gliadin genes is pseu-
dogenes [29, 31].
All sequences were submitted to GenBank database
(GQ999806–GQ999831). Each allele was translated (with a
BioEdit dedicated option) into the corresponding protein.
Deduced aminoacid sequences showed a high degree of
similarity (about 75% and 86% for S and L isoforms, resp.).
Protein alignment displayed the two epitope motifs (p31–
49 and p56–75) in all aminoacid sequences, although with
a large series of variants (Table 2; Figure 2). Polymorphisms
49, DQ2-Glia-α2, DQ2-Glia-α3 and DQ8-Glia-α1) and a
deletion of Q at p2 (in DQ2-Glia-α2) or at p4 (in DQ2-
Glia-α1). Namely, (1) p31–49 variants included L1→P, P6→
Q, Y13→D, P16→A, and P18→T; (2) DQ2-Glia-α1 variants
included a deletion of Q at position 4 (observed only in
epitopes from locus Gli-B2) and the substitution Y9→H; (3)
DQ2-Glia-α2 variants included the substitution P8→S (all
in epitopes from locus Gli-A2), Y7→H and a deletion of Q
at position 2, epitopes showing the deletion at p2were from
locus Gli-B2; (4) DQ2-Glia-α3 variants included R2→P or
R2→S, P3→T, Y7→I and P8→S or P8→L; (5) DQ8-Glia-α1
variants included the aminoacid substitutions G2→V, S3→F,
Q5→W or Q5→R, and Q9→L.
Celiac disease, a prototype of T-cell-mediated diseases, is
caused by a combination of adaptive and innate immune
responses. Generally speaking, T (and B) cells are part of
the adaptive immune system, which is characterized by
the ability to recognize and remember previous specific
antigenic pathogens and adapt its response with time. T
cells recognize antigens in the context of MHC molecules,
normallydisplayed bya set of specialized cellscalledantigen-
antigen complex is not sufficient to induce a protective T-cell
immune response; in fact, T cells have to recognize antigen
in the context of an activated innate immune system (i.e.,
the nonspecific immune system and first line of defense).
The Scientific World Journal5
Table 2: p31–49, DQ2-Glia-α1, DQ2-Glia-α2, DQ2-Glia-α3, and DQ8-Glia-α1 epitopes variants detected in durum wheats under study.
Canonical sequences (cs), p31–49: L1G2Q3Q4Q5P6F7P8P9Q10Q11P12Y13P14Q15P16Q17P18F19; DQ2-Glia-α1: P1(F/Y)2P3Q4P5Q6L7P8Y9; DQ2-Glia-α2:
P1Q2P3Q4L5P6Y7P8Q9; DQ2-Glia-α3: F1R2P3Q4Q5P6Y7P8Q9; DQ8-Glia-α1: Q1G2S3F4Q5P6S7Q8Q9N10. In bold underlined: aminoacid substitution;
hyphen: aminoacid deletion.
In celiac disease, gliadin acts as an antigen recognized by
the CD4+T cells and, moreover, has the ability to induce
an activation of the innate immune response as well .
Hence, although the adaptive immune system is central
to the development of celiac disease, adaptive immune re-
sponses are, however, controlled by an earlier activation of
Key steps underlying the intestinal inflammatory response
to CD include (1) a direct response of the epithelium via
the innate immune system to toxic proteins in wheat gluten,
(2) modification of wheat gluten proteins by tissue trans-
glutaminase, (3) the role of HLA-DQ2 and HLA-DQ8 in
presenting toxic wheat proteins to T cells, and (4) activation
of T and B cells .
Several T-cell stimulatory α-gliadin peptides have been
identified, DQ2-Glia-α1 [P(F/Y)PQPQLPY], DQ2-Glia-α2
(PQPQLPYPQ), DQ2-Glia-α3 (FRPQQPYPQ), and DQ8-
Glia-α1 (QGSFQPSQQN), showing a large diversity of natu-
ral variants, many of which have T-cell stimulatory activity
. An additional α-gliadin peptide, known as p31–49,
has been reported to activate the innate immune system by
playing an important role as danger signal. It is not the
target of gluten-specific T cells but does induce changes
associated with CD on administration in vivo and during
biopsy challenges in vitro. It has also been shown that prein-
cubation of biopsy specimens of CD patients with the 31–49
peptide enabled T-cell activation. These effects were found
to correlate with the induction of IL-15, a cytokine that is
crucial for the activation and survival of memory T cells
and induces epithelial changes. Moreover, IL-15 production
by enterocytes could have an effect on the adaptive immune
response to gluten .
In the present study, we characterized α-gliadin from
Graziella Ra and Kamut in order to investigate (by molecular
analyses and inspection of translated aminoacid sequences)
the hypothesis suggesting that ancient grains might show
lower immunogenic properties and therefore the possibility
to introduce them in the diet of wheat-sensitive people,
including celiac patients. To this aim, α-gliadin main toxic
peptides related to CD, p31–49 (LGQQQPFPPQQPYPQ-
PQPF) and p56–75 (LQLQPFPQPQLPYPQPQLPY), were
analysed in two ancient durum wheats and in four durum
varieties (Cappelli, Flaminio, Grazia, and Svevo) by two dif-
ferent approaches. A first level of analysis was performed
by ELISA to evaluate wheat toxicity by specific mAbs raised
against the immune-reactive peptides. Subsequently, amino-
acid mutations (substitutions and/or deletions) in the toxic
motifs, including DQ2-Glia-α3 and DQ8-Glia-α1, were ex-
By using a commercially available gluten test kit, we de-
termined that Graziella Ra and Kamut have relatively greater
amount of gliadin than Cappelli, Grazia, Flaminio, and
Svevo. Taking into account that gluten, about 80% of the
entire protein reservoir in wheat, is composed by gliadins
and glutenins, present results would validate the producers’
claims that ancient wheats are endowed with kernels usually
bigger and richer in proteins than modern wheats. Inter-
estingly, Cappelli showed an amount of gliadin that is in-
between ancient and modern accessions, which supports the
placing it as a traditional wheat. When employing a two-
step indirect ELISA with PN3 and CDC-5 mAbs, p31–49
and p56–75 were observed in all accessions. In particular,
as α-gladin amount), thus challenging the “low-immuno-
genicity” hypothesis. These findings point out that, in our
study, ancient wheats have greater amounts of both total
and α-gliadin than modern accessions; moreover, taking into
account that α-gliadins from ancient wheats showed a strong
and specific binding reaction to anti-p31–49 and anti-p56–
as modern ones.
A large series of α-gliadin epitope variants, mainly con-
sisting of one or two aminoacid substitutions (Table 2) were
detected in all the accessions (including ancient ones). Al-
though their T-cell stimulatory capacity would need to be
further investigated, nevertheless, the immunogenic prop-
erties of (at least) some of them may be discussed in the
light of recently published data. In our study, in fact, we
6The Scientific World Journal
210220230240250260270 280 290 300
p31−49 Signal peptide
Figure 2: Alignment of deduced L and S α-gliadin isoforms of each wheat accession under study. The five epitopes known to be
involved in CD, p31–49 (LGQQQPFPPQQPYPQPQPF), DQ2-Glia-α1 [P(F/Y)PQPQLPY], DQ2-Glia-α2 (PQPQLPYPQ), DQ2-Glia-α3
(FRPQQPYPQ), and DQ8-Glia-α1 (QGSFQPSQQN) are shown in red. In light grey: aminoacid substitution; hyphen and light grey:
aminoacid deletion. For each α-gliadin isoform the signal peptide is in light blue.
did encounter several variants which, according to Mitea
et al. , have a diminished or abolished T-cell stimulatory
capacity. These include (1) aminoacid deletion at p4in DQ2-
Glia-α1 (Flaminio, Grazia, Graziella Ra, Kamut, and Svevo);
(2) a single substitution of the proline for a serine residue
(P→S) at p8in DQ2-Glia-α2 (Cappelli, Grazia and Kamut);
(3) an arginine to proline substitution (R→P) at p2in DQ2-
Glia-α3 (Flaminio, Grazia, Graziella Ra, Kamut and Svevo);
(4) a single serine to phenylalanine substitution (S→F) at
p3 (Grazia and Svevo S); a single glutamine to arginine
The Scientific World Journal7
substitution (Q→R) at p5(Cappelli, Grazia) in DQ8-Glia-
α1. As far as concerns the other epitope variants reported in
the present paper, several have never been described before
while some have been described but never tested for their
T-cell stimulatory capacity. Granted that accessions under
study would deserve a deeper investigation to verify and
evaluate their immunogenic capacity, on the other hand,
ELISA results (with PN3 and CDC-5 mAbs) demonstrated
that detected polymorphisms do not (or little) seem to affect
the binding of the monoclonal antibodies to their targets.
In fact, both mAbs gave intense positive reactions for all
wheats (including ancient ones). Such a finding might be
due to the fact that these variants are not contained within
the motif recognized by the mAb or, alternatively, although
being inside the region, they do not significantly influence
the binding reaction. For instance, PN3, which has been
shown to bind in the region QQQPFP of the peptide p31–
49 , in the present study apparently reacted to the
QQQQFP variant as well. As far as concerns the effect of
DQ2-Glia-α1 and DQ2-Glia-α2 variants on CDC-5 binding
reaction, the issue remains unclear since, unfortunately, the
exact sequence within the T-cell stimulating peptide p56–75
identified by the antibody CDC-5 is not known at present.
Further studies will be necessary to address this question
and evaluate how sensitive CDC-5 is in discriminating DQ2-
we can provide the reader with indirect evidence on the T-
Graziella Ra, and Kamut α-gliadins, however, even if the
above discussed variants were confirmed, by T-cell prolif-
eration assays, to have weak (or absent) T-cell stimulatory
properties, all wheats herein examined would still not be safe
for CD patients since, besides α-gliadins, many other gluten
proteins (i.e., γ- and ω-gliadins, HMW and LMW glutenins)
contain stimulatory peptides (relevant to CD pathogenesis)
recognized by a heterogeneous repertoire of intestinal T-cell
In conclusion, our results demonstrate that (1) the
of total and α-gliadin than modern accessions; (2) α-gliadins
from such ancient wheats show a strong and specific binding
reaction to anti-p31–49 and anti-p56–75 mAbs; (3) the
(putatively) less toxic variants of p31–49, DQ2-Glia-α1 and
DQ2-Glia-α2 epitopes detected in all accessions, including
ancient wheats, do not seem to affect mAbs/epitope binding
reactions. Therefore, we suggest that Graziella Ra and Kamut
are potentially as toxic as modern wheats with reference
to CD and strongly recommend that they should not be
introduced in the diet of celiac patients.
Both authors equally contributed to this work.
This research was supported by Regione Marche (CIPE
20/04—DGR 438/2005) funds to M. S. Colomba and A.
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