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ABSTRACT: Wheat kernel albumins/globulins (A/G) and gluten proteins are responsible for baker's asthma and food allergy in atopic subjects. Although no commercial genetically modified wheats are currently being grown, they are under study and the allergenicity of GM products is a major concern. In order to establish the expected and unexpected effects of genetic transformation on allergenicity and also to carry out a safety assessment of genetic transformation, two GM wheat lines (bread and pasta wheat) transformed with endogenous genes were compared to their untransformed counterparts (wt), first by an allergenomic approach, and second, using ELISA with sera from patients suffering from food allergy to wheat and baker's asthma. The 2D immunoblots performed on sera from patients suffering from food allergy and baker's asthma on the A/G fraction of the four lines (two GM and two wt) revealed comparable IgE-binding profiles. A total of 109 IgE-binding spots were analyzed by mass spectrometry, and most of the proteins identified had already been described as allergens or potential allergens. Only two IgE-binding proteins were specific to one GM line. The concentration of specific IgE against the A/G fractions of GM wheat lines and their wt genotypes differed for some sera. BIOLOGICAL SIGNIFICANCE: The originality of our paper is to relate the transformation of wheat lines with their potential allergenicity using patient sera, such focus has never been done before in wheat and should be of interest to the researches working in this field. Another interesting point of this paper is the study of two types of allergies (respiratory and food) on two wheat genotypes and their GM which reveals that some allergens already known in respiratory allergy could be involved in children suffering from wheat food allergy. In this paper we used a classical 2D proteomic analysis and the protein identifications were performed by mass spectrometry after spot picking and in gel trypsin hydrolysis. Concerning the LC-MS/MS analyses classical software and parameters were used as described in Material and methods. We worked on wheat which is actually not fully sequenced that was a difficulty; we therefore searched against two databanks (proteins and ESTs) in order to compare the results. Moreover all proteins reported in our paper were identified with at least three unique peptides. The identified proteins were checked for their potential allergenicity. In order to have a best interpretation of protein identified in terms of potential allergens, BLAST alignments were performed by using an allergen databank (SDAP). This allows the determination of the cross-reactivity of these identified proteins with known allergens of other species and also the prediction of a potential allergenicity.
Journal of proteomics 02/2013; 80C:281-291. · 5.07 Impact Factor
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M Suliman,
A L Chateigner-Boutin,
M Francin-Allami,
A Partier,
B Bouchet,
J Salse,
C Pont,
J Marion,
H Rogniaux,
D Tessier,
F Guillon, C Larré
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ABSTRACT: Plant cell walls are complex structures critical for plant fitness and valuable for human nutrition as dietary fibre and for industrial uses such as biofuel production. The cell wall polysaccharides in wheat endosperm consist of two major polymers, arabinoxylans and beta-glucans, as well as other minor components. Most of these polysaccharides are synthesized in the Golgi apparatus but the mechanisms underlying their synthesis have yet to be fully elucidated and only a few of the enzymes involved have been characterized. To identify actors involved in the wheat endosperm cell wall formation, we used a subcellular fractionation strategy to isolate Golgi-enriched fractions from endosperm harvested during active cell wall deposition. The proteins extracted from these Golgi-enriched fractions were analysed by LC-MS/MS. We report the identification of 1135 proteins among which 64 glycosyltransferases distributed in 17 families. Their potential function in cell wall synthesis is discussed. In addition, we identified 63 glycosylhydrolases, some of which may be involved in cell wall remodeling. Several glycosyltransferases were validated by showing that when expressed as fusion proteins with a fluorescent reporter, they indeed accumulate in the Golgi apparatus. Our results provide new candidates potentially involved in cell wall biogenesis in wheat endosperm.
Journal of proteomics 11/2012; · 5.07 Impact Factor
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S Denery-Papini,
M Bodinier, C Larré,
C Brossard,
F Pineau,
S Triballeau,
M Pietri,
F Battais,
T Mothes,
E Paty,
D-A Moneret-Vautrin
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ABSTRACT: Gluten proteins can be modified by deamidation to enhance their solubility and technological applications. However, severe allergic reactions have been reported after the consumption of food products containing deamidated gluten (DG) in subjects tolerant to wheat. This work aimed to characterize allergen profiles for these patients in comparison with those of patients allergic to wheat and to identify IgE-binding epitopes.
Sera were obtained from 15 patients allergic to DG and from nine patients allergic to wheat proteins (WP). IgE-binding profiles were characterized both in ELISA and in a humanized rat basophilic leukaemia (RBL) cell model. Epitopes were mapped on γ- and ω2-gliadin sequences by Pepscan, and effect of glutamine/glutamic acid substitutions was studied.
Compared to the heterogeneous pattern of allergens detected by IgE from patients allergic to WP, responses of patients allergic to DG were homogeneous. In ELISA, all the sera displayed IgE binding to deamidated γ- and ω2-gliadins and deamidated total gliadins, frequently with high concentrations. These modified proteins induced RBL degranulation with most of the sera from DG-allergic patients. A consensus epitope was found on native γ- and ω2-gliadins (QPQQPFPQ); it was repeated several times in their sequences. The substitution of two or three glutamines of this epitope into glutamic acid at positions Q3 or Q4 and Q8 (QPEEPFPE) increased its recognition the best.
Allergy to DG is a separate entity from wheat allergy. It can be evidenced by strong IgE binding to deamidated gliadins or peptides of the type QPEEPFPE.
Allergy 06/2012; 67(8):1023-32. · 6.27 Impact Factor
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F Guillon, C Larré,
F Petipas,
A Berger,
J Moussawi,
H Rogniaux,
A Santoni,
L Saulnier,
F Jamme,
M Miquel,
L Lepiniec,
B Dubreucq
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ABSTRACT: A detailed and comprehensive understanding of seed reserve accumulation is of great importance for agriculture and crop improvement strategies. This work is part of a research programme aimed at using Brachypodium distachyon as a model plant for cereal grain development and filling. The focus was on the Bd21-3 accession, gathering morphological, cytological, and biochemical data, including protein, lipid, sugars, starch, and cell-wall analyses during grain development. This study highlighted the existence of three main developmental phases in Brachypodium caryopsis and provided an extensive description of Brachypodium grain development. In the first phase, namely morphogenesis, the embryo developed rapidly reaching its final morphology about 18 d after fertilization (DAF). Over the same period the endosperm enlarged, finally to occupy 80% of the grain volume. During the maturation phase, carbohydrates were continuously stored, mainly in the endosperm, switching from sucrose to starch accumulation. Large quantities of β-glucans accumulated in the endosperm with local variations in the deposition pattern. Interestingly, new β-glucans were found in Brachypodium compared with other cereals. Proteins (i.e. globulins and prolamins) were found in large quantities from 15 DAF onwards. These proteins were stored in two different sub-cellular structures which are also found in rice, but are unusual for the Pooideae. During the late stage of development, the grain desiccated while the dry matter remained fairly constant. Brachypodium exhibits some significant differences with domesticated cereals. Beta-glucan accumulates during grain development and this cell wall polysaccharide is the main storage carbohydrate at the expense of starch.
Journal of Experimental Botany 01/2012; 63(2):739-55. · 5.36 Impact Factor
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ABSTRACT: Wheat is an important part of the daily diet of millions of people. However, this staple food is also responsible for food allergies. Ancient cultivars of wheat are gaining interest today but nothing is known about their allergenicity. Many wheat proteins have been reported as causative food allergens, including some prolamin-type gluten proteins, and salt soluble proteins of the albumin/globulin (A/G) type. The objective of this work is to obtain information about the allergenicity of the salt soluble A/G fraction of an ancient diploid cultivar compared with a standard hexaploid bread wheat cultivar using 20 sera from patients with wheat allergy. Differences in the IgE reactivity of sera towards the two genotypes were quantified by ELISA. Qualitative differences in IgE-binding proteins were searched after 1D or 2D electrophoresis. For most of the sera, the concentration in A/G specific IgE was higher for the hexaploid T. aestivum (cv Récital) than for the diploid T. monococcum (cv Engrain). The analysis of 2D spots revealed by immunoblotting leads to the identification by mass spectrometry of 39 IgE-binding proteins, some of them unknown until now as wheat allergens. Numerous allergens were identified, differences observed between Engrain and Récital will be discussed.
Journal of proteomics 04/2011; 74(8):1279-89. · 5.07 Impact Factor
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ABSTRACT: Seed storage proteins are of great importance in nutrition and in industrial transformation because of their functional properties. Brachypodium distachyon has been proposed as a new model plant to study temperate cereals. The protein composition of Brachypodium grain was investigated by separating the proteins on the basis of their solubility combined with a proteomic approach. Salt-soluble proteins as well as salt-insoluble proteins separated by two-dimensional gel electrophoresis revealed 284 and 120 spots, respectively. Proteins from the major spots were sequenced by mass spectrometry and identified by searching against a Brachypodium putative protein database. Our analysis detected globulins and prolamins but no albumins. Globulins were represented mainly by the 11S type and their solubility properties corresponded to the glutelin found in rice. An in silico search for storage proteins returned more translated genes than expressed products identified by mass spectrometry, particularly in the case of prolamin type proteins, reflecting a strong expression of globulins at the expense of prolamins. Microscopic examination of endosperm cells revealed scarce small-size starch granules surrounded by protein bodies containing 11S globulins. The presence of protein bodies containing glutelins makes B. distachyon closer to rice or oat than to wheat endosperm.
Journal of Experimental Botany 06/2010; 61(6):1771-83. · 5.36 Impact Factor
