Molecular Characterization and Variation of the Celiac Disease

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Article

Molecular characterization and variation of the celiac disease epitope domains among #-gliadin genes in Aegilops tauschii YuGe Li, HuiHui Liang, Sheng-Long Bai, Yun Zhou, Guiling Sun, Ya-Rui Su, An-Li Gao, DaLe Zhang, and SuoPing Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00338 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 11, 2017

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Journal of Agricultural and Food Chemistry

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Molecular characterization and variation of the celiac disease

2

epitope domains among α-gliadin genes in Aegilops tauschii

3

Yu-Ge Li1,2+, Hui-Hui Liang1,2+, Sheng-Long Bai2, Yun Zhou1,2, Guiling Sun1,2,

4

Ya-Rui Su1, An-Li Gao1, Da-Le Zhang1,2*, Suo-Ping Li1,2*

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Abstract: To explore the distribution and quantity of toxic epitopes in α-gliadins from

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Aegilops tauschii, a total of 133 complete α-gliadin coding sequences were obtained,

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including 69 pseudogenes with at least one premature stop codon and 64 genes with

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complete open reading frames (ORFs). Plenty of deletions and single amino acid

9

substitutions were found in the 4 celiac disease (CD) toxic epitope domains through

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multiple alignments, in which the sequence of DQ2.5-glia-α2 demonstrated the most

11

significant changes. Interestingly, 7 of the 59 α-gliadins were free of any kind of

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intact CD toxic epitopes, providing potential gene resources for low CD toxicity

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breeding of common wheat. Analysis of the neighbor-joining tree demonstrates that 2

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of the totally 7 α-gliadins cluster within the homologs of Triticum (A genome), and

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the other 5 group with those of Aegilops Sitopsis (B genome). This result implies that

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the 7 α-gliadin genes may be originated from the ancestor species of Ae. tauschii,

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evolved by homoploid hybrid of Triticum and Aegilops Sitopsis. The remaining 52

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α-gliadins form a separate clade from other homologs of A and B genomes,

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suggesting a recent rapid gene expansion by gene duplication associated with the

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species adaptation.

1

School of Life Science, Henan University, Kaifeng, 475004, Henan, People’s Republic of China. Institute of Plant Stress Biology, Henan University, Kaifeng, 475004, People’s Republic of China. + Equal contributors * Corresponding authors: [email protected] (D.L. Zhang), [email protected] (S.P. Li) 1 2

ACS Paragon Plus Environment


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Keywords: Aegilops tauschii; α-gliadin; celiac disease; toxic epitope; phylogenetic

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relationships

23

Celiac disease (CD) is a lifelong T cell-mediated autoimmune disease

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characterized by an aberrant inflammatory response to dietary gluten in genetically

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susceptible individuals, which immunologically induces small intestinal mucosal

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damage including villous atrophy and crypt hyperplasia, etc.1 Recently, the

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development and application of serological and immunological techniques, featured

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by higher sensitivity and stronger specificity, establish CD to be a worldwide disease.

29

Besides the individuals neglected from proper treatment for ambiguous symptoms, the

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global prevalence of CD has already reached to 0.9%, and is still on the rise.2, 3

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Wheat gluten proteins, which are abundant of prolines and glutamines, are

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widely regarded as a main external stimulation factor to induce the CD.4 These gluten

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proteins generate plenty of peptides with immune activity in vivo, which are believed

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to increase the binding affinity to HLA-DQ2.5 and HLA-DQ8 by transglutaminase 2

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(TG2)-mediated

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immunoreaction.5-8 Up to now, altogether 31 CD toxic epitopes with immune activity

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in vivo or in vitro, composed of 9 amino acid core sequences, have been detected

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from common wheat and its related wild species.9,

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epitopes exhibit high frequencies and immune activities.11,

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DQ2.5-glia-γ1 (PQQSFPEQQ) is located almost on each γ-gliadin, while the other 4

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toxic epitopes, DQ2.5-glia-α1a (PFPQPQLPY), DQ2.5-glia-α2 (PQPQLPYPQ),

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DQ2.5-glia-α3 (FRPQQPYPQ) and DQ8-glia-α1 (QGSFQPSQQ), are distributed on

deamidation,

inducing

specific

CD+T

10

cell

stimulatory

Among them, 5 CD toxic

2


12

Specifically, the

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α-gliadins with various combinations. Several studies revealed that peptides from

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α-gliadins induced stronger T cell responses in large majority of patients compared

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with γ-gliadins.13, 14 Therefore, the a-gliadins are generally considered to be the most

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relevant gluten fraction able to stimulate CD.10, 12, 15

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The α-gliadins are encoded by the Gli-2 loci on the short arms of chromosomes

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6A, 6B, and 6D from hexaploid wheat (Triticum aestivum L.). As a multigene family,

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the sequences of α-gliadin genes are highly conserved on both ends.16 The number of

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α-gliadin genes is highly variable among wheat and its ancestors for the duplication

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and deletion of chromosome segments during evolutionary process. Therefore, Gli-2

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loci may contain 25 to 100 or even up to 150 copies in an individual haploid

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genome.17 However, almost 50% of the α-gliadin genes are pseudogenes due to

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nonsense or frame-shift mutations, in which the high base substitution rate, especially

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C→T substitution, contributes to the appearance of stop codons.18 As a result, only a

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few α-gliadins have been detected by protein electrophoresis.

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In addition, the quantity and distribution of 4 toxic epitopes mentioned above

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could be exploited to associate α-gliadins with specific chromosome.10,15,19

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Specifically, the α-gliadins derived from chromosome 6A of common wheat usually

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contain toxic epitopes DQ2.5-glia-α1a and DQ2.5-glia-α3, with incomplete toxic

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epitopes DQ2.5-glia-α1 or DQ2.5-glia-α2. The α-gliadins from chromosome 6B do

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not contain any of the T cell epitopes, and a few of them only contains DQ2.5-glia-α1.

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The α-gliadins encoded by chromosome 6D were found to contain all of the 4 T cell

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epitopes in various combinations,15 and some α-gliadins even may contain one 3



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repeated DQ2.5-glia-α2, thus to form highly immunostimulatory alpha1α-33mer

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fragment (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF).14 Consequently, the

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α-gliadins from chromosome 6D are deemed to have strong capacity to stimulate

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CD+T cells due to containing toxic epitopes with the largest type and quantity.

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As the diploid progenitor of bread wheat, Aegilops tauschii Cosson (DD,

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2n=2x=14) is an annual, self-pollinated plant with high genetic variability level for

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disease-resistance, productivity traits and abiotic stress resistance.20 Fertile Crescent is

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regarded as the diversity center and origin of Ae. tauschii, spreading from northern

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Syria and Turkey to western China. It is found in all the regions, Ae. tauschii has

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adapted to diverse environments including sandy seashore, margins of deserts, stony

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hills, steppes, wastelands, roadsides and humid temperate forests,21 thus forming

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abundant genetic background. In China, Ae. tauschii is mainly distributed in Yili area

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of Xinjiang and middle reaches of the Yellow River (including Shanxi and Henan

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provinces).22 The genetic variation type of Ae. tauschii (L1 lines) is more abundant

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than that of wheat D genome, since limited areas of Ae. tauschii (L2 lines) are

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involved in the origin of common wheat.23 Therefore, like many wild crop progenitors,

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Ae. tauschii is considered to be a valuable germplasm resource for the improvement

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of common wheat.24 Regarding the origin of wheat D genome, it was reported to be

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derived from the homoploid hybrid speciation of A and B genomes about 5 to 6

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million years ago, which were diverged from a common ancestor ~7 million years

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ago.25

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Ae. tauschii contains a higher quality of storage proteins with extensive allelic 4


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variations.26 Unfortunately, the CD epitope domains among its α-gliadins have been

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rarely investigated. Recently, Xie et al.18 reported the molecular cloning and sequence

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analysis on 4 α-gliadin genes in this species for the first time. To obtain more

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comprehensive information of CD epitope domains, novel α-gliadin genes were

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cloned from Ae. tauschii accession T006 to investigate the molecular characterization

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and variations of CD epitope domains in this study, which provides valuable gene

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resources for the quality breeding of common wheat.

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Material and methods

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Plant material

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The diploid Ae. tauschii ssp. tauschii accession T006 was originally derived from

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Henan province, which was preserved and cultivated in Plant Germplasm Resources

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and Genetic Engineering Laboratory, Henan University.

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PCR amplification and molecular cloning of α-gliadin genes

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Genomic DNA was extracted from young leaves according to the approach reported

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by Zhang et al. 27. One pair of allelic specific PCR (AS-PCR) primers was designed

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using Primer Premier 5 based on the conservative sequence of α-gliadin genes: P-1:

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5’-ATGAAGACCTTTCTCATCCT-3’

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5’-TCAGTTAGTACCGAAGATGC-3’. PCR reactions and molecular cloning

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programs were conducted with the method described by Yan et al.28. PCR

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amplifications were performed in 50 µl reaction volume containing 2.5 U La Taq

107

polymerase (TaKaRa), 60 ng of template DNA, 25µl 2 × GC buffer I (MgCl2 plus),

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0.4 mM dNTP, 0.5 µM of each primer, and making up to 50 µl with ddH2O. The

and

5


P-2:


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reaction was carried out in a S1000TM Thermal Cycler (Bio-Rad Corp., USA) using

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the following protocol: pre-denaturation at 94 °C for 3 min, cycled 30 times at 94 °C

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for 45 s, 62 °C for 45 s and 72 °C for 1 min, and a final extension at 72 °C for 10 min.

112

To ensure the accuracy of the obtained sequence, an enhanced annealing temperature

113

and La Tag DNA polymerase (Takara, Japan) with high fidelity were used in the PCR

114

reactions. Each clone was sequenced twice.

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Sequence alignment, toxic epitope identification, and secondary structure

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prediction

117

Multiple alignments of the deduced amino acid sequences of complete α-gliadin genes

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were carried out using the multiple sequence alignment software Clustal X 2.0.29

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Identification of four toxic epitopes: DQ2.5-glia-α1a (PFPQPQLPY), DQ2.5-glia-α2

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(PQPQLPYPQ), DQ2.5-glia-α3 (FRPQQPYPQ) and DQ8-glia-α1 (QGSFQPSQQ) in

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the α-gliadin was determined through the strategy reported by Van Herpen et al.15.

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Prediction of secondary structure of deduced amino acid sequences was carried out by

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PSIPRED server (http://bioinf.cs.ucl.ac.uk/psipred/) according to Xie et al.18.

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Phylogenetic analysis

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The neighbor-joining trees of different genes were constructed by the Molecular

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Evolutionary Genetics Analysis software MEGA 7.0.21.30 The bootstrap values in the

127

phylogenetic

128

Jones-Taylor-Thornton (JTT) + Gamma Distributed (G) model was selected, with

129

gamma parameter of 8.

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Results

tree

were

evaluated

based

on

1000

6


replications.

The

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Cloning and molecular characterization of α-gliadin genes in Ae. tauschii

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The amplified fragments (about 900bp) were obtained from the genomic DNA of Ae.

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tauschii accession T006 by AS-PCR (Fig. 1). The purified products were further

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cloned and sequenced, from which altogether 133 complete α-gliadin coding

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sequences were acquired, including 64 full-ORF genes with sequence length of

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843~909 bp and 69 pseudogenes with at least one stop codon. These pseudogenes

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were temporarily named Gli2-AT-1~Gli2-AT-69, most of which were formed due to

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mutations from C to T, i.e., a glutamine codon (CAG or CAA) was mutated into a

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stop

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Gli2-AT-70~Gli2-AT-133, with nucleotide similarities of themselves varied from 86.3

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to 99.2%. On the other hand, the sequence identities with the published α-gliadin

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genes from Triticeae were established to be ranging from 95.8 to 100%. Analysis of

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sequence identities demonstrates that all cloned genes contain typical structural

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features of previously reported α-gliadin genes, including no introns and ending with

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stop codon TGA. As the functional proteins cannot be expressed by pseudogenes,

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only 64 full-ORF genes were analyzed in the following work.

codon

(TAG

or

TAA).

Sixty-four

full-ORF

genes

were

called

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Five synonymous mutation genes were observed in the multiple alignments of

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the deduced amino acid sequences from the 64 full-ORF genes. Then, a phylogenetic

149

tree was constructed based on the deduced amino acid sequences of the 59 α-gliadin

150

genes to explore the sequence divergence of the α-gliadin in this study. As can be seen

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from Fig. 2, the 59 α-gliadin genes were obviously clustered into three groups, in

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which 51 α-gliadin genes from Ae. tauschii were contained in group I, accounting for 7



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a high proportion of 86.4%. This reveals the similar sequence composition of the

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studied α-gliadin genes. In comparison with group I, obvious difference could be

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found in the same phylogenetic tree for groups II and III. For simplicity, a typical

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sequence Gli2-AT-86 from group I and all the sequences from groups II and III

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(Gli2-AT-85, Gli2-AT-87, Gli2-AT-88, Gli2-AT-73, Gli2-AT-93, Gli2-AT-107,

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Gli2-AT-119, and Gli2-AT-126) were deposited in GenBank with accession numbers

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JX828228-JX828231 and KY434321- KY434325.

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To explore the specific difference among the sequences from groups II-III and

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those from group I, multiple alignments were conducted based on the deduced amino

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acid sequences of 8 α-gliadin genes from the former groups and the selected typical

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sequence Gli2-AT-86 from group I (Fig. 3). The result showed that 9 α-gliadins

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generally shared the typical structural features, including signal peptide with 20 amino

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acid residues (S), N-terminal repetitive domain(R), Polyglutamine region Ⅰ(Q1), the

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first non-repetitive region (NR1), Polyglutamine region Ⅱ(Q2), and the second

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non-repetitive region (NR2).17 While apparently, the amino acid sequences of 8

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α-gliadin genes from groups II and III exhibited strikingly difference with that of

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Gli2-AT-86 in amino acid composition. Signal peptide domain was relatively

170

conservative in the full sequences of 9 α-gliadin genes, displaying the main mutations

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from alanine (A) to serine (S) and threonine (T) to isoleucine (I). Comparatively, a

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large amount of single amino acid substitution and deletions could be observed in

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other domains, including primary mutations from leucine (L) to valine (V), serine (S)

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to leucine (L), glutanine (Q) to glutamic acid (E) and phenylalanine (F) to valine (V) 8


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as well as the insertion of ‘ST’ amino acid fragment. Particularly, the sequences of

176

Gli2-AT-73,

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prominent difference with that of Gli2-AT-86 in sequence composition, as revealed by

178

the variation in the glutamine number of two polyglutamine regions and a deletion of

179

PQLPYPQP amino acid fragment occurred in their N-terminal repetitive domain (Fig.

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3).

181

Identification and variations of celiac disease epitopes

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Generally, the position of CD toxic epitopes, viz. DQ2.5-glia-α1a (PFPQPQLPY),

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DQ2.5-glia-α2 (PQPQLPYPQ), DQ2.5-glia-α3 (FRPQQPYPQ) and DQ8-glia-α1

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(QGSFQPSQQ) was conservative in most α-gliadin subunits.18,

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DQ2.5-glia-α1a, DQ2.5-glia-α2 and DQ2.5-glia-α3 were present in the N-terminal

186

repetitive domain, and DQ8-glia-α1 was located in the second non-repetitive region.

187

The variations of the CD epitope domains of 59 α-gliadins were listed in Table 1,

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from which various deletions and single amino acid substitutions could be observed in

189

the four CD toxic epitopes. The DQ8-glia-α1 displayed more conservative property,

190

and only those of 7 α-gliadins were substituted by single amino acid, mainly

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occurring at the fifth amino acid residue (Q) of DQ8-glia-α1. Relatively, the sequence

192

of DQ2.5-glia-α2 with high risk for celiac disease changed significantly, and

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altogether those of 44 α-gliadins (74.6%) were involved in deletion and single amino

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acid substitution, in which the deletion of QLPYPQ amino acid fragment occurred

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most frequently. From another aspect, only 6 amino acid residues (Arg, Gln, Leu, Phe,

196

Pro and Tyr) were substituted in the 4 CD toxic epitopes, and their frequencies were

Gli2-AT-93,

Gli2-AT-107,

Gli2-AT-119,

9


Gli2-AT-126

31

exhibited

In detail,


197

graphed in Fig. 4. Among them, proline (P) and glutanine (Q) demonstrated relative

198

high substitution frequencies of 47.3% and 25.5%.

199

In addition, a total of 7 combinations of CD toxic epitopes were detected in this

200

study, since that α-gliadin may contain one or several types of CD toxic epitopes.

201

As shown in Fig. 5, the combination distribution of CD toxic epitopes exhibited a

202

significant difference in 59 α-gliadins. The α-gliadins containing 3-4 CD toxic

203

epitopes (except for DQ2.5-glia-α2) reached to 39, accounting for 66.1%.

204

Interestingly, 7 α-gliadins (Gli2-AT-73, Gli2-AT-85, Gli2-AT-87, Gli2-AT-93,

205

Gli2-AT-107, Gli2-AT-119, and Gli2-AT-126) were free of any kind of intact CD toxic

206

epitopes in this study (details shown in Fig. 3). Compared with those of Gli2-AT-85

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and Gli2-AT-87, the toxic peptide of Gli2-AT-73, Gli2-AT-93, Gli2-AT-107,

208

Gli2-AT-119, and Gli2-AT-126 were of similar characteristic in variant types.

209

Mutation from glutamine (Q) to serine (S) and a deletion of PQLPYPQP amino acid

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segment were occurred in the regions of DQ2.5-glia-α1a and DQ2.5-glia-α2.

211

Mutations from arginine (R) to proline (P) and from glutamine (Q) to glutamic acid (E)

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were observed in the domains of DQ2.5-glia-α3 and DQ8-glia-α1. Besides, one more

213

mutation from glutamine (Q) to histidine (H) was detected in the region of

214

DQ8-glia-α1 for Gli2-AT-73. Comparatively, little differences were found in

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Gli2-AT-85 and Gli2-AT-87. For the former, 4 amino acid substitutions and 1 long

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deletion of LPYPQPQ amino acid fragment were identified in the region of 4 CD

217

toxic epitopes, while 3 amino acid substitutions and 2 single amino acid deletions

218

were detected in the latter α-gliadin. 10


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The 19-residue motif (LGQQQPFPPQQPYPQPQPF) in the N-terminal

220

repetitive domain was deemed to be active for celiac disease.32 In addition, short

221

motif (LGQGSFRPSQQN) in the second non-repetitive region associated with

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adenovirus type 12 infections.33 In this work, the sequences of Gli2-AT-73,

223

Gli2-AT-87, Gli2-AT-93, Gli2-AT-107, Gli2-AT-119, and

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free of the above two specific peptides, indicating potential values in these α-gliadin

225

genes for lower risk of celiac disease.

Gli2-AT-126 were found

226

Furthermore, the predicted secondary structures of the mature protein subunits of

227

the 59 deduced α-gliadins were intensively analyzed (Table S1). As a result, all

228

α-gliadins showed high α-helix content, with relatively conservative positions, which

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mainly presented in the regions of polyglutamine I (Q1), the first non-repetitive (NR1)

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and the second non-repetitive (NR2). In detail, the number of α-helix in the 59

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α-gliadins ranged from 5 to 7 (23.8-38.7%), most of which appearing in NR1.

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Nevertheless, β-strands were only observed in the NR2 regions of Gli2-AT-78,

233

Gli2-AT-93, Gli2-AT-95, Gli2-AT-103, Gli2-AT-106 and Gli2-AT-107, ranging from

234

0.7-1.2%. Of all the 59 α-gliadins, α-helixes could be found in all the five regions of

235

R, Q1, Q2, NR1, NR2 for Gli2-AT-80 and Gli2-AT-116. The number and distribution

236

of the α-helix and β-strand are apparently uneven in the different mature a-gliadins.

237

And comparatively, the most amino acid residues (81) were observed in α-helix and

238

β-strand of Gli2-AT-107 (Fig. S1), accounting for 39.4%.

239

Phylogenetic relationships among the α-gliadin genes from Aegilops and Triticum

240

genomes 11



241

To determine the origin of the α-gliadin genes in this study, another neighbor-joining

242

tree of the deduced amino acid sequences was constructed, from which 59 α-gliadin

243

genes were obtained from Ae. tauschii accession T006 and the other 40 genes were

244

acquired from Triticum (T. monococcum and T. urartu) and Aegilops Sitopsis (Ae.

245

speltoides, Ae. bicornis, Ae. searsii, and Ae. sharonensis) registered in the Genbank.

246

As shown in Fig. 6, the neighbor-joining tree shows three main subgroups. Five

247

α-gliadins of Ae. tauschii D genome (Gli2-AT-73, Gli2-AT-93, Gli2-AT-107,

248

Gli2-AT-119, and Gli2-AT-126) and the sequences from Aegilops Sitopsis were

249

classified to group I (bootstrap value 80%), implying that the 5 α-gliadin sequences

250

have a close genetic relationship with that of Aegilops Sitopsis. Also as an indicative

251

of close relationship, two α-gliadins (Gli2-AT-87 and Gli2-AT-85) from Ae. tauschii D

252

genome and the sequences from T. monococcum and T. urartu were designated to

253

group II, with a bootstrap support value of 87%. Comparatively, the other 52

254

α-gliadins from Ae. tauschii D genome were separately identified to group III, with

255

bootstrap value of 82%, revealing distant genetic relationship with the

256

above-mentioned 7 α-gliadins. Ae. tauschii-specific gene duplication can be inferred

257

to explain the rapid gene expansion of these 52 α-gliadin genes.

258

Discussion

259

It is an effective approach to explore and utilize the desirable genes from Ae. tauschii

260

to improve the common wheat, and concurrently, to enrich the monotonous genetic

261

background due to breeding.20 As indicated by the analysis from celiac lesion-derived

262

T-cell lines and monoclonal antibody, extensive natural variations exist in the toxic 12


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epitopes of α-gliadins from common wheat and its ancient species.14, 34, 35 The more

264

variety and quantity of toxic epitopes in gluten proteins, the higher incidence and risk

265

of CD. On the contrary, less toxic epitopes in gluten proteins are relative safe and

266

low-toxic for most CD patients.15, 36 Xie et al.,18 cloned and identified 4 α-gliadin

267

genes (Gli-At1, Gli-At2, Gli-At3, Gli-At4) from Ae. tauschii accessions T15, T43 and

268

T26 provided by GenBank (Braunschweig, Germany). Molecular characterization of

269

the CD epitope domains in these 4 α-gliadins implies the quantity inconformity of

270

their toxic epitopes, in which only intact DQ2.5-glia-α3 was discovered in Gli-At4. So

271

far, most researches focused on Ae. tauschii from Transcaucasus and northern Iran,

272

owing to the deep consensus that the Ae. tauschii in these regions (mainly for L2 line)

273

may involve in the origin of wheat D genome.24 Relatively, little is known about

274

genetic and phenotypic properties of Ae. tauschii (mainly for L1 line) from the eastern

275

and southern populations (i.e., those from Syria, Afghanistan, Pakistan, Central Asia,

276

and China).37 In this study, a total of 133 complete α-gliadin coding sequences were

277

obtained in Ae. tauschii accession T006 from Huanghuai area, China, including 64

278

full-ORF

279

(Gli2-AT-1-Gli2-AT-69). Considering that an individual haploid genome may contain

280

25-100 α-gliadin genes, even up to 150 copies,17 the characterization of the α-gliadin

281

genes obtained from Ae. tauschii D genome, which is highly representative, could be

282

fully explored in this work. Analysis of sequence identities reveals typical structural

283

features of α-gliadin in all cloned genes. The phylogenetic tree constructed based on

284

59 α-gliadin sequences (except for 5 synonymous mutations) displays prominent

genes

(Gli2-AT-70-Gli2-AT-133)

and

13


69

pseudogenes


285

difference in their amino acid sequences.

286

The properties of altered peptide ligands identified through polyclonal T cells in

287

vitro suggested that single amino acid substitution for toxic epitopes may even abolish

288

their capacity to stimulate IFN-γ from CD4 T cells, though the capacity to stimulate

289

the T cell response could be partly retained or reduced by some modifications.38 In

290

this case, plenty of deletions and single amino acid substitutions were observed in the

291

4 CD toxic epitopes from 59 α-gliadins, in which DQ2.5-glia-α2 with high risk for

292

CD exhibited sequence difference with the highest frequency, compared with the

293

other 3 toxic epitopes. Similarly, the highest variation frequency of DQ2.5-glia-α2

294

epitopes had also been observed in wheat cultivars and landraces.39 Moreover, 7

295

combinations of CD toxic epitopes were detected in 59 α-gliadins, in which 39

296

α-gliadins (accounting for 66.1%) contained all the 4 CD toxic epitopes or 3 CD toxic

297

epitopes (except for DQ2.5-glia-α2). This implies that the a-gliadins from Ae. tauschii

298

have strong capacity to stimulate CD4 T cells.35 Interestingly, none intact CD toxic

299

epitope could be detected in the 7 α-gliadins (Gli2-AT-73, Gli2-AT-85, Gli2-AT-87,

300

Gli2-AT-93, Gli2-AT-107, Gli2-AT-119, and Gli2-AT-126), with plenty of single

301

amino acid substitutions and long deletions of amino acid segment in 4 toxic epitope

302

domains. These α-gliadin genes may be exploited as potential resources for decreasing

303

and preventing celiac diseases. In addition, Gli2-AT-107 contained the highest ratio of

304

amino acid residues, which further generated the secondary structures, implying that

305

this gene could be favorable for constructing a good gluten structure and exhibiting

306

superior dough quality.40, 41 14


Page 14 of 32

Page 15 of 32


307

Based on such recognition, the quantity and distribution of the 4 toxic epitopes

308

could be applied to associate α-gliadins with specific chromosome.13, 15 To be specific,

309

the α-gliadins from chromosome 6B were identified free of any intact T cell epitopes.

310

Therefore, the above mentioned 7 α-gliadins from Ae. tauschii (D genome) should be

311

ascribed to B genome. Furthermore, the neighbor-joining tree constructed by 59

312

α-gliadin genes advanced in this study and the other 40 genes acquired from Triticum

313

and Aegilops Sitopsis displays that two α-gliadins, Gli2-AT-85 and Gli2-AT-87, have a

314

close relationship with T. monococcum and T. urartu (genetically close to wheat A

315

genome) and five α-gliadins, Gli2-AT-73, Gli2-AT-93, Gli2-AT-107, Gli2-AT-119, and

316

Gli2-AT-126, cluster with those from Aegilops Sitopsis, which had been supposed to

317

be donor species of wheat B genome.42, 43 Recently, the phylogenetic history of the A,

318

B, and D genome lineages were reevaluated using the genome-wide sample of 275

319

gene trees based on the genome sequences of hexaploid bread wheat subgenomes

320

(denoted TaA, TaB, and TaD) and 5 diploid relatives (T. monococcum, T. urartu, Ae.

321

sharonensis, Ae. speltoides and Ae. tauschii).24 This result implies that the present-day

322

bread wheat genome originated from multiple rounds of hybrid speciation (homoploid

323

and polyploid), in which the D genome was generated through homoploid

324

hybridization of A and B genomes. It is thus concluded that 7 α-gliadin genes in this

325

study might be retained from the species of Triticum and Aegilops Sitopsis in the

326

formation of Ae. tauschii. While the other 52 α-gliadin genes may derive from rapid

327

gene expansion occurring after the

328

Sitopsis, which probably provide adaptive benefit to the evolution of Ae. tauschii in

homoploid hybrid of

15


Triticum

and

Aegilops


329

various hash environment.

330

To sum up, Ae. tauschii has abundant α-gliadin genes, and contains extensive

331

natural variation in toxic epitope domains. Though typical D genome characterization

332

is found in majority of α-gliadins with toxic epitopes, absence of intact toxic epitope

333

is also observed in a few α-gliadins, which shows close relationship with A and B

334

genome. These potential gene resources are of great significance and could arouse

335

enthusiasm in the application for low CD toxicity breeding of common wheat.

336

Funding

337

This work was supported by National Natural Science Foundation of China (Grant

338

Nos. 31401379, 31571649, and U1604116), Project of Young Teachers in Henan

339

Province (Grant No.2015GGJS-019) and Project of Major Science and Technology in

340

Henan Province (Grant No.161100110400)

341

References

342

(1) Plugis, N. M.; Khosla, C. Therapeutic approaches for celiac disease. Best Pract.

343

Res. Clin. Gastroenterol., 2015, 29, 503-510.

344

(2) Kang, J. Y.; Kang, A. H.; Green, A.; Gwee, K. A.; Ho, K. Y. Systematic review:

345

worldwide variation in the frequency of coeliac disease and changes over time.

346

Aliment. Pharm. Therap., 2013, 38, 226-245.

347

(3) Lionetti, E.; Gatti, S.; Pulvirenti, A.; Catassi, C. Celiac disease from a global

348

perspective. Best Pract. Res. Clin. Gastroenterol., 2015, 29, 365-379.

349

(4) Vriezinga, S. L.; Schweizer, J. J.; Koning, F.; Mearin, M. L. Coeliac disease and

350

gluten-related disorders in childhood. Nat. Rev. Gastro. Hepat., 2015, 12, 527-536. 16


Page 16 of 32

Page 17 of 32


351

(5) Mitea, C.; Salentijn, E. M. J.; van Veelen, P.; Goryunova, S. V.; van der Meer, I.

352

M.; van den Broeck, H. C.; Mujico, J. R.; Monserrat, V.; Gilissen, L. J. W. J.;

353

Drijfhout, J. W.; Dekking, L.; Koning, F.; Smulders, M. J. M. A universal approach to

354

eliminate antigenic properties of alpha-gliadin peptides in celiac disease. PLoS ONE,

355

2010, 5, e15637.

356

(6) Arentz-Hansen, E. H.; McAdam, S. N.; Molberg, O.; Kristiansen, C.; Sollid, L. M.

357

Production of a panel of recombinant gliadins for the characterization of T cell

358

reactivity in coeliac disease. Gut., 2000, 46, 46-51.

359

(7) Camarca, A.; Anderson, R. P.; Mamone, G.; Fierro, O.; Facchiano, A.; Costantini,

360

S.; Zanzi, D.; Sidney, J.; Auricchio, S.; Sette, A.; Troncone, R.; Gianfrani, C.

361

Intestinal T cell responses to gluten peptides are largely heterogeneous: implications

362

for a peptide based therapy in celiac disease. J. Immunol., 2009, 182, 4158-4166.

363

(8) Koning, F. The molecular basis of celiac disease. J. Mol. Recognit., 2003, 16,

364

333-336.

365

(9) Sollid, L. M.; Qiao, S. W.; Anderson, R. P.; Gianfrani, C.; Koning, F.

366

Nomenclature and listing of celiac disease relevant gluten T-cell epitopes restricted by

367

HLA-DQ molecules. Immunogenetics, 2012, 64, 455-460.

368

(10) Shewry, P. R.; Tatham, A. S. Improving wheat to remove coeliac epitopes but

369

retain functionality. J. Cereal Sci., 2015, 64, 26-41.

370

(11) Molberg, O.; Uhlen, A. K.; Jensen, T.; Flaete, N. S.; Fleckenstein, B.;

371

Arentz-Hansen, H.; Raki, M.; Lundin, K. E. A.; Sollid, L. M. Mapping of gluten

17



372

T-cell epitopes in the bread wheat ancestors: implications for celiac disease.

373

Gastroenterology, 2005, 128, 393-401.

374

(12) Vaccino, P.; Becker, H. A.; Brandolini, A.; Salamini, F.; Kilian, B. A catalogue of

375

Triticum monococcum genes encoding toxic and immunogenic peptides for celiac

376

disease patients. Mol. Genet. Genomics, 2009, 281, 289-300.

377

(13) Vader, L.W.; Stepniak, D. T.; Bunnik, E. M.; Kooy, Y. M.; de Haan, W.; Drijfhout,

378

J. W.; van Veelen, P. A.; Koning, F. Characterization of cereal toxicity for celiac

379

disease patients based on protein homology in grains. Gastroenterology, 2003, 125,

380

1105-1113.

381

(14) Molberg, Ø.; Solheim, F. N.; Jensen, T.; Lundin, K. E. A.; Arentz-Hansen, E. H.;

382

Anderson, O. D.; Kjersti, U. A.; Sollid, L. M. Intestinal T-cell responses to

383

high-molecular-weight glutenins in celiac disease. Gastroenterology, 2003, 125,

384

337-344.

385

(15) van Herpen, T. W. J. M.; Goryunova, S. V.; van der Schoot, J.; Mitreva, M.;

386

Salentijn, E.; Vorst, O.; Schenk, M. F.; van Veelen, P. A.; Koning, F.; van Soest, L. J.

387

M.; Vosman, B.; Bosch, D.; Hamer, R. J.; Gilissen, L. J. W. J.; Smulders, M. J. M.

388

Alpha-gliadin genes from the A, B, and D genomes of wheat contain different sets of

389

celiac disease epitopes. BMC Genomics, 2006, 7, 1-13.

390

(16) Anderson, O. D.; Litts, J. C.; Gautier, M. F.; Greene, F. C. Nucleic acid sequence

391

and chromosome assignment of a wheat storage protein gene. Nucleic. Acids. Res.,

392

1984, 12, 8129-8144.

18


Page 18 of 32

Page 19 of 32


393

(17) Anderson, O. D.; Litts, J. C.; Greene, F. C. The a-gliadin gene family. I.

394

Characterization of ten new wheat a-gliadin genomic clones, evidence for limited

395

sequence conservation of flanking DNA, and Southern analysis of the gene family.

396

Theor. Appl. Genet., 1997, 95, 50-58.

397

(18) Xie, Z. Z.; Wang, C. Y.; Wang, K.; Wang, S. L.; Li, X. H.; Zhang, Z.; Ma, W. J.;

398

Yan, Y. M. Molecular characterization of the celiac disease epitope domains in

399

α-gliadin genes in Aegilops tauschii and hexaploid wheats (Triticum aestivum L.).

400

Theor. Appl. Genet., 2010, 121, 1239-1251.

401

(19) Salentijn, E. M. J.; Mitea, D. C.; Goryunova, S. V.; van der Meer, I. M.;

402

Padioleau, I.; Gilissen, L. J. W. J.; Koning, F.; Smulders, M. J. M. Celiac disease

403

T-cell epitopes from gamma-gliadins: immunoreactivity depends on the genome of

404

origin, transcript frequency, and flanking protein variation. BMC Genomics, 2012, 13,

405

277-289.

406

(20) Sukhwinder-Singh, Chahal, G. S.; Singh, P. K.; Gill, B. S. Discovery of desirable

407

genes in the germplasm pool of Aegilops tauschii Coss. Indian. J. Genet., 2012, 72,

408

271-277.

409

(21) Tanaka, M.; Tsujimoto, H. Natural habitat of Aegilops squarrosa in Xinjiang

410

Uygur, China. Wheat Inf. Serv., 1991, 73, 33-35.

411

(22) Wei, H. T.; Li, J.; Peng, Z. S.; Lu, B. R.; Zhao, Z. J.; Yang, W. Y. Relationships of

412

Aegilops tauschii revealed by DNA fingerprints: The evidence for agriculture

413

exchange between China and the West. Prog. Nat. Sci., 2008, 18, 1525-1531.

19



414

(23) Wang, J.; Luo, M. C.; Chen, Z.; You, F. M.; Wei, Y.; Zheng, Y.; Dvorak, J.

415

Aegilops tauschii single nucleotide polymorphisms shed light on the origins of wheat

416

D-genome genetic diversity and pinpoint the geographic origin of hexaploid wheat.

417

New Phytol., 2013, 198, 925-937.

418

(24) Gill, B. S.; Raupp, W. J. Direct genetic transfers from Aegilops squarrosa L. to

419

hexaploid wheat. Crop Sci., 1987, 27, 445-450.

420

(25) Marcussen, T.; Sandve, S. R.; Heier, L.; Spannagl, M.; Pfeifer, M. The

421

International Wheat Genome Sequencing Consortium; Jakobsen, K. S.; Wulff, B. B.

422

H.; Steuernagel, B.; Mayer, K. F. X.; Olsen, O. A. Ancient hybridizations among the

423

ancestral genomes of bread wheat. Science, 2014, 345, 1250092.

424

(26) Yan, Y. M.; Hsam, S. L. K.; Yu, J. Z.; Jiang, Y.; Zeller, F. J. Genetic

425

polymorphisms at Gli-Dt gliadin loci in Aegilops tauschii as revealed by acid

426

polyacrylamide gel and capillary electrophoresis. Plant Breeding, 2003, 122, 120-124.

427

(27) Zhang, D. L.; He, T. T.; Liang, H. H.; Huang, L. Y.; Su, Y. Z.; Li, Y. G.; Li, S. P.

428

Flour quality and related molecular characterization of high molecular weight glutenin

429

subunit genes from wild emmer wheat accession TD-256. J. Agric. Food Chem., 2016,

430

64, 5128-5136.

431

(28)Yan, Y. M.; Jiang, Y.; An, X. L.; Peia, Y. H.; Li, X. H.; Zhang, Y. Z.; Wang, A.

432

L.; He, Z.; Xi, X.; Bekes, F.; Ma, W. Cloning, expression and functional analysis of

433

HMW glutenin subunit 1By8 gene from Italy pasta wheat (Triticum turgidum L. ssp.

434

durum). J. Cereal Sci., 2009, 50, 398-406.

20


Page 20 of 32

Page 21 of 32


435

(29) Larkin, M. A.; Blackshields, G.; Brown, N. P.; Chenna, R.; McGettigan, P. A.;

436

McWilliam, H.; Valentin, F.; Wallace, I. M.; Wilm, A.; Lopez, R.; Thompson, J. D.;

437

Gibson, T. J.; Higgins, D. G. Clustal W and Clustal X version 2.0. Bioinformatics,

438

2007, 23, 2947-2948.

439

(30) Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular evolutionary genetics

440

analysis version 7.0 for bigger datasets. Mol. Biol. Evol., 2016, 33, 1870-1874.

441

(31) Cornell, H. J.; Willsjohnson, G. Structure-activity relationships in coeliac-toxic

442

gliadin peptides. Amino. Acids., 2001, 21, 243-53.

443

(32) Wieser, H. Comparative investigations of gluten proteins from different wheat

444

species I. qualitative and quantitative composition of gluten protein types. Eur. Food

445

Res. Technol., 2001, 213, 183-186.

446

(33) Kasarda, D. D.; D'Ovidio, R. Deduced amino acid sequence of an alpha-gliadin

447

gene from spelt wheat (spelta) includes sequences active in celiac disease. Cereal

448

Chem., 1999, 76, 548-551.

449

(34) Spaenij-Dekking, L.; Kooy-Winkelaar, Y.; van Veelen, P.; Drijfhout, J. W.; Jonker,

450

H.; van Soest, L.; Smulders, M. J.; Bosch, D.; Gilissen, L. J.; Koning, F. Natural

451

variation in toxicity of wheat: potential for selection of nontoxic varieties for celiac

452

disease patients. Gastroenterology, 2005, 129, 797-806.

453

(35) Pizzuti, D.; Buda, A.; D'Odorico, A.; D'Inca, R.; Chiarelli, S.; Curioni, A.;

454

Martines, D. Lack of intestinal mucosal toxicity of Triticum monococcum in celiac

455

disease patients. Scand. J. Gastroentero., 2006, 41, 1305-1311.

21



456

(36) Koining, F. Celiac disease: quantity matters. Semin. Immunopathol., 2012, 34,

457

541-549.

458

(37) Matsuoka, Y.; Nishioka, E.; Kawahara, T.; Takumi, S. Genealogical analysis of

459

subspecies divergence and spikelet-shape diversification in central Eurasian wild

460

wheat Aegilops tauschii Coss. Plant Syst. Evol., 2009, 279, 233-244.

461

(38) Anderson, R. P.; van Heel, D. A.; Tye-Din, J. A.; Jewell, D. P.; Hill, A. V. S.

462

Antagonists and non-toxic variants of the dominant wheat gliadin T cell epitope in

463

coeliac disease. Gut., 2006, 55, 485-491.

464

(39) Kaur, A.; Bains, N. S.; Sood, A.; Yadav, B.; Sharma, P.; Kaur, S; Garg, M; Midha,

465

V; Chhuneja, P. Molecular characterization of α-gliadin gene sequences in Indian

466

wheat cultivars vis-a`-vis celiac disease eliciting epitopes. J. Plant Biochem.

467

Biotechnol., 2017, 26, 106-112.

468

(40) Guo, X. H.; Hu, J. L.; Wu, B. H.; Wang, Z. Z.; Wang, D.; Liu, D. C.; Zheng, Y. L.

469

Special HMW-GSs and their genes of Triticum turgidum subsp. dicoccoides accession

470

D141 and the potential utilization in common wheat. Genet. Resour. Crop. Evol. 2016,

471

63, 833-844.

472

(41) Jin, M.; Xie, Z.; Li, J.; Jiang, S.; Ge, P.; Subburaj, S.; Li, X.; Zeller, F. J.; Hsam,

473

S. L. K.; Yan, Y. Identification and molecular characterization of HMW glutenin

474

subunit 1By16* in wild emmer. J. Appl. Genet. 2012, 53, 249-258.

475

(42) Haider, N. Evidence for the origin of the B genome of bread wheat based on

476

chloroplast DNA. Turk. J. Agric. For., 2012, 36, 13-25. 22


Page 22 of 32

Page 23 of 32


477

(43) Liu, B.; Segal, G.; Rong, J. K.; Feldman, M. A chromosome-specific sequence

478

common to the B genome of polyploid wheat and Aegilops searsii. Plant Syst. Evol.,

479

2003, 241, 55-66.

23



Page 24 of 32

480

Table 1 Variations of the CD epitope domains in α-gliadin genes from Aegilops

481

tauschii

Epitope

Risk for celiac disease

Expression in cis or trans position

DQ2.5-glia-α1a

High

cis, trans

DQ2.5-glia-α2

High

cis, trans

DQ2.5-glia-α3

High

cis, trans

DQ8-glia-α1

Low

cis

Sequences

Number of α-gliadin gene

PFPQPQLPY PFPQPQQPY PFP - SQLPY PFLQPQLPY PFPQPRLPY PFPQPQ - PF PQPQLPYPQ PQP - - - - - PQPQLLYPQ PHPQLPYPQ LQPQLPYPQ PHPQLSYPQ P - PQLPYPQ SQP - - - - - FRPQQPYPQ FPPQQPYPQ FRPRQPYPQ FRPQQPHPQ LRPQQPYPQ FSPQQPYPQ FRPQQLYPQ QGSFQPSQQ QGSFEPSQQ QGFFQPSQQ QGSFRPSQQ QGSFEPSHQ

43 7 5 2 1 1 15 22 15 3 1 1 1 1 48 5 2 1 1 1 1 52 4 1 1 1

482

Note: Sequences highlighted in blue are the intact epitopes, in pink are the epitope

483

variants, in red are the mutational amino acid residues. Dashes represent deletion of

484

amino acids residues.

24


Page 25 of 32


485

Figure Captions

486

Fig. 1 PCR amplification of α-gliadin genes from Ae. tauschii accession T006. M:

487

DL2000 marker; 1: the amplified product of primers P1 and P2.

488

Fig. 2 Phylogenetic tree constructed based on the deduced amino acid sequences of

489

the 59 α-gliadin genes. The group I, II and III are distinguished in red, blue and brown,

490

respectively. The bootstrap values less than 60% are not indicated.

491

Fig. 3 Multiple alignment of the deduced amino acid sequences in the 9 α-gliadin

492

genes. Dashes represent the deletion of amino acids residues. Dots indicate the

493

identical amino acid residues. Pink segments of the aligned sequences show the

494

position of the T-cell stimulatory epitopes.

495

Fig. 4 Frequency of amino acid residues substitution of 4 toxic epitope domains in 59

496

α-gliadins. Arg: arginine; Gln: glutamine; Leu: leucine; Phe: phenylalanine; Pro:

497

proline; Tyr: tyrosine.

498

Fig. 5 Distribution of toxic epitope combinations in 59 α-gliadins. α1a:

499

DQ2.5-glia-α1a; α2: DQ2.5-glia-α2; α3: DQ2.5-glia-α3; α1: DQ8-glia-α1; N: none of

500

four toxic epitopes.

501

Fig. 6 Neighbor-joining tree of the deduced amino acid sequences from the 59

502

α-gliadin genes in this study and 40 α-gliadin genes in Triticum and Aegilops species.

503

Groups I, II and II are distinguished with strings in brown, blue and red colors. The 7

504

α-gliadins without any intact toxic epitope from Ae. tauschii accession T006 are

505

marked by boxes. The bootstrap values less than 60% are not indicated.

25



506 507

Fig. 1 PCR amplification of α-gliadin genes from Ae. tauschii accession T006. M:

508

DL2000 marker; 1: the amplified product of primers P1 and P2.

26


Page 26 of 32

Page 27 of 32


509 510

Fig. 2 Neighbor-joining tree constructed based on the deduced amino acid sequences

511

of the 59 α-gliadin genes. The group I, II and III are distinguished in red, blue and

512

brown, respectively. The bootstrap values less than 60% are not indicated. 27



513 514

Fig. 3 Multiple alignment of the deduced amino acid sequences in the 9 α-gliadin

515

genes. Dashes represent the deletion of amino acids residues. Dots indicate the

516

identical amino acid residues. Pink segments of the aligned sequences show the

517

position of the T-cell stimulatory epitopes.

28


Page 28 of 32

Page 29 of 32


518 519

Fig. 4 Frequency of amino acid residues substitution of 4 toxic epitope domains in 59

520

α-gliadins. Arg: arginine; Gln: glutamine; Leu: leucine; Phe: phenylalanine; Pro:

521

proline; Tyr: tyrosine.

29



522 523

Fig. 5 Distribution of toxic epitope combinations in 59 α-gliadins. α1a:

524

DQ2.5-glia-α1a; α2: DQ2.5-glia-α2; α3: DQ2.5-glia-α3; α1: DQ8-glia-α1; N: none of

525

four toxic epitopes.

30


Page 30 of 32

Page 31 of 32


526 527

Fig. 6 Neighbor-joining tree of the deduced amino acid sequences from the 59

528

α-gliadin genes in this study and 40 α-gliadin genes in Triticum and Aegilops species.

529

Groups I, II and II are distinguished with strings in brown, blue and red colors. The 7

530

α-gliadins without any intact toxic epitope from Ae. tauschii accession T006 are

531

marked by boxes. The bootstrap values less than 60% are not indicated.

532

31



533

TOC Graphic

534

32


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Molecular Characterization and Variation of the Celiac Disease

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