Immunomodulatory properties of protein hydrolysates
Kiewiet, Mensiena Berentje Geertje
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Publication date: 2018
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Kiewiet, M. B. G. (2018). Immunomodulatory properties of protein hydrolysates. Rijksuniversiteit Groningen.
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8
wheat hydrolysate for clinical nutrition
M.B.G. Kiewiet1, R. Dekkers2, M. P. van Gool2, L.H. Ulfman2,
A. Groeneveld2, M. M. Faas1,3, P. de Vos1
1Immunoendocrinology, Division of Medical Biology, Department of Pathology and
Medical Biology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands
2FrieslandCampina, Stationsplein 4, 3818 LE Amersfoort, The Netherlands 3Department of Obstetrics and Gynecology, University of Groningen, University
Abstract
Wheat hydrolysates are used for medical nutrition to provide undernourished patients a readily digestible protein source for instance to recover from chemotherapy induced damage to the intestine. Another potential route for hydrolysates to interfere with chemotherapy induced intestinal damage and inflammation is via the immune system since there is evidence that Toll-like receptors (TLRs) are involved in this process. Here we determined the capacity of three wheat hydrolysates to modulate immunity by interfering with TLR signaling. We studied possible inhibiting effects on TLR2, TLR4, and TLR9. All wheat hydrolysates had TLR modulating effects but only one had strong TLR2 inhibiting effects, both attenuating TLR2/1 and TLR2/6 signaling in a reporter cell system. Furthermore, this TLR2 blocking hydrolysate reduced IL-6 production in human dendritic cells. Application of Reversed Phase- Ultra High Performance Liquid Chromatography combined with mass-spectrometry revealed that the presence of peptide WQIPEQSR is associated with the TLR2 inhibiting capacity of wheat hydrolysates. Our study demonstrates TLR2 inhibiting capacities of a wheat hydrolysate. As not all hydrolysates have TLR2 inhibiting capacity, our technology platform with TLR expressing cells can be instrumental in identification of these bioactive hydrolysates. As chemotherapy induced intestinal inflammation is often TLR2 dependent, our findings provide a good start for further research to investigate whether these hydrolysates might contribute to the management of mucositis in cancer patients receiving chemotherapy.
8
Introduction
Nutritional interventions with hydrolysates are used to prevent adverse clinical outcomes in undernourished patients [1]. About 30% of hospitalized patients are at risk for undernutrition, which leads to prolonged hospital stay, increased readmissions and increased mortality [2,3]. This malnutrition is not always caused by decreased appetite, but also by malabsorption due to intestinal issues, such as inflammation [4]. Malabsorption may be the consequence of disease, or develop as a side-effect of pharmaceutical intervention. For example, the majority of cancer patients treated with chemotherapeutics develops gastrointestinal mucositis or ileitis, resulting in malnutrition [5].
Clinical nutrition to treat undernourished patients with intestinal issues contains high amounts of proteins [6], that are hydrolyzed to facilitate uptake in the damaged intestine [7]. Wheat hydrolysates are of special interest for this type of nutritional products, due to their relatively high peptide-bound glutamine content [8], compared to for example cow’s milk hydrolysates. Glutamine supplementation was found to decrease the length of a hospital stay, complications and mortality in critical ill patients [9].
Besides their nutritional value, hydrolysates have been recognized to actively modulate the immune system [10]. Hydrolysis of intact proteins leads to the release of bioactive peptides which are normally hidden in the intact protein. Hydrolysates from different protein sources were found to induce many different immune effects, including a range of anti-inflammatory effects [11-14]. These effects can be induced via Toll-like receptors (TLRs), since we previously showed immune effects of cow’s milk hydrolysates via TLRs activation or inhibition [15]. For wheat, this has not been studied to the same extend, but since wheat hydrolysates also showed similar immune effects [16], wheat hydrolysates could potentially modulate immunity via TLR signaling.
A category of intestinal disorders in which the immunomodulating effects of wheat hydrolysates may be of special interest is mucositis and ileitis induced by chemotherapy. Current estimates are that 40-100% of all patients on chemotherapy suffer from mucositis and ileitis [17-20]. Currently, there is no cure for this side effect of chemotherapy but it is known that TLR signaling is involved [21]. It starts with injury of epithelial and underlying cells by the chemotherapeutic drug, which results in the release of Damage Associated Molecular Patterns (DAMPs) by dying intestinal cells. These DAMPs are intracellular components such as heat-shock proteins and DNA-fragments [22] that stimulate TLRs and induce immune activation [23] causing mucosal damage and barrier dysfunction [24]. Especially TLR2, TLR4, and TLR9 activation has been found to play a role in intestinal mucositis development, and a deficiency in these receptors was found to be protective against mucositis [25,26].
Here we studied the possible inhibiting effects on TLR2, TLR4, and TLR9 activation of three wheat hydrolysates, which might contain TLR modulating proteins and peptides. We further studied the effects of the most potential hydrolysate in more detail by investigating the inhibiting effects of the TLR2/1 and TLR2/6 dimers and by studying effects on dendritic cell cytokine production. We also identified the bioactive fraction within the hydrolysate, and determined possible bioactive peptides from this fraction. Our technology platform containing TLR expressing reporter cells might lead to identification of specific wheat hydrolysates that not only serve as a nutrient source, but can also serve as bioactive food component.
Materials and methods
Tested materials
Wheat hydrolysates were provided by FrieslandCampina (Amersfoort, the Netherlands). The hydrolysates were produced by a two-step digestion of the source materials. Molecular weight distributions of the hydrolysates were obtained allowing identification of differences in protein and peptide composition. The samples were tested for endotoxins by using the Limulus amebocyte lysate assay (LAL) according to the manufacturer’s instructions (ThermoFisher Scientific, Waltham, US). Endotoxin concentrations in the samples had no significant activating effect on the cells applied.
Inhibition assay using HEK-XBlueTM-hTLR2, 4, and 9 reporter cells
To test whether the wheat hydrolysates are able to inhibit TLR2, 4, and 9 activation induced by known ligands, the samples were tested on a HEK-XBlueTM-hTLR2, 4, and 9 (Invivogen, Toulouse,
France) reporter cell assay. To quantify TLR activation, the cell line contains both a TLR construct and a construct for Secreted Embryonic Alkaline Phosphatase (SEAP), which was coupled to the nuclear factor κB/Activating protein-1 (NF-κB/AP-1) promoter. NF-κB/AP-1 is a known downstream target of TLR receptors [27,28].
Cells were cultured following the manufacturer’s instructions, and as described before [27]. For the assay, cells were seeded in a flat bottom 96 wells plate at a concentration described in table 1 (180 μL/well). Cells were stimulated with 2 mg/mL wheat hydrolysate, and the respective activating ligand (table 1) at the same time, and incubated for 24 hours (37 °C, 95% oxygen, 5% CO2). TLR ligand alone was used as a positive control. Medium was used as a negative control.
After incubation, Quanti-Blue detection medium was used to analyze the cell supernatant as described before [27]. Absorbance (650 nm) was quantified using a VersaMax microplate reader (Molecular Devices GmbH, Biberach an der Riss, Germany) and SoftMax Pro Data Acquisition & Analysis Software to determine SEAP activity, which represents activation of NF-κB/AP-1. The median and range for each sample were plotted as the fold-change compared to the positive control, which were TLR ligand stimulated cells. The positive controls were set at 1.
Since the most striking inhibiting effect was observed by Wheat 1 on TLR2, we further investigated this effect. The dose dependency of the overall TLR2 inhibition was tested by stimulating HEK-XBlueTM-hTLR2 cells with graded concentrations of wheat hydrolysate and HKLM
(table 1), after which cells were incubated for 24 hours (37 °C, 95% oxygen, 5% CO2) and analyzed.
The TLR2 receptor forms heterodimers with TLR1 and TLR6 in order to recognize a broader range of ligands [29] and stimulate different immune pathways [30,30,31]. In order to investigate whether TLR2 inhibition is mediated via the TLR2/TLR1 or TLR2/TLR6 heterodimer, the above experiment was repeated using the TLR2/TLR1 specific tri-acetylated lipopeptide P3CSK4 (25 ng/ mL) [32] and the TLR2/TLR6 specific di-acetylated lipopeptide FSL-1 (25 ng/mL) [33] as activating ligands instead of HKLM.
Cell line Cell density for seeding Positive control (concentration in well)
HEK-Blue human TLR2 2.8*105cells/mL (180 µl/well)) Heat killed Listeria monocytogenes (107cells/mL) P3CSK4 (25 ng/mL)
FSL-1 (25 ng/mL)
HEK-Blue human TLR4 1.4*105cells/mL (180 µl/well) Escherichia coli K12 Lipopolysaccharide (10 ng/mL)
HEK-Blue human TLR9 4.5*105cells/mL (180 µl/well) Type B CpG oligonucleotide (ODN 2006, 0,25 μM)
Table 1. Cell densities and ligands used in the different reporter cell line assays.
Direct stimulation of dendritic cells (DCs) with wheat hydrolysate
To investigate the direct effects of wheat hydrolysate on human DCs, cytokine production was measured after stimulation of immature DCs with the wheat hydrolysate for 24 hours. DCs were purchased from MatTek Corporation (Ashland, MA, USA).
Stimulations were performed by seeding 6x104/well freshly thawed DCs in each well of a 96
wells plate (in 200 μL). Cells were precultured for 24 h before starting the experiment as described in the manufacturer’s instructions. Then, cells were exposed to 2 mg/mL hydrolysate and 107
cells/mL HKLM, after which cells were incubated for 24 h (37 °C, 5% CO2). HKLM alone was used as a positive control, medium as a negative control. To assess the role of TLR2 inhibition in the effects observed, instead of the hydrolysate, cells were treated with 5 μg/mL TLR2 blocking antibody (PAb-hTLR2, Invivogen, Toulouse, France) for 30 min, after which HKLM was added. Supernatant was collected and stored at -80 °C for cytokine measurements.
Assessment of cytokine expression
The levels of IL-1β, IL-1RA, IL-10, IL-12, IL-6, IL-8, MCP-1/CCL2, MIP-1α/CCL3, RANTES/CCL5, TNFα, and TSLP in the DC supernatant were measured using a custom-made ProcartaPlex® multiplex immunoassay (Affymetrix, CA, USA). The immunoassay was performed according to the manufacturer’s protocol. Briefly, cytokine standards were resuspended, and serial dilutions were prepared. Antibody magnetic bead mix was added to the plate. After washing, standards and samples were added (50 μL/well), the plate was sealed, and incubated while shaking (30 min at room temperature (RT), overnight at 4 °C, and again 30 min at RT). After washing the plate twice, detection antibodies were added (25 μL/well) and the plate was incubated for 30 min at RT on a plate shaker. After incubation, the plate was washed twice and 50 μL/well streptavidin-phycoerythrin was added. Again, the plate was incubated at RT for 30 min while shaking. To prepare the plate for analysis, the plate was washed, and 120 μL/well of reading buffer was added. After shaking the plate for 5 min at RT fluorescence was measured using a Luminex 100 System. The data obtained were analyzed using StarStation software.
Fractionation of the hydrolysate
The hydrolysate was fractionated based on size using an Amicon stirred cell (Merck, Nottingham, UK) with a capacity of 50 mL. Before use, the ultracentrifugal unit was sterilized, and the collecting tubes were cleaned with 70% ethanol. Filter membranes with MWCO’s of 3 kD (regenerated
cellulose), 1 kD (regenerated cellulose) and 0.5 kD (cellulose acetate) (all Merck, Nottingham, UK) were used to prepare fractions containing peptides and proteins >3 kD, peptides between 3 and 1 kD, peptides between 1 and 0.5 kD and peptides <0.5 kD. To remove glycerine from the membranes before use, the membranes were soaked in sterile H2O for 1 hour, while changing the
sterile H2O three times.
Then, the hydrolysate was dissolved in 50 mL sterile water at a concentration of 40 mg/mL. To remove undissolved particles that could block the filtration membranes, the hydrolysate was centrifuged at 4000g for 5 min, after which the supernatant was used for further processing. Two mL of the supernatant was stored at -20 °C. The rest of the supernatant was first filtered using the 3 kD filter under continuous stirring, by applying a N2 pressure (3,5 bar). The permeate was
collected in a 50 mL tube, until approximately 80% of the sample was filtered. Then, the filtration was stopped, and the retentate was stirred for 15 min to remove proteins from the membrane. The retentate was also collected in a 50 mL tube. The described filtration steps were repeated for the collected 3 kD permeate using the 1 kD filter and for the collected 1 kD permeate using the 0.5 kD filter. Sterile water was filtered in the same manner to check for contamination during the filtration steps. The filtration process was performed 7 times and fractions were pooled in order to collect enough volume of the sample for subsequent experiments. The protein concentration in the pooled fractions was measured using a Pierce BCA protein assay, following manufacturer’s instructions (Thermoscientific, Pittsburgh, USA). Fractions with a concentration less than 20 mg/ mL were concentrated using a SpeedVac centrifugal evaporator (ThermoScientific, Pittsburgh, USA) for 4 hours. The filtered sterile water was concentrated in the same manner. The protein concentration of the concentrated protein fractions were measured again, and were now 20 mg/ mL or higher. Fractions were stored at -20 °C before testing in the HEK-XBlueTM-hTLR2 cell lines as
described above.
Peptide analysis on Reversed Phase Ultra High Performance Liquid Chromatography (RP-UHPLC) coupled to mass spectrometry (MS)
In order to investigate which individual peptides could be responsible for TLR modulating effects, the peptide composition of the specific hydrolysate was fractionated and analyzed with RP-UHPLC coupled to MS. To this end, wheat fraction samples (3-1 kD, 1-0.5 D and <0.5 kD) were diluted 4x with Mili-Q water. The diluted samples were centrifuged (16.110 g for 10 min), after which the supernatant was transferred to an HPLC vial. The obtained fractions were analyzed on an H class Acquity UPLC system (Waters, Milford, MA, USA) equipped with a BEH C18 column (1.7 μm, 2.1×100 mm, Waters) with an Acquity BEH C18 guard precolumn. The UPLC system was coupled to an Acquity 145 UPLC® PDA detector (Waters). Separation was carried out using the following elution profile at a flowrate of 0.350 mL/min: 5% ACN isocratic for 2 min; 5-42% ACN in 37 min; isocratic cleaning step of 90% ACN for the duration of 3 min; and re-equilibration to starting conditions for 6 min. Ultraviolet (UV) data was acquired using MassLynx software (Waters). The mass spectra of the peptides were determined with Electron Spray Ionization Time of Flight Mass Spectrometry (ESI -Q-TOF-MS), using an online SYNAPT G2-Si high definition mass spectrometer (Waters) coupled to the RP-UHPLC. The system was calibrated with sodium iodide. The capillary voltage was set to 3 kV with the source operation in positive ion mode and the
source temperature at 150 °C. The sample cone was operated at 40 V. Nitrogen was used as desolvation gas (500 °C, 800 L/h) and cone gas (200 L/h). MS and MS/MS (Resolution method) data were collected between m/z 100-3000 with a scan time of 0.3 seconds. Online lock mass data (Angiotensin II, Mw 523.7751 Da) were collected and the correction was applied during data reprocessing. The data were analyzed using Unifi software (Waters).
The mass tolerance for the accepted annotation was ≤10 ppm between the theoretical mass and the measured mass. A generic method was used in which non-specific enzyme specificity was selected. A list with intact wheat storage proteins used in the analysis is included as supplementary file S1. The peptides were annotated by MS and confirmed by MS/MS through the presence of fragment ions with an assigned intensity of at least 50%.
Further analysis was performed by comparing the peptide compositions of the fractions. This was done by investigating which peptides were exclusively present in a fraction with TLR modulating effects. When peptides were present in all fractions, it was determined whether the relative abundance was higher in the specific TLR modulating fraction compared to fractions with lesser effects.
Statistical analysis
Statistical analysis was performed using Graphpad Prism. Normal distribution of the data was tested using the Kolmogorov-Smirnov test. When data were normally distributed, which was the case for the cytokine production levels by the stimulated DCs, values were expressed as mean ± standard deviation (SD). One-way ANOVA, followed by t-tests was used to identify differences. When data were not normally distributed, values were expressed as median with range. Significant differences were in that case assessed using a Kruskal-Wallis test followed by a Dunn’s post test. A
p-value of <0.05 was considered to indicate a statistical significant difference. Results
Characteristics of the wheat hydrolysates
The wheat protein hydrolysates were obtained by a two-step hydrolysis. Three different wheat hydrolysates with different peptide compositions were investigated, as illustrated by the different molecular weight distributions of the hydrolysates as shown in table 2. Wheat 1 differed most from the other wheat hydrolysates as it was found that Wheat 1 contained more proteins bigger
samples source Molecular weight distribution (%) >10,000 Da 10,000 - 5,000 Da 5,000 - 2,000 Da 2,000 - 1,000 Da 1,000 – 500 Da < 500 Da Wheat 1 Wheat gluten 17 15 20 14 12 22 Wheat 2 Wheat gluten 1 1 5 9 18 66 Wheat 3 Wheat gluten 1 1 4 7 13 74
Table 2. Overview of the characteristics of the hydrolysates studied
Wheat 1 hydrolysate strongly inhibits HKLM induced TLR2 activation in a dose dependent manner
In order to test the TLR inhibiting capacity of the wheat hydrolysates, HEK-XBlueTM-hTLR2, 4, and
9 reporter cells were stimulated with 2 mg/mL hydrolysate and the corresponding ligands to induce TLR activation. After 24 hours of stimulation, TLR activation was determined.
Wheat hydrolysates showed TLR inhibiting capacities after TLR stimulation with a known ligand (figure 1). Wheat 1 had the most pronounced effects. Besides a small, but statistically significant inhibition of TLR4 and TLR9 (TLR4 p<0.001; TLR9 p<0.01), it showed a strong TLR2 inhibiting effect (p<0.0001). Wheat 2 only inhibited TLR4 (p<0.01), while Wheat 3 inhibited TLR4 and 9 (TLR4 p<0.001;TLR9 p<0.01).
Figure 1. NF-κB/AP-1 activation in HEK-XBlueTM-hTLR2, 4, and 9 cells after simultaneous stimulation with its relevant ligand and 2 mg/mL of the wheat hydrolysates. Wheat hydrolysates were able to inhibit TLR activation. Hydrolysate Wheat 1 inhibited activation of TLR2, 4, and 9, with a strong inhibition of TLR2. Wheat 2 inhibited TLR4, while Wheat 3 inhibited TLR 4 and 9. Significant differences were determined by using the Kruskal-Wallis test followed by the Dunn’s test. Significant differences compared to the negative control were indicated by * (p<0.05), ** (p<0.01),*** (p<0.001) or by **** (p<0.0001), significant differences compared to the positive control were indicated by # (p<0.05), ## (p<0.01),### (p<0.001) or by #### (p<0.0001).
Since the strongest inhibiting effect was induced by Wheat 1 on TLR2, in further experiments, we only used Wheat 1 and studied its effect on TLR2 in more detail. To study the dose response effect of Wheat 1 on TLR2 inhibition, HEK-XBlueTM-hTLR2 cells where incubated with graded
concentrations of Wheat 1 and HKLM (107 cells/mL) (figure 2). TLR inhibition was found to be
decreased in a dose dependent fashion. A concentration of 1 mg/mL was the minimal hydrolysate concentration that was still able to induce a significantly decreased TLR2 activation (p<0.05). The maximal TLR2 inhibition was observed at a concentration of 5 mg/mL, with a median of 0.25 (0.17-0.60), which corresponds to a decrease in TLR2 activation of 75%.
Figure 2. NF-κB/AP-1 activation in HEK-XBlueTM-hTLR2 cells after simultaneous stimulation with 107 cells/ mL HKLM and graded concentrations of Wheat 1. Wheat 1 showed TLR2 inhibition in a dose dependent way. Significant differences were determined by using the Kruskal-Wallis test followed by the Dunn’s test. Significant differences compared to the negative control were indicated by * (p<0.05), ** (p<0.01),*** (p<0.001) or by **** (p<0.0001), significant differences compared to the positive control were indicated by # (p<0.05), ## (p<0.01),### (p<0.001) or by #### (p<0.0001).
Wheat 1 was able to inhibit both TLR2/TLR1 and TLR2/TLR6 signaling
TLR2 is able to form a heterodimer with either TLR1 or TLR6, which leads to different downstream immune responses [30,31]. In order to determine the heterodimer specificity of the TLR2 inhibiting effect of Wheat 1, HEK-XBlueTM-hTLR2 reporter cells were incubated with different concentrations
of Wheat 1 together with either the TLR2/TLR1 ligand P3CSK4 [32] or the TLR2/TLR6 ligand FSL-1 [33].
As shown in figure 3, Wheat 1 was able to inhibit both P3CSK4 and FSL-1 induced TLR2 activation. Both for P3CSK4 and FSL-1 stimulated cells, 1 mg/mL was the lowest hydrolysate concentration that was still able to induce a statistically significant decreased TLR2 activation compared to ligand stimulated cells (p<0.05). Independent of the TLR2 activating ligand used, a hydrolysate concentration of 6 mg/mL induced the strongest TLR2 inhibition. The TLR2 activation
dropped to 0.17 (0.09-0.23) for P3CSK4 and to 0.22 (0.13- 0.32) for FSL-1, indicating a 83 and 78% TLR2 inhibition, respectively.
Figure 3. NF-κB/AP-1 activation in HEK-XBlueTM-hTLR2 cells after simultaneous stimulation with either the TLR2/1 ligand P3CSK4 or the TLR2/6 ligand FSL-1 and graded concentrations of Wheat 1. Wheat 1 showed TLR2 inhibition in a dose dependent way, both in 25 ng/mL P3CSK4 and 25 ng/mL FSL-1 stimulated cells. Significant differences were determined by using the Kruskal-Wallis test followed by the Dunn’s test. Significant differences compared to the negative control were indicated by * (p<0.05), ** (p<0.01),*** (p<0.001) or by **** (p<0.0001), significant differences compared to the positive control were indicated by # (p<0.05), ## (p<0.01),### (p<0.001) or by #### (p<0.0001).
Wheat 1 decreased HKLM induced IL-6 production in DCs
Next, we investigated the effects of Wheat 1 on HKLM stimulated human DCs. DCs are important in orchestrating intestinal immune responses [34,35]. Figure 4 shows the effects of Wheat 1 on the production of the regulatory cytokine IL-10, the proinflammatory cytokine IL- 12 and the pleiotropic cytokine IL-6 of HKLM stimulated DCs, which are associated with DC activation [36]. Effects on other cytokines measured are shown in supplementary file 2.
The production of IL-12 was significantly increased after DC stimulation for 24 hours with either Wheat 1 alone or HKLM alone compared to the negative control (both p<0.05). When DCs were treated with a combination of TLR2 activating HKLM and Wheat 1, IL-12 production was also significantly increased compared to unstimulated cells (p<0.05), and this effect was even significantly higher compared to DCs treated with HKLM alone. The TLR2 blocking antibody had no effect on the IL-12 production of HKLM stimulated cells.
For IL-10 a similar effect was observed. Stimulation of DCs with Wheat 1 alone increased 10 production significantly (p<0.05), while HKLM stimulation had no significant effect on IL-10 production by DCs. DCs treated with a combination of Wheat 1 and HKLM did result in an increased IL-10 production, which was significantly different from both the unstimulated cells and HKLM treated cells (both p<0.05). Again, the TLR2 blocking antibody did not affect HKLM
stimulated DC IL-10 production.
For IL-6 another effect was observed. Stimulation with Wheat 1 alone did not change IL-6 production of DCs, while HKLM stimulation significantly increased IL-6 production compared to the IL-6 production of untreated cells (p<0.05). Interestingly, when DCs were treated with a combination of HKLM and Wheat 1, IL-6 production was significantly decreased compared to DCs stimulated with HKLM alone (p<0.05). This effect could be induced via TLR2, since the IL-6 production in DCs treated with HKLM after preincubation with the TLR2 blocking antibody was similar to DCs treated with HKLM and Wheat 1.
Figure 4. Cytokine production in DCs stimulated with Wheat 1, HKLM, or a combination. Wheat 1 (2 mg/mL) increased IL-12 and IL-10 production in DCs, either alone or in combination with HKLM. Wheat 1 was able to inhibit HKLM induced IL-6 production. Preincubation of DCs with a TLR2 antibody showed the same effect. Statistically significant differences compared to the negative control were determined by using t-tests and indicated by *.
The peptide fraction containing peptides smaller than 0.5 kD has the strongest TLR2 inhibiting effects
To investigate which peptide(s) in Wheat 1 are responsible for the TLR inhibiting effects, size based fractions were produced (>3 kD, 3-1 kD, 1-0.5 kD and <0.5 kD), and tested for TLR2 inhibiting effects in HKLM stimulated HEK-XBlueTM-hTLR2 reporter cells.
As shown in figure 5, it was found that only the fractions 1-0.5 kD and <0.5 kD showed a significantly reduced TLR2 activation compared to cells treated with HKLM alone (both p<0.05). The fraction containing the peptides smaller than 0.5 kD showed the strongest TLR2 inhibiting effect.
Figure 5. NF-κB/AP-1 activation in HEK-XBlueTM-hTLR2 cells after simultaneous stimulation with 107 cells/ mL HKLM and different size based fractions of Wheat 1. Only the two smallest fractions, 1-0.5 kD and <0.5 kD, showed TLR2 inhibition in HKLM stimulated cells. Significant differences were determined by using the Kruskal-Wallis test followed by the Dunn’s test. Significant differences compared to the negative control were indicated by * (p<0.05), ** (p<0.01),*** (p<0.001) or by **** (p<0.0001), significant differences compared to the positive control were indicated by # (p<0.05), ## (p<0.01),### (p<0.001) or by #### (p<0.0001).
Peptides with a potential TLR2 inhibiting effect were identified in the fraction <0.5 kD To identify the specific peptides responsible for the TLR2 inhibition in the fractions 1-0.5 kD and <0.5 kD, RP-UHPLC coupled to MS was applied. The analysis also included fraction 3-1 kD, since this fraction contained no inhibiting effect. In the fraction 3-1 kD 1300 peptides were identified. In the fraction 1-0.5 kD 930 peptides were identified, and in the fraction <0.5 kD 862 peptides were identified.
Differences in peptide composition can be observed between fractions, by comparing UV absorbance profiles at 214 nm (figure 6). The two TLR2 inhibiting fractions showed similar patterns in the peptide area (~0-23 min). The 3-1 kD fraction, which did not inhibit TLR2, showed a different pattern. Most distinct differences are present at lower retention times, until about 10 min, where several peaks had a higher UV response compared to the 3-1 kD fraction. The mass spectrum (figure 7) shows similar differences between fractions, although a higher peak in the 1-0.5 kD and <0.5 kD fractions compared to the 3-1 kD fraction was also observed here at a retention time between 20 and 21 min. It should be noted that a relative intensity is given in the mass spectrum and that the higher peak can also be due to higher ionization affinity instead of quantity of a specific peptide.
The next step was to identify which peptides were uniquely present in TLR2 inhibiting fractions (<0.5 kD and 1-0.5 kD), by comparing the different peptides in the three fractions, where only those were selected of having an assigned intensity of 50% or higher. The peptides IFWGIPA and IAPVGIF were present in all fractions, and did not differ in relative abundance (response). The peptides MHILLP, TTIAPFGIFGTN, ILQQQL, and VCSVSQIIMRQ were unique for the <0.5 kD fraction and absent in both the 1-0.5 kD and 3- 1 kD fraction. Interestingly, the peptide WQIPEQSR was present in both TLR2 inhibiting fractions, but not in the 3-1 kD fraction without TLR2 inhibiting effects (table 3). The relative abundance (response) of the peptide WQIPEQSR was similar in both
Figure 6. UV chromatogram of the peptide fractions 3-1 kD, 1-0.5 kD and <0.5 kD of Wheat 1. Specifi c peaks were increased in the TLR2 inhibiting fractions 1-0.5 kD and <0.5 kD compared to the not inhibiting 3-1 kD fraction, mainly at lower retention times. Also some new peaks are formed in these fractions.
Figure 7. Mass spectrum (total ion count) of the peptide fractions 3-1 kD, 1-0.5 kD and <0.5 kD of Wheat 1. Relative intensity of several peaks was higher in the TLR2 inhibiting fractions 1-0.5 kD and <0.5 kD compared to the not inhibiting 3-1 kD fraction, and some new peaks are present in these fractions.
peptide Size
(Dalton) Source protein Present in fraction
3-1 kD 1-0.5
kD <0.5 kD
MHILLP 723 ɣ-gliadin no no yes
TTIAPFGIFGTN 1238 α/β gliadin no no yes
ILQQQL 742 α/β -gliadin,
α-gliadin no no yes
VCSVSQIIMRQ 1263 Avidin like
A2,A3, A7 no no yes
WQIPEQSR 1043 α/β gliadin no yes yes
Table 3. A list of possible TLR2 inhibiting peptides
Discussion
Wheat hydrolysates are used for medical nutrition to provide undernourished patients a readily digestible protein source for instance to recover from chemotherapy induced damage to the intestine. Another potential beneficial effect of hydrolysates on chemotherapy induced intestinal damage and inflammation is via the modulation of the immune system. There is evidence that Toll-like receptors (TLRs) are involved in the induction of mucositis and ileitis [25] and some hydrolysates have been found to be capable of TLR modulation [15]. Therefore, we studied TLR inhibiting effects of three wheat hydrolysates used in medical nutrition.
The inhibiting capacities of the three wheat hydrolysates were first studied for TLR2, 4, and 9, which are all associated with mucositis development [25,26]. They all had TLR inhibitory effects but to a varying extent and the effect was highly hydrolysate dependent. Wheat 1 showed a strong TLR2 inhibiting effect, which was not observed for the other hydrolysates. TLR2 is the most studied TLR in relation with mucositis [25,37,38]. Its blockade can prevent doxorubicin induced mucositis [25]. Doxorubicin is one of the most potent and commonly applied chemotherapeutic drugs [39], but its application is sometimes limited by its toxicity [40]. Our data suggest that wheat hydrolysates might be instrumental in doxorubicin treated patients by increasing its therapeutic potential due to its TLR2 inhibiting effects. Since TLR2 regulation of the intestinal damage was found to be chemotherapeutic drug specific [23], the effects of wheat hydrolysates might differ between chemotherapies.
TLR2 has the unique capacity to form dimers with TLR1 or TLR6 [29]. Depending on the dimer formed, more pro- or anti-inflammatory responses are induced [30,31]. To better understand the effects of TLR2 inhibition by Wheat 1, we investigated whether the inhibiting effect was specific for TLR2/1 or TLR2/6 or a combination thereof. It was found that Wheat 1 inhibited both TLR2/1 and TLR2/6 signaling (figure 3), indicating that Wheat 1 blocks the TLR2 ligand binding site itself, in such a way that it does not interfere with the TLR2/1 and TLR2/6 dimerization. TLR2 has been shown before to be able to bind to proteins and (synthetic) peptides [41-43]. The TLR2 ligand binding site has a large binding surface with many insertions and β-sheets to which many proteins can bind [44]. More research is needed however to further study the binding mechanism of hydrolysate peptides to TLR2.
Stimulating DCs with Wheat 1, HKLM or a combination had distinct effects on the production of the cytokines IL-12, IL-10, and IL-6 (figure 4). TLR2 activation by HKLM had a pronounced effect on IL-6 production, which is known to be merely TLR2 dependent [45,46]. Although IL-6 is a pleiotropic cytokine, it is known to enhance pro-inflammatory events during intestinal injury. In mucositis, IL-6, together with TNFα and IL-1β, was found to be significantly increased both in blood and intestinal tissue in animal models and patients [47-49]. Especially IL-6 levels correlate with the severity of the inflammation [50], and reduction of IL-6 attenuates intestinal inflammation [51]. Here we found that Wheat 1 was able to inhibit the HKLM induced IL-6 production in DCs. Therefore, administration of wheat hydrolysate might be a new way to provide nutrients and simultaneously inhibit IL-6, although more research is needed to confirm this. IL-10 and IL-12, nor any of the other cytokines measured, were inhibited by Wheat 1 which confirms the TLR2 specificity of the inhibiting effect of Wheat 1, as regulation of these cytokines dependents more on other TLR types such as TLR4 [52,53].
In order to be able to create reproducible TLR2 inhibiting wheat hydrolysates we designed a strategy to identify the unique peptide sequences in Wheat 1 that might be responsible for the TLR2 inhibiting effects. We obtained size based fractions of the hydrolysate and detected most of the TLR2 inhibitory effects in the smallest fraction (<0.5 kD). Five possible TLR2 inhibiting peptides in this fraction were identified by analyzing the fractions using RP-UPLC-MS and comparing their peptide composition. Since only the peptide WQIPEQSR was present in both fractions showing TLR inhibition and since it was absent in the fraction without TLR2 effects, we propose that this peptide is most likely essential for TLR2 inhibition. Modulation of TLR signaling by small molecules, which do not resemble PAMPs, is of recent interest [43,54]. Molecules have for example been found to bind the ligand site [55], but also intracellular regions [56], or interfere with dimerization and the downstream pathway [57]. Therefore, more research on the exact interacting mechanism of the wheat peptides might lead to creation of specific wheat formulations for treatment of intestinal disorders.
In summary, in this study we identified a wheat hydrolysate with a strong TLR2 inhibitory effect, which resulted in HKLM induced IL-6 inhibition in DCs. Since TLR2 and IL-6 are recognized to be crucial in the development of intestinal mucositis, future research should focus on testing whether wheat hydrolysates can be applied in clinical nutrition to attenuate mucositis. Although more studies are required to identify the exact mechanism by which peptides can block TLR2, we were also able to show that the presence of the peptide sequence WQIPEQSR in the hydrolysate fractions is associated with TLR2 inhibition. Our technology platform with TLR expressing cells can be instrumental in identification of these bioactive hydrolysates.
Supporting information S1: List of reference proteins
Search strategy Uniprot: ‘triticum aestivum’+‘storage proteins’,resulted in 72 hits. Excluded enzymes and unknown fragments
Glutenin, high molecular weight subunit DY10
EGEASRQLQCERELQESSLEACRQVVDQQLAGRLPWSTG LQMRCCQQLRDVSAK-CRSV A VSQV ARQYEQTVVPPKGGSFYPGETTPLQQLQQGIFWGTS SQTVQGYYPGVT-SPRQGSYYPGQASPQQPGQGQQPGKWQEPGQGQQWYYPTSLQQPGQGQ QIGKGQ-QGYYPTSLQQPGQGQQGYYPTSLQHTGQRQQPVQGQQPEQGQQPGQWQQGYYPT SPQQLGQGQQPRQWQQSGQGQQGHYPTSLQQPGQGQQGHYLASQQQPGQGQQGHYPASQQQP-GQGQQGHYPASQQQPGQGQQ GHYPASQQEPGQGQQGQIPASQQQPGQGQQGHYPASLQQPG-QGQQGHYPTSLQQLGQGQQTGQPGQKQQPGQGQQTGQGQQPE QEQQPGQGQQGYYPTSLQQP-GQGQQQGQGQQGYYPTSLQQPGQGQQGHYPASLQQPGQGQPGQRQQPGQGQHPEQGKQPGQG QQGYYPTSPQQPGQGQQLGQGQQGYYPTSPQQPGQGQQPGQGQQGHCPTSPQQSGQAQQPGQGQQIGQVQ-QPGQGQQGYYPTS VQQPGQGQQSGQGQQSGQGHQPGQGQQSGQEQQGYDSPYHVSAEQQAASPMVAKAQ-QPATQLPTVCRMEGGDALSASQ
Glutenin, high molecular weight subunit DX5
EGEASEQLQCERELQELQERELKACQQVMDQQLRDISPE CHPVVVSPVAGQYEQQIVVPPKGGSFYP-GETTPPQQLQQRIFWGIPALLKRYYPSVTCPQ QVSYYPGQASPQRPGQGQQPGQGQQGYYPTSP-QQPGQWQQPEQGQPRYYPTSPQQSGQLQ QPAQGQQPGQGQQGQQPGQGQPGYYPTS-SQLQPGQLQQPAQGQQGQQPGQAQQGQQPGQGQQPGQGQQGQQPGQGQQPGQGQ QGQQLGQGQQGYYPTSLQQSGQGQPGYYPTSLQQLGQGQSGYYPTSPQQPGQGQQPGQLQQPAQG-QQPGQGQQGQQPGQGQQG QQPGQGQQPGQGQPGYYPTSPQQSGQGQPGYYPTSSQQPTQSQ-QPGQGQQGQQVGQGQQAQQPGQGQQPGQGQPGYYPTSPQQ SGQGQPGYYLTSPQQSGQGQQP-GQLQQSAQGQKGQQPGQGQQPGQGQQGQQPGQGQQGQQPGQGQPGYYPTSPQQSGQGQQPG QWQQPGQGQPGYYPTSPLQPGQGQPGYDPTSPQQPGQGQQPGQLQQPAQGQQGQQLAQGQQG-QQPAQVQQGQRPAQGQQGQQ PGQGQQGQQLGQGQQGQQPGQGQQGQQPAQGQQGQQPGQG-QQGQQPGQGQQGQQPGQGQQPGQGQPWYYPTSPQESGQGQQ PGQWQQPGQGQPGYYLTSPL-QLGQGQQGYYPTSLQQPGQGQQPGQWQQSGQGQHWYYPTSPQLSGQGQRPGQWLQPGQGQQG YYPTSPQQPGQGQQLGQWLQPGQGQQGYYPTSLQQTGQGQQSGQGQQGYYSSYHVSVEHQAASLKVAKAQ-QLAAQLPAMCRL EGGDALSASQ Avenin-like b1
QLETTCSQGFGQYQQQQQPGQRQLLEQMKPCV AFLQQQCRPL RMPFLQTQVEQLSSCQIVQHQCCQQLAQIPERIR-CHAIHSVVEAIMQQQSQQQWQERQQQ AQHKSMRMLLENLSLMCNIYVPVQCQQQQQMGQQQQQQQLQEQLT-PCATFLQHQCSPVTV PFPQIPVDQPTSCQNVQHQCCRQLSQIPEQFRCQAIHNV AEAIRQQQPQQQWQGMYQPQQ PAQHESIRMSLQALRSMCNIYIPVQCPAPTAYNIPMVATCTSGAC
Alpha/beta-gliadin
VRVPVPQLQPQNPSQQQPQEQVPLVQQQQFPGQQQPFPPQ QPYPQPQPFPSQQPYLQLQPFPQPQLPYPQPQL-PYPQPQLPYPQPQPFRPQQPYPQSQPQ YSQPQQPISQQQQQQQQQQQQKQQQQQQQQILQQILQQQLIPCRD-VVLQQHSIAYGSSQV LQQSTYQLVQQLCCQQLWQIPEQSRCQAIHNVVHAIILHQQQQQQQQQQQQPLSQVSFQQ PQQQYPSGQGSFQPSQQNPQAQGSVQPQQLPQFEEIRNLALETLPAMCNVYIPPYCTIAP VGIFGTN
Glutenin, low molecular weight subunit 1D1
RCIPGLERPWQQQPLPPQQTFPQQPLFSQQQQQQLFP QQPSFSQQQPPFWQQQPPFSQQQPILPQQPPFSQ-QQQLVLPQQPPFSQQQQPVLPPQQSP FPQQQQQHQQLVQQQIPVVQPSILQQLNPCKVFLQQQCSPVAMPQR-LARSQMLQQSSCHV MQQQCCQQLPQIPQQSRYEAIRAIIYSIILQEQQQVQGSIQSQQQQPQQLGQCVSQPQQQ SQQQLGQQPQQQQLAQGTFLQPHQIAQLEVMTSIALRILPTMCSVNVPLYRTTTSVPFGV GTGVGAY
Glutenin, high molecular weight subunit 12
EGEASRQLQCERELQESSLEACRQVVDQQLAGRLPWSTG
LQMRCCQQLRDVSAKCRSV A VSQV ARQYEQTVVPPKGGSFYPGETTPLQQLQQGIFWGTS SQTVQGYYPS-VTSPRQGSYYPGQASPQQPGQGQQPGKWQEPGQGQQWYYPTSLQQPGQGQ QIGKGKQGYYPTSLQ-QPGQGQQIGQGQQGYYPTSPQHTGQRQQPVQGQQIGQGQQPEQGQ QPGQWQQGYYPTSPQQLG-QGQQPGQWQQSGQGQQGHYPTSLQQPGQGQQGHYLASQQQPAQGQQGHYPASQQQPGQGQQGH YPASQQQPGQGQQGHYPASQQEPGQGQQGQIPASQQQPGQGQQGHYPASLQQPGQQGHYPTSL-QQLGQGQQIGQPGQKQQPGQG QQTGQGQQPEQEQQPGQGQQGYYPTSLQQPGQGQQQGQGQ-QGYYPTSLQQPGQGQQGHYPASLQQPGQGQGQPGQRQQPGQG QHPEQGQQPGQGQQGYYPTSP-QQPGQGQQLGQGQQGYYPTSPQQPGQGQQPGQGQQGHCPMSPQQTGQAQQLGQGQQIGQVQQ PGQGQQGYYPTSLQQPGQGQQSGQGQQSGQGHQPGQGQQSGQEKQGYDSPYHVSAEQQAASPMVAKAQQPAT-QLPTVCRMEG GDALSASQ
Glutenin, low molecular weight subunit
PTDUCD1 METSCIPGLERPWQEQPLPPQHTLFPQQQPFPQQQQPPFS QQQPSFLQQQPILPQLPFSQQQQPVLPQ-QSPFSQQQLVLPPQQQYQQVLQQQIPIVQPSV LQQLNPCKVFLQQQCNPVAMPQRLARSQMLQQSSCHVMQQQC-CQQLPQIPEQSRYDVIRA
ITYSIILQEQQQGFVQAQQQQPQQLGQGVSQSQQQSQQQLGQCSFQQPQQQLGQQPQQQQ VLQGTFLQPHQIAH-LEVMTSIALRTLPTMCSVNVPLYSSTTSVPFSVGTGVGAYL
Glutenin, high molecular weight subunit
PW212 EGEASEQLQCERELQELQERELKACQQVMDQQLRDISPE CHPVVVSPVAGQYEQQIVVPK-GGSFYPGETTPPQQLQQRIFWGIPALLKRYYPSVTSPQQ VSYYPGQASPQRPGQGQQPGQGQQS-GQGQQGYYPTSPQQPGQWQQPEQGQPGYYPTSPQQ PGQLQQPAQGQQPGQGQQGRQPG-QGQPGYYPTSSQLQPGQLQQPAQGQQGQQPGQGQQGQQPGQGQQPGQGQQGQQPGQGQQ PGQGQQGQQLGQGQQGYYPTSLQQSGQGQPGYYPTSLQQLGQGQSGYYPTSPQQPGQGQQPGQLQQPAQGQQPEQGQQGQQPG QGQQGQQPGQGQQPGQGQPGYYPTSPQQSGQGQPGYYPTSSQQPTQSQQPGQGQQGQQVGQGQQAQQPGQGQQPGQGQPGYYP TSPLQSGQGQPGYYLTSPQQSGQGQQPGQLQQSAQGQKGQQPGQGQQPGQGQQGQQPGQGQQGQQPGQGQPGYYPTSPQQSGQ GQQPGQWQQPGQGQPGYYPTSPLQPGQGQPGYDPTSPQQPGQGQQPGQLQQPAQGQQGQQLAQGQQGQQPAQVQQGQQPAQG
QQGQQLGQGQQGQQPGQGQQPAQGQQGQQPGQGQQGQQPGQGQQPGQGQPWYYPTSPQESGQGQQPGQWQQPGQWQ-8
QPGQG QPGYYLTSPLQLGQGQQGYYPTSLQQPGQGQQPGQWQQSGQGQHGYYPTSPQLSGQGQRPGQWLQPGQGQQGYYPTSP-QQSGQ GQQLGQWLQPGQGQQGYYPTSLQQTGQGQPGYYLTSPLQLGQGQQGYYPTSLQQPGQGQQPGQWQQSGQGQHGYYPTSPQLSGQGQRPGQWLQPGQGQQGYYPTSP-QQSGQGQQGYYSSYHVSVEHQAASLKVAKAQQLAAQL- GQQLGQWLQPGQGQQGYYPTSLQQTGQGQQSGQGQQGYYSSYHVSVEHQAASLKVAKAQQLAAQL-PAMCRLEGGDALSASQ
Glutenin, low molecular weight subunit
QISQQQQAPPFSQQQQPPFSQQQQPPFSQQQQSPFSQQQQQ PPFAQQQQPPFSQQPPISQQQQPPFSQQQ-QPQFSQQQQPPYSQQQQPPYSQQQQPPFSQQ QQPPFSQQQQQPPFTQQQQQQQQQQPFTQQQQPPFS-QQPPISQQQQPPFLQQQRPPFSRQ QQIPVIHPSVLQQLNPCKVFLQQQCIPV AMQRCLARSQMLQQSICHVM-QQQCCQQLRQIP EQSRHESIRAIIYSIILQQQQQQQQQQQQQQGQSIIQYQQQQPQQLGQCVSQPLQQLQQQ LGQQPQQQQLAHQIAQLEVMTSIALRTLPTMCNVNVPLYETTTSVPLGVGIGVGVY Avenin-like a1 QLYTTCSQGYGQCQQQPQPQPQPQPQMNTCAAFLQQCSQTP HVQTQMWQASGCQLVRQQCCQPLAQISEQARC-QAVCSVAQIIMRQQQGQSFGQPQQQVPV EIMRMVLQTLPLMCRVNIPQYCTTTPCSTITPAIYSIPMTATCAGGAC Alpha/beta-gliadin VRFPVPQLQPQNPSQQQPQEQVPLVQQQQFLGQQQPFPPQ QPYPQPQPFPSQLPYLQLQPFPQPQLPYSQPQP-FRPQQPYPQPQPQYSQPQQPISQQQQQ QQQQQQQQQQQQQILQQILQQQLIPCMDVVLQQHNIAHGRSQVLQ-QSTYQLLQELCCQHL WQIPEQSQCQAIHNVVHAIILHQQQKQQQQPSSQVSFQQPLQQYPLGQGSFRPSQQNPQA QGSVQPQQLPQFEEIRNLALQTLPAMCNVYIPPYCTIAPFGIFGTN Avenin-like a5 QLDTTCSQGYGQCQQQPQQQVNTCSALLQQCSPTPYVQSQM WQASGCQLMRQQCCQPLAQISEQARCHA VCGV AQVIMRQQQGQSFGQPQQQQGQSFSQPQ QQVPIEIRRMVLQTLPSMCNVNIPQYCTTTPCSTITQTPYNVPMATT-CVGGTC Alpha/beta-gliadin A-II VRVPVPQLQLQNPSQQQPQEQVPLVQEQQFQGQQQPFPPQ QPYPQPQPFPSQQPYLQLQPFPQPQLPYPQPQP-FRPQQPYPQPQPQYSQPQQPISQQQQQ QQQQQQQQQQILQQILQQQLIPCRDVVLQQHNIAHGSSQVLQESTY-QLVQQLCCQQLWQI PEQSRCQAIHNVVHAIILHQQHHHHQQQQQQQQQQPLSQVSFQQPQQQYPSGQGFFQPSQ QNPQAQGSFQPQQLPQFEEIRNLALQTLPAMCNVYIPPYCTIAPFGIFGTN Alpha/beta-gliadin A-III VRVPVPQLQPQNPSQQQPQEQVPLMQQQQQFPGQQEQFPP QQPYPHQQPFPSQQPYPQPQPFPPQLPYPQTQP-FPPQQPYPQPQPQYPQPQQPISQQQAQ QQQQQQQTLQQILQQQLIPCRDVVLQQHNIAHASSQVLQQSSYQQL-QQLCCQQLFQIPEQ SRCQAIHNVVHAIILHHHQQQQQQPSSQVSYQQPQEQYPSGQVSFQSSQQNPQAQGSVQP QQLPQFQEIRNLALQTLPAMCNVYIPPYCSTTIAPFGIFGTN Alpha/beta-gliadin A-IV VRVPVPQLQPQNPSQQQPQKQVPLVQQQQFPGQQQPFPPQ QPYPQQQPFPSQQPYMQLQPFPQPQLPYPQPQL-PYPQPQPFRPQQSYPQPQPQYSQPQQP ISQQQQQQQQQQQQQQQILQQILQQQLIPCRDVVLQQHSIAHGSSQVL-QQSTYQLVQQFC CQQLWQIPEQSRCQAIHNVVHAIILHQQQQQQQQQQQQQQQPLSQVCFQQSQQQYPSGQG SFQPSQQNPQAQGSVQPQQLPQFEEIRNLALETLPAMCNVYIPPYCTIAPVGIFGTN
Alpha/beta-gliadin clone PW1215 VPVPQPQPQNPSQPQPQGQVPLVQQQQFPGQQQQFPPQ QPYPQPQPFPSQQPYLQLQPFPQPQPFPPQLPYP-QPPPFSPQQPYPQPQPQYPQPQQPIS QQQAQQQQQQQQQQQQQQQQQQILQQILQQQLIPCRDVVLQQHNIA-HARSQVLQQSTYQP LQQLCCQQLWQIPEQSRCQAIHNVVHAIILHQQQRQQQPSSQVSLQQPQQQYPSGQGFFQ PSQQNPQAQGSVQPQQLPQFEEIRNLALQTLPRMCNVYIPPYCSTTIAPFGIFGTN Alpha/beta-gliadin A-V VRVPVPQLQPQNPSQQQPQEQVPLVQQQQFPGQQQQFPPQ QPYPQPQPFPSQQPYLQLQPFPQPQPFPPQLPYP-QPQSFPPQQPYPQQQPQYLQPQQPIS QQQAQQQQQQQQQQQQQQQILQQILQQQLIPCRDVVLQQHNIAHASS-QVLQQSTYQLLQQ LCCQQLLQIPEQSQCQAIHNV AHAIIMHQQQQQQQEQKQQLQQQQQQQQQLQQQQQQQQQQPSSQVSFQQP-QQQYPSSQVSFQPS QLNPQAQGSVQPQQLPQFAEIRNLALQTLPAMCNV YIPPHCSTTIAPFGISGTN Alpha/beta-gliadin A-I VRVPVPQLQPQNPSQQQPQEQVPLVQQQQFLGQQQPFPPQ QPYPQPQPFPSQQPYLQLQPFLQPQLPYSQPQPFRPQQPYPQPQPQYSQPQQPISQQQQQ QQQQQQQQQQQQQ-QIIQQILQQQLIPCMDVVLQQHNIVHGKSQVLQQSTYQLLQELCCQH LWQIPEQSQCQAIHNVVHAIILHQQQKQQQ-QPSSQVSFQQPLQQYPLGQGSFRPSQQNPQ AQGSVQPQQLPQFEEIRNLARK Alpha/beta-gliadin clone PW8142 PVPQLQPKNPSQQQPQEQVPLVQQQQFPGQQQQFPPQQPY PQPQPFPSQQPYLQLQPFPQPQPFLP-QLPYPQPQSFPPQQPYPQQRPKYLQPQQPISQQQ AQQQQQQQQQQQQQQQQQILQQILQQQLIPCRDVVLQ-QHNIAHASSQVLQQSTYQLLQQL CCQQLLQIPEQSRCQAIHNVVHAIIMHQQEQQQQLQQQQQQQLQQQQQ-QQQQQQQPSSQV SFQQPQQQYPSSQGSFQPSQQNPQAQGSVQPQQLPQFAEIRNLALQTLPAMCNVYIPPHC STTIAPFGIFGTN Avenin-like b5
QLETTCSQGFRQYQQQQQPGQRQLLEQMRPCV AFLQQQCRPL RMPFLQTQVEQLSSCQIVQYQCCQQLAQIPE-QIRCHAIHNVVEAIMQQQSQQQRQERQQQ AQHKSMRMLLETLYLMCNIYVPIQCQQQQQLGQQQQQQLQEQLTP-CATFLQHQCSPVTVP FPQIPVDQPTSCQNVQHQCCRQLSQIPEQFRCQAIHNVAEAIRQQQPQQQWQGMYQPQQP AQLESIRMSLQALRSMCSIYIPVQCPAPTAYNIPMVATYTGGAC Avenin-like a2 QLYTTCSQGYGQCQQQPQPQPQPQPQMNTCAAFLQQCIQTP YVQSQMWQASGCQLMRQQCCQPLAQISEQARC-QA VCSVSQIIMRQQQGQRFGQPQQQQGQ SFGQPQQQVPVEIMRMVLQTLPSMCSVNIPQYCTTTPCSTITPAIYSIP-MTATCAGGAC Avenin-like a3 QLYTTCSQGYGQCQQQPQPQPQMNTCAAFLQQCIQTPYVQS QMWQASGCQLMRQQCCQPLAQISEQARCQA VCSVSQIIMRQQQGQRFGQPQQQQGQSFGQ PQQQVPVEIMRMVLQTLPSMCSVNIPQYCTTTPCSTITPAIYSIPMTAT-CAGGAC
8
Avenin-like a4
QLDTTCSQGYGQCQQQPQQQVNTCSALLQQCSPTPYVQSQM WQASGCQLMRQQCCQPLAQISEQARCQA VCSV AQVIMRQQQGQSFGQPQQQVQSFSQPQH QVPIEITRMVLQTLPSMCNVNIPQYCTTTPCRTITQTPYNIPMSATCVG-GTC
Avenin-like b10
QLETTCSQGFGQSQQQQQPGQRQLLEQMKPCV AFLQQKCSPL RMPFLQTQVEQLSSCQIVQYQCCQQLAQIPERTR-CHAIHIVVEAIIQQQSQQQWQEPQQQ AQHKSMRMLLENLSLMCNIYVPVQCQQQQQLGQQQQQQLQEQLTPCT-TFLQQQCSPVTVP FPQIPVDQPTSCQNVQHQCCRQLSQIPEQFRCQAIHNVAEAIRQQQPQQQWQGMYQPQQP AQLESIRMSLQALRSMRSIYIPVQCPAPTTYNIPLVATYTGGAC
Avenin-like b2
QLETTCSQGFGQSQQQQQPGQRQLLEQMKPCV AFLQQKCSPL RMPFLQTQVEQLSSCQIVQYQCCQQLAQIPER-TRCHAIHIVVEAIIQQQSQQQWQEPQQQ AQHKSMRMLLENLSLMCNIYVPVQCQQQQQMGQQPQQQQLQEQLT-PCATFLQHQCSPVTV PFPQIPVDQPTSCQNVQHQCCRQLSQIPEQFRCQAIHNV AEAIRQQQPQQQWQGMYQPQQ PAQHESIRMSLQALRSMCNIYIPVQCPAPTAYNIPMVATCTSGAC
Avenin-like b8
QLETTCSQGFGQSQQQQQPGQRQLLEQMKPCV AFLQQKCGPL RMPFLQTQVEQLSSCQIVQYQCCQQLAQIPER-TRCHAIHIVVEAIIQQQSQQQWQEPQQQ AQHKSMRMLLENLSLMCNIYVPVQCQQQQQLGQQQQQQLQEQLT-PCTTFLQQQCSPVTVP FPQIPVDQPTSCQNVQHQCCRQLSQIPEQFRCQAIHNVAEAIRQQQPQQQWQGMYQPQQP AQLESIRMSLQALRSMCSIYIPVQCPAPTTYNIPLVATYTGGAC Avenin-like a6 QLYTTCSQGYGQCQQQPQPQPQPQPQPQMNTCSAFLQQCSQ TAYVQSQMWQASGCQLMRQQCCQPLAQISE-QARCQAVCSVAQIIMRQQQGQRFGQPQQQQ GQSFSQPQQQVPVEIMGMVLQTLPSMCSVNIPQYCTTTPCSTIA-PAIYNIPMTATCAGGA C Avenin-like b3
QLETTCSQGFGQSQQQQQPGQRQLLEQMKPCV AFLQQKCSPL RMPFLQTQVEQLSSCQIVQYQCCQQLAQIPERTR-CHAIHIVVEAIIQQQSQQQWQEPQQQ AQHKSMRMLLENLSLMCNIYVPVQCQQQQQLGQQQQQQLQEQLTPCT-TFLQQQCSPVTVP FPQIPVDQPTSCQNVQHQCCRQLSQIPEQFRCQAIHNVAEAIRQQQPQQQWQGMYQPQQP AQLESIRMSLQALRSMCNIYIPVQCPAPTTYNIPLVATYTGGAC
Avenin-like b6
QLETTCSQGFGQSQQQQQPGQRQLLEQMKPCV AFLQQKCSPL RMPFLQTQVEQLSSCQIVQYQCCQQLAQIPERTR-CHAIHIVVEAIIQQQSQQQWQEPQQQ AQHKSMRMLLENLSLMCNIYVPVQCQQQQQLGQQQQQQLQEQLTPCT-TFLQQQCSPVTVP FPQIPVDQPTSCQNVQHQCCRQLSQIPEQFRCQAIHNVAEAIRQQQPQQQWQGMYQPQQP AQLESIRMSLQALRSMCSIYIPVQCPAPTTYNIPLVATYTGGAC
Avenin-like a7
QLYTTCSQGYGQCQQQPQPQPQMNTCSAFLQQCIQTPYVQS QMWQASSCQLMRQQCCQPLAQISEQARCQAVCS-VSQIIMRQQQGQRFGQPQQQQGQSFSQ PQQQVPVEIMRMVLQTLPSMCSVNIPQYCTTTPCSTITPAIYSIPMTAT-CAGGAC
Avenin-like b4
QLETTCSQGFRQYQQQQQPGQRQLLEQMRPCV AFLQQQCRPL RMPFLQTQVEQLSSCQIVQYQCCQQLAQIPE-QIRCHAIHNVVEAIMQQQSQQQRQERQQQ AQHKSMRMLLENLSLMCNIYVPIQCQQQQQLGQQQQQQLQEQLTP-CATFLQHQCSPVTVP FPQIPVDQPTSCQNVQHQCCRQLSQIPEQFRCQAIHNVAEAIRQQQPQQQWQGMYQPQQP AQLESIRMSLQALRSMCSIYIPVQCPAPTAYNIPMVATYTGGAC
Avenin-like b9
QLETTCSQGFGQSQQQQQPGQRQLLEQMKPCV AFLQQKCSPL RMPFLQTQVEQLSSCQIVQYQCCQQLAQIPER-TRCHAIHIVVEAIIQQQSQQQWQEPQQQ AQHKSMRMLLENLSLMCNIYVPVQCQQQQQLGQQQQQQLQEQLT-PCTTFLQQQCSPVTVP FPQIPVDQPTSCQNVQYQCCRQLSQIPEQFRCQAIHNVAEAIRQQQPQQQWQGMYQPQQP AQLESIRMSLQALRSMCSIYIPVQCPAPTTYNIPLVATYTGGAC
Avenin-like b11
QLDTTCSQGFRQYQQQQQPGQRQLLEQMRPCV AFLQQQCRPL RMPFLQTQVEQLSSCQIDQYQCCQQLAQIPE-QIRCHAIHNVVEAIMQQQSQQHRQERQQQ AQHKSMRMLLETLYLMCNIYVPIQCQQQQQLGQQQQQQLQEQLTP-CATFLQHQCSPVTVP FPQIRVDQPTSCQNVQHQCCRQLSQIPEQYRCQAIHNVAEAIRHQQPQQQCQGMYQPQQP AKLESIRMSLQALRSMCRIYIPVQCPAPTAYNIPMVATYTGGAC Avenin-like b7 QLETTCSQGFGQSQQQQQPGQRQLLEQMKPCAAFLQQKCSPL RMPFLQTQVEQLSSCQIVQYQCCQQLAQIPERTR-CHAIHIVVEAIIQQQSQQQWQEPQQQ AQHKSMRMLLENLSLMCNIYVPVQCQQQQQLGQQQQQQLQEQLTPCT-TFLQQQCSPVTVP FPQIPVDQPTSCQNVQHQCCRQLSQIPEQFRCQAIHNVAEAIRQQQPQQQWQGMYQPQQP AQLESIRMSLQALRSMCSIYIPVQCPAPTTYNIPLVATYTGGAC Gamma-gliadin NIQVDPSGQVQWLQQQLVPQLQQPLSQQPQQTFPQPQQTFP HQPQQQVPQPQQPQQPFLQPQQPFPQQPQQP-FPQTQQPQQPFPQQPQQPFPQTQQPQQPF PQQPQQPFPQTQQPQQPFPQLQQPQQPFPQPQQQLPQPQQPQ-QSFPQQQRPFIQPSLQQQ LNPCKNILLQQSKPASLVSSLWSIIWPQSDCQVMRQQCCQQLAQIPQQLQCAAIHSVVHS IIMQQQQQQQQQQGIDIFLPLSQHEQVGQGSLVQGQGIIQPQQPAQLEAIRSLVLQTLPS MCNVYVPPECSIMRAPFA-SIVAGIGGQ Gamma-gliadin B NMQADPSGQVQWPQQQPFLQPHQPFSQQPQQIFPQPQQTFP HQPQQQFPQPQQPQQQFLQPRQPFPQQPQQ-PYPQQPQQPFPQTQQPQQPFPQSKQPQQPF PQPQQPQQSFPQQQPSLIQQSLQQQLNPCKNFLLQQCKPVSLVS-SLWSIILPPSDCQVMR QQCCQQLAQIPQQLQCAAIHSVVHSIIMQQEQQEQLQGVQILVPLSQQQQVGQGILVQGQ GIIQPQQPAQLEVIRSLVLQTLPTMCNVYVPPYCSTIRAPFASIVASIGGQ
8
Gamma-gliadin B-I
SCISGLERPWQQQPLPPQQSFSQQPPFSQQQQQPLPQ QPSFSQQQPPFSQQQPILSQQPPFSQQQQPVLPQQSPFS-QQQQLVLPPQQQQQQLVQQQI PIVQPSVLQQLNPCKVFLQQQCSPV AMPQRLARSQMWQQSSCHVMQQQCCQ-QLQQIPEQS RYEAIRAIIYSIILQEQQQGFVQPQQQQPQQSGQGVSQSQQQSQQQLGQCSFQQPQQQLG QQPQQQ-QQQQVLQGTFLQPHQIAHLEAVTSIALRTLPTMCSVNVPLYSATTSVPFGVGTG VGAY Gamma-gliadin NMQVDPSGQVQWPQQQPFPQPQQPFCQQPQRTIPQPHQTFH HQPQQTFPQPQQTYPHQPQQQFPQTQQPQQP-FPQPQQTFPQQPQLPFPQQPQQPFPQPQQ PQQPFPQSQQPQQPFPQPQQQFPQPQQPQQSFPQQQQPAIQSFL-QQQMNPCKNFLLQQCN HVSLVSSLVSIILPRSDCQVMQQQCCQQLAQIPQQLQCAAIHSVAHSIIMQQEQQQGVPI LRPLFQLAQGLGIIQPQQPAQLEGIRSLVLKTLPTMCNVYVPPDCSTINVPYANIDAGIG GQ Gamma-gliadin NIQVDPSGQVQWPQQQPFPQPQPFSQQPQQAFLQPQHTFPL QPQQVFPQPQQPQQQFPQPQQPQQPFPQPQ-QPQLPFPQQPQQPFPQPQQPQQPFPQSQQP QQPFPQPQQQFPQPQQPQQSFPQQQPPLIQPYLQQQMNPCK-NYLLQQCNPVSLVSSLVSM ILPRNDCQVMQQQCCQQLAQIPRQLQCTAIHSVVHAIIMQQEQQGIQILRPLFQLVQG-QG IIQPQQPAQYEVIRSLVLRTLPNMCNVYVRPDCSTINAPFASIVAGIGGQ
Low-molecular-weight glutenin storage protein QISQQQQQPPFSQQQQPQFSQQPPFSQQQQQPP FAQQQQISQQQQQPPFSQQQQPQFSQQPPFSQQQQQPPFSQQISQQQQQPPFSQQQQPQFSQQPPFSQQQQQPPISQQQQISQQQQQPPFSQQQQPQFSQQPPFSQQQQQPPFSQQQQPQFSQQQQISQQQQQPPFSQQQQPQFSQQPPFSQQQQQPPYSQQQQISQQQQQPPFSQQQQPQFSQQPPFSQQQQQPPYSQQQQISQQQQQPPFSQQQQPQFSQQPPFSQQQQQPPFSQQQQ PPFSQQQQ-QPPFTQQQQQQQQQQPFTQQQQPPFSQQPPISQQQQPPFSQQQQPQFSQQQQ IPVIHPSVLQQLNPCKVFLQQQ-CIPV AMQRCLARSQMLQQSICHVMQQQCCQQLRQIPEQ SRHESIRAIIYSIILQQQQQQQQQQQQGQSIIQYQQQQPQQLGQCVSQPQQQLQQQLGQQ PQQQQLAHGTFLQPH-QIAQLEVMTSIAPRTLPTMCSVNVPLYETTTSVPLGVGIGVGVY Gamma-gliadin NMQVDPSGQVQWPQQQPFLQPHQPFSQQPQQIFPQPQQTFP HQPQQQFPQPQQPRQQFLQPRQPFPQQPQQ-PYPQQPQRPFPQTQQPQQPFPQSKQPQQPF PQPQQPQQSFPQQQPSLIQQSLQQQLNPCKDFLLQQCKPVSLVS-SLWSIILPPSDCQVMR QQCCQQLAQIPQQLQCAAIHSVVHSTIMQQEQQEQLQGVQILGPLSQQQQVGQGILVQGQ GIIQPQQPAQLEVIGSLVLQTLPTMCNVHVPPYCSTIRAPFASIVASIGGQE LMW-GS P-21 QMETRCIPGLERPWQQQPLPPQQTFPQQPLFSQQQQQQLFP QQPSFSQQQPPFWQQQPPFSQQQPILPQQPPFS-QQQQLVLPQQPPFSQQQQPVLPPQQSP FPQQQQQHQQLVQQQIPVVQPSILQQLNPCKVFLQQQCSPVAMPQR-LARSQMLQQSSCHV MQQQCCQQLPQIPQQSRYEAIRAIIYSIILQEQQQVQGSIQSQQQQPQQLGQCVSQPQQQ SQQQLGQQPQQQQLAQGTFLQPHQIAQLEVMTSIALRILPTMCSVNVPLYRTTTSVPFGV GTGVGAY Alpha gliadin VRVSVPQLQPQNPSQQQPQEQVPLVQQQQFLGQQQPFPPQ QPYPQLQPFPSQQPYLQLQPFPQPQLPYSQPQP-FRPQQPYPQPQPQYSQPQQPISQQQQQ QQQQQQQQQQQQQQQQILQQILQQQLIPCMDVVLQQHNIVHGRS-QVLQQSTYQLLRELCC QHLWQIPEQSQCQAIHNVVHAIILHQQQKQQQQPSSQVSFQQPLQQYPLGQGSFRPSQQN PQAQGSVQPQQLPQFEEIRNLALQTLPAMCNVYIPPYCTIAPFGIFGTN
Alpha-gliadin
VRVPVPQLQPQNPSQQQPQEQVPLVQQQQFLGQQQPFPPQ QPYPQPQPFPSQQPYLQLQPFLQPQLPYSQPQP-FRPQQPYPQPQPQYSQPQQPISQQQQQ QQQQQQQQQQQQQQIIQQILQQQLIPCMDVVLQQHNIVHGKSQVL-QQSTYQLLQELCCQH LWQIPEQSQCQAIHNVVHAIILHQQQKQQQQPSSQVSFQQPLQQYPLGQGSFRPSQQNPQ AQGSVQPQQLPQFEEIRNLALQTLPAMCNVYIPPYCTIAPFGIFGTN
Omega gliadin storage protein
ARELNPSNKELQSPQQSFSYQQQPFPQQPYPQQPYPSQQPY PSQQPFPTPQQQFPEQSQQPFTQPQQPTPIQPQ-QPFPQQPQQPQQPFPQPQQPFPWQPQQ PFPQTQQSFPLQPQQPFPQQPQQPFPQPQLPFPQQSEQIIPQQLQ-QPFPLQPQQPFPQQP QQPFPQPQQPIPVQPQQSFPQQSQQSQQPFAQPQQLFPELQQPIPQQPQQPFPLQPQQPF PQQPQQPFPQQPQQSFPQQPQQPYPQQQPYGSSLTSIGGQ
Low-molecular-weight glutenin storage protein
QMETRCIPGLERPWQQQPLPPQQTFPQQPLFSQQQQQQLFP QQPSFSQQQPPFWQQQPPFSQQQPILPQQPPFS-QQQQLVLPQQPPFSQQQQPVLPPQQSP FPQQQQQHQQLVQQQIPVVQPSILQQLNPCKVFLQQCSPVAMPQR-LARSQMLQQSSCHVM QQQCCQQLPQIPQQSRYEAIRAIIYSIILQEQQQVQGSIQSQQQQPQQLGQCVSQPQQQS QQQLGQQPQQQQLAQGTFLQPHQIAQLEVMTSIALRILPTMCSVNVPLYRTTTSVPFGVG
TGVGAY
High molecular weight glutenin subunit
EGEASGQLQCERELQEHSLKACRQVVDQQLRDVGPECQP VGGGPVARQYEQ-QVVVPPKGGSFYPGETTPPQQLQQSILWGVPALLRRYYLSVASPQQVS YYPGQASS-RRPGQGQQEYYLTSPQQSGQWQQPGQGQSGYYPTSPQQSGQKQPGYYPTSPW QPEQLQQPTQGQQRQQPGQGQQLRQGQQGQQSGQGQPRYYPTSSQQPGQLQQLVQGQQGQQPERG-QQGQQSGQGQQLGQGQQ GQQPGQKQQSGQGQQGYYPISPQQLGQGQQSGQGQLGYYPTSPQ-QSGQGQSGYYPTSAQQPGQLQQSTQEQQLGQEQQDPQSGQ GRQGQQSGQRQQDQQSGQGQ-QPGQRQPGYYSTSPQQLGQGQPRYYPTCPQQPGQEQQPRQLQQPEHGQQGQQPEQGQQGQQQR QGEHGQQPGQGQQGQQPGQGQPGYYPTSPQQSGQGQPGYYPTSPQQSGQLQQPAQGQQPGQEQ-QGQQPGQGQQPGQGQPGYYP TSPQQPGQEQQLEQWQQSGQGQPGHYPTSPLQPGQGQPGYYPTSP-QQIGQGQQPGQLQQPTQGQQGQQPGQGQQGQQPGEGQQG QQPGQGQQPGQGQPGYYPTSLQ-QSGQGQQPGQWQQPGQGQPGYYPTSSLQPEQGQQGYYPTSQQQPGQGPQPGQWQQSGQGQQ GYYPTSSQQSGQGQQPGQWLQPGQWLQSGYYLTSPQQLGQGQQPRQWSQPRQGQQGYYPTSPQQSGQGQQLG-QGQQGYYPTSP QQSGQGQQGYDSPYYVSAEHQAASLKVAKAQQLAAQLPAMCRLEGGDALLASQ
Low-molecular-weight glutenin storage protein
QMETSCIPGLERPWQQQPLQQKETFPQQPPSSQQQQPFPQQ PPFLQQQPSFSQQPLFSQKQQPVLPQQPAFSQ-QQQTVLPQQPAFSQQQHQQLLQQQIPIV HPSILQQLNPCKVFLQQQCSPVAMPQHLARSQMWQQSSCNVMQQ-QCCQQLPRIPEQSRYE AIRAIIFSIILQEQQQGFVQPQQQQPQQSVQGVYQPQQQSQQQLLQCSFQQPQQQLGQQP QQQQVQKGTFLQPHQIARLEVMTSIALRTLPTMCSVNVPLYSSITSAPLGVGSRVGAY
Alpha-gliadin storage protein
VRFPVPQLQPQNPSQQLPQEQVPLVQQQQFLGQQQPFPPQ QPYPQPQPFPSQLPYLQLQPFPQPQLPYSQPQP-FRPQQPYPQPQPQYSQPQQPISQQQQQ QQQQQQQQQQQQQILQQILQQQLIPCMDVVLQQHNIAHGRSQVLQ-QSTYQLLQELCCQHL WQIPEQSQCQAIHNVVHAIILHQQQKQQQQPSSQVSFQQPLQQYPLGQGSFRPSQQNPQA QGSVQPQQLPQFEEIRNLALQTLPAMCNVYIPPYCTIAPFGIFGTN
Low-molecular-weight glutenin storage protein QMETSCISGLERPWQQQPLPPQQSFSQQPPFSQQQQQPLPQ QPSFSQQQPPFSQQQPILSQQPPFSQQQQPVLPQQSPFSQQQQLVLPPQQQQQQLVQQQI PIVQPSVLQQLNPCK-VFLQQQCSPV AMPQRLARSQMWQQSSCHVMQQQCCQQLQQIPEQS RYEAIRAIIYSIILQEQQQGFVQPQQQQPQ-QSGQGLSQSQQQSQQQLGQCSFQQPQQQLG
QQPQQQQQQVLQGTFLQPHQIAHLEAVTSIALRTLPTMCSVNVPLYSATTSVRFGVGTGV GAY Alpha-/beta-gliadin storage protein
VRVPVPQLQPQNPSQQQPQEQVPLVQQQQFPGQQQPFPPQ QPYPQPQPFPSQQPYLQLQPFPQPQLPYPQPQP-FRPQQPYPQPQPQYSQPQQPISQQQQQ QQQQQQQQQILQQILQQQLIPCMDVVLQQHNIAQGRSQVLQQSTYQL-LQELCCQHLWQIP EQSQCQAIHNVVHAIILHQQHHHHQQQQQQQQQQPLSQVSFQQPQQQYPSGQGFFQPSQQ NPQAQGSFQPQQLPQFEAIRNLALQTLPAMCNVYIPPYCTIAPFGIFGTN
Alpha-gliadin storage protein
VRWPVPQLQPQNPSQQQPQEQVPLVQQQQFLGQQQPFPPQ QPYPQPQPFPSQQPYLQLQPFSQPQLPYSQPQP-FRPQQPYPQPQPQYSQPQQPISQQQQQ QQQQQQQQQQQQEQQILQQILQQQLTPCMDVVLQQHNIARGRSQ-VLQQSTYQLLQELCCQ HLWQIPEKLQCQAIHNVVHAIILHQQQQKQQQPSSQVSFQQPQQQYPLGQGSFRPSQQNP QAQGSVQPQQLPQFEEIRNLALQTLPAMCNVYIPPYCTIAPFGIFGTN
Alpha-/beta-gliadin storage protein
VRVPVPQLQPQNPSQQQPQEQVPLVQQQQFLGQQQPFPPQ QPYPQPQPFPSQQPYLQLQPFPQPQLPYPQPQP-FRPQQPYPQPQPQYSQPQQPISQQQQQ QQQQQQQQQQILQQILQQQLIPCMDVVLQQHNIVHGRSQVLQQSTY-QLLRELCCQHLWQI PEQSQCQAIHNVVHAIILHQQQKQQQQPSSQVSFQQPLQQYPLGQGSFRPSQQNPQAQGS VQPQQLPQFEEIRNLALQTLPAMCNVYIPPYCTIAPFGIFGTN Storage protein SHDDEDDRRGGHSLQQCVQRCRQERPRYSHARC VQECRDDQQQHGRHEQEEEQGRGRGWHGEGEREEEH-GRGRGRHGEGEREEEHGRGRGRHG EGEREEERGRGHGRHGEGEREEERGRGRGRHGEGEREEEEGRGRGR-RGEGERDEEQGDSR RPYVFGPRSFRRIIQSDHGFVRALRPFDQVSRLLRGIRDYRVAIMEVNPRAFVVPGFTDA DGVGYVAQGEGVLTVIENGEKRSYTVKEGDVIVAPAGSIMHLANTDGRRKLVIAKILHTI SVPGKFQFLSVKPLLASLSKR-VLRAAFKTSDERLERLFNQRQGQEKTRSVSIVRASEEQL RELRREAAEGGQGHRWPLPPFRGDSRDTFNLLEQRPKIAN-RHGRLYEADARSFHALANQD VRVAVANITPGSMTAPYLNTQSFKLAVVLEGEGEVQIVCPHLGRESESEREHGKGRRREE EEDDQRQQRRRGSESESEEEEEQQRYETVRARVSRGSAFVVPPGHPVVEISSSQGSSNLQ VVCFEINAERNERVWLAG-RNNVIGKLGSPAQELTFGRPAREVQEVFRAQDQDEGFVAGPE QQSREQEQEQERHRRRGDRGRGDEAVETFLRMATGAI
Seed storage protein NMQVDPSGQVPWPQQQPFPQPHQPFSQQPQQTFPQPQQTFP HQPQQQFSQPQQPQQQFIQPQQPFPQQPQ-QTYPQRPQQPFPQTQQPQQPFPQSQQPQQPF PQPQQQFPQPQQPQQSFPQQQPSLIQQSLQQQLNPCKNFLLQ-QCKPVSLVSSLWSMILPR SDCQVMRQQCCQQPAQIPQQLQCAAIHSIVHSIIMQQEQQEQRQGVQILVPLSQQQQVGQ GTLVQGQGIIQPQQPAQLEVIRSLVLQTLATMCNVYVPPYCSTIRAPFASIVAGIGGQ High-molecular-weight glutenin EGEASEQLQCDRELQELQERELKACQQVMDQQLRDISPECHPVVVSPVAGQYEQQIVVP PKGGTFYPGETTPPQQLQ-QRIFWGIPALLKRYYPSVTCPQQVSYYPGQASPQRSRDITSS SYHVSVEHQAASLKVAKAQQLAAQLPAMCRLEGGDAL-SASQ
Alpha/beta-gliadin storage protein
VRVPVPQLQPKNPSQQQPQEQVPLVQQQQFPGQQQQFPPQQPY PQPQPFPSQQPYLQLQPFPQPQPFLPQL-PYPQPQSFPPQQPYPQQRPMYLQPQQPISQQQ AQQQQQQQQQQQQQQQQQILQQILQQQLIPCRDVVLQ-QHNIAHASSQVLQQSTYQLLQQL CCQQLLQIPEQSRCQAIHNVVHAIIMHQQEQQQQLQQQQQQQLQQQQQ-QQQQQQQPSSQV SFQQPQQQYPSSQGSFQPSQQNPQAQGSVQPQQLPQFAEIRNLALQTLPAMCNVYIPPHC STTIAPFGIFGTN
Alpha-gliadin storage protein
VRVPVPQLQPKNPSQQQPQEQVPLVQQQQFPGQQQQFPPQQPY PQPQPFPSQQPYLQLQPFPQPQPFLPQLPYP-QPQSFPPQQPYPQQRPKYLQPQQPISQQQ AQQQQQQQQQQQQQQQQQILQQILQQQLIPCRDVVLQQHNIA-HASSQVLQQSTYQLLQQL CCQQLLQIPEQSRCQAIHNVVHAIIMHQQEQQQQLQQQQQQQLQQQQQQQQQQQ-QPSSQV SFQQPQQQYPSSQVSFQPSQLNPQAQGSVQPQQLPQFAEIRNLALQTLPAMCNVYIPPHC STTIAPFGIFGTN
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