IDP Force Fields Applied to Model PPII-Rich 33-mer Gliadin Peptides.

The 33-mer gliadin peptide and its deamidated metabolite, 33-mer DGP, are the immunodominant peptides responsible for the adaptive immune response in celiac disease (CD). CD is a complex autoimmune chronic disorder triggered by gluten ingestion that affects the small intestine and affects ∼1% of the global population. The 33-mers are polyproline II-rich (PPII) and intrinsically disordered peptides (IDPs), whose structures remain elusive. We sampled the conformational ensembles of both 33-mer peptides via molecular dynamics simulations employing two force fields (FFs) (Amber ff03ws and Amber ff99SB-disp) specifically validated for other IDPs. Our results show that both FFs allow the extensive exploration of the conformational landscape, which was not possible with the standard FF GROMOS53A6 reported before. Clustering analysis of the trajectories showed that the five largest clusters (78-88% of the total structures) present elongated, semielongated, and curved conformations in both FFs. Large average radius of gyration and solvent-exposed surfaces characterized these structures. While the structures sampled are similar, the Amber ff99SB-disp trajectories explored folded conformations with a higher probability. In addition, PPII secondary structure was preserved throughout the trajectories (58-73%) together with a non-negligible content of ß structures (11-23%), in agreement with previous experimental results. This work represents the initial step in studying further the interaction of these peptides with other biologically relevant molecules, which could lead to finally disclose the molecular events that lead to CD.

Molecular and Structural Parallels between Gluten Pathogenic Peptides and Bacterial-Derived Proteins by Bioinformatics Analysis.

Gluten-related disorders (GRDs) are a group of diseases that involve the activation of the immune system triggered by the ingestion of gluten, with a worldwide prevalence of 5%. Among them, Celiac disease (CeD) is a T-cell-mediated autoimmune disease causing a plethora of symptoms from diarrhea and malabsorption to lymphoma. Even though GRDs have been intensively studied, the environmental triggers promoting the diverse reactions to gluten proteins in susceptible individuals remain elusive. It has been proposed that pathogens could act as disease-causing environmental triggers of CeD by molecular mimicry mechanisms. Additionally, it could also be possible that unrecognized molecular, structural, and physical parallels between gluten and pathogens have a relevant role. Herein, we report sequence, structural and physical similarities of the two most relevant gluten peptides, the 33-mer and p31-43 gliadin peptides, with bacterial pathogens using bioinformatics going beyond the molecular mimicry hypothesis. First, a stringent BLASTp search using the two gliadin peptides identified high sequence similarity regions within pathogen-derived proteins, e.g., extracellular proteins from Streptococcus pneumoniae and Granulicatella sp. Second, molecular dynamics calculations of an updated α-2-gliadin model revealed close spatial localization and solvent-exposure of the 33-mer and p31-43 peptide, which was compared with the pathogen-related proteins by homology models and localization predictors. We found putative functions of the identified pathogen-derived sequence by identifying T-cell epitopes and SH3/WW-binding domains. Finally, shape and size parallels between the pathogens and the superstructures of gliadin peptides gave rise to novel hypotheses about activation of innate immunity and dysbiosis. Based on our structural findings and the similarities with the bacterial pathogens, evidence emerges that these pathologically relevant gluten-derived peptides could behave as non-replicating pathogens opening new research questions in the interface of innate immunity, microbiome, and food research.

Optimized Chitosan-Coated Gliadin Nanoparticles Improved the Hesperidin Cytotoxicity over Tumor Cells

Abstract Hesperidin is a natural compound which is found in citric fruits and presents antitumor and antimicrobial activities. However, the in vivo efficacy of Hesperidin is reduced due to its low oral bioavailability. Protein-based nanoparticles have been applied to improve biological parameters of drugs and natural compounds. Gliadin is a monomeric protein present in wheat. In this study, gliadin-based nanoparticles containing hesperidin were obtained by desolvation technique and a Taguchi orthogonal array design was employed to optimize the formulation. The independent variables were set as concentration of CaCl2 (0.5; 1 or 2%) and stabilizing agent (Pluronic F68, Tween 80 or sodium caseinate). The dependent variables consisted of mean diameter, polydispersity index, zeta potential, and encapsulation efficiency. The results showed significant effects on the dependent variables when 1% CaCl2 and Pluronic F68 were used. The optimized formulation was coated with chitosan to increase the physical stability of the nanoparticles. The final nanoparticles presented a mean diameter of 321 nm and polydispersity index of 0.217, and spherical shape. After coating, the Zeta potential was +21 mV, and the encapsulation efficiency was 73 %. The in vitro release assay showed that about 98% of the drug was released from the nanoparticles after 48 h. Moreover, the nanoparticles reduced hesperidin cytotoxicity on healthy cells (Vero cells) and improved the cytotoxicity on tumor cells (HeLa, PC-3 and Caco-2 cells). Results showed that the chitosan-coated gliadin nanoparticles are potential carriers for hesperidin delivery for cancer treatment.

Digestion of Intact Gluten Proteins by Bifidobacterium Species: Reduction of Cytotoxicity and Proinflammatory Responses.

Celiac disease (CD) is a chronic illness characterized by an inflammatory process triggered by gluten protein intake. Recent evidence has suggested that the lower relative abundance of bifidobacteria in the intestinal lumen may be associated with CD. Herein, we assessed the effect of the Bifidobacterium species Bifidobacterium bifidum, Bifidobacterium longum, Bembidion breve, Bifidobacterium animalis alone, and also a Bifidobacterium consortium on the digestion of intact gluten proteins (gliadins and glutenins) and the associated immunomodulatory responses elicited by the resulting peptides. The cytotoxicity and proinflammatory responses were evaluated through the activation of NF-kB p65 and the expression of cytokines TNF-α and IL-1ß in Caco-2 cell cultures exposed to gluten-derived peptides. The peptides induced a clear reduction in cytotoxic responses and proinflammatory marker levels compared to the gluten fragments generated during noninoculated gastrointestinal digestion. These results highlight the possible use of probiotics based on bifidobacteria as a prospective treatment for CD.

p31-43 Gliadin Peptide Forms Oligomers and Induces NLRP3 Inflammasome/Caspase 1- Dependent Mucosal Damage in Small Intestine.

Celiac disease (CD) is a chronic enteropathy elicited by a Th1 response to gluten peptides in the small intestine of genetically susceptible individuals. However, it remains unclear what drives the induction of inflammatory responses of this kind against harmless antigens in food. In a recent work, we have shown that the p31-43 peptide (p31-43) from α-gliadin can induce an innate immune response in the intestine and that this may initiate pathological adaptive immunity. The receptors and mechanisms responsible for the induction of innate immunity by p31-43 are unknown and here we present evidence that this may reflect conformational changes in the peptide that allow it to activate the NLRP3 inflammasome. Administration of p31-43, but not scrambled or inverted peptides, to normal mice induced enteropathy in the proximal small intestine, associated with increased production of type I interferon and mature IL-1ß. P31-43 showed a sequence-specific spontaneous ability to form structured oligomers and aggregates in vitro and induced activation of the ASC speck complex. In parallel, the enteropathy induced by p31-43 in vivo did not occur in the absence of NLRP3 or caspase 1 and was inhibited by administration of the caspase 1 inhibitor Ac-YVAD-cmk. Collectively, these findings show that p31-43 gliadin has an intrinsic propensity to form oligomers which trigger the NLRP3 inflammasome and that this pathway is required for intestinal inflammation and pathology when p31-43 is administered orally to mice. This innate activation of the inflammasome may have important implications in the initial stages of CD pathogenesis.

Celiac Disease: Historical Standpoint, New Perspectives of Treatments and Contemporary Research Techniques.

Celiac disease (CD) is an inflammatory syndrome that affects mainly the intestine, but also other organs. This ailment is also affected by the physicochemical behavior of gluten as such. From the medical standpoint, this pathology results from a combination of genetic and environmental factors. At the same time, gliadin (the alcohol-soluble fraction of gluten) along with other related oligomers, such as 33-gliadin, present high immunogenicity and are responsible for triggering of this disease. Within CD characterization, there are mainly two different approaches to carry out this study; one focuses on its chronic phase, while the other deals with its initial stages. Although the chronic phase of CD has been well characterized, the initiation of the inflammatory process is still unclear. As this process is apparently related to the aggregation of the oligomers involved in CD, the initiation of the disease could be explained by means of clarifying their self-assembly behavior. Thus, this work addresses the clinical explanation, within the chronic approach, attempting to combine it with the physicochemical techniques used for characterization of proteins aggregates as well.

Self-organization of gliadin in aqueous media under physiological digestive pHs.

Here we showed that gliadin, a complex protein system related to celiac disease and other human diseases, is spontaneously self-organized in a very dilute solution at pH 3.0 and 7.0 in water under low ionic strength (10mM NaCl). The spontaneous self-organization at pH 3.0 increases the apparent solubility due to the formation of finite sized aggregates, such as those formed in the micellization of amphiphilic molecules. Switching the pH from 3.0 to 7.0 lead to a phase separation, however part of the nano-particles are stable remaining disperse in water after centrifugation. Also, beside the pH change led to changes in protein composition and concentration, we determined that the secondary structure of both system is the same. Moreover, Tyrs are slightly more buried and Trps are slightly more exposed to water at pH 7.0 than those at pH 3.0. Electron microscopy techniques showed that both gliadin systems are composed of nanostructures and in the case of pH 7.0 amorphous microaggregates were found, too. Only nanostructures at pH 3.0 showed a micromolar binding affinity to Nile red probe, suggesting the presence of accessible hydrophobic patches which are not more accessible at pH 7.0. All our results suggest that gliadin is able to self-organized at pH 3.0 forming protein micelles type nanostructures (ζ=+13, 42 ± 1.55 mV), meanwhile at 7.0 the decrease of superficial charge to ζ of +4, 78 ± 0.48 mV led to the formation of stable colloidal nanoparticles, unable to interact with Nile red probe. Our findings may open new perspectives for the understanding of gliadin ability to avoid proteolysis, to reach and cross the intestinal lumen and to trigger different immunological disorders.

Self-assembly of 33-mer gliadin peptide oligomers.

The 33-mer gliadin peptide, LQLQPF(PQPQLPY)3PQPQPF, is a highly immunogenic peptide involved in celiac disease and probably in other immunopathologies associated with gliadin. Herein, dynamic light scattering measurements showed that 33-mer, in the micromolar concentration range, forms polydisperse nano- and micrometer range particles in aqueous media. This behaviour is reminiscent of classical association of colloids and we hypothesized that the 33-mer peptide self-assembles into micelles that could be the precursors of 33-mer oligomers in water. Deposition of 33-mer peptide aqueous solution on bare mica generated nano- and microstructures with different morphologies as revealed by atomic force microscopy. At 6 µM, the 33-mer is organised in isolated and clusters of spherical nanostructures. In the 60 to 250 µM concentration range, the spherical oligomers associated mainly in linear and annular arrangements and structures adopting a "sheet" type morphology appeared. At higher concentrations (610 µM), mainly filaments and plaques immersed in a background of nanospherical structures were detected. The occurrence of different morphologies of oligomers and finally the filaments suggests that the unique specific geometry of the 33-mer oligomers has a crucial role in the subsequent condensation and organization of their fractal structures into the final filaments. The self-assembly process on mica is described qualitatively and quantitatively by a fractal diffusion limited aggregation (DLA) behaviour with the fractal dimension in the range of 1.62 ± 0.02 to 1.73 ± 0.03. Secondary structure evaluation of the oligomers by Attenuated Total Reflection FTIR spectroscopy (ATR-FTIR) revealed the existence of a conformational equilibrium of self-assembled structures, from an extended conformation to a more folded parallel beta elongated structures. Altogether, these findings provide structural and morphological information about supramolecular organization of the 33-mer peptide, which might offer new perspectives for the understanding and treatment of gliadin intolerance disorders.

Transamidation of gluten proteins during the bread-making process of wheat flour to produce breads with less immunoreactive gluten.

Due to an increasing incidence of celiac disease (CD) and other gluten-related disorders, different gluten-free breads have been developed using starches and additives as a substitute for gluten. Thus, patients miss not only the taste and aroma of wheat bread but also risk their sensitive intestines. Therefore, modifying gluten to avoid an immune response in CD and its application to baking is in progress. The aim of the study was to enzymatically modify gluten on wheat flour, during bread-making avoiding the use of additives, to reduce immunoreactivity, preserving its properties. Microbial transglutaminase (mTG) or chymotrypsin (ChT) was used to bind lysine or valine to gluten proteins in a model system. The best conditions were directly applied to wheat flour for bread-making with and without punching at 45 min. Subsequently, the rheological properties of the doughs, specific volume of the loaves, immunoreactive gluten content and modification of the extracted proteins were evaluated. ChT-treated breads presented a better appearance with a more homogeneous crumb, higher specific volume values (3.34-4.25 cm(3) g(-1)) and higher reactive gluten reduction (up to 71%) than the mTG-treated ones (1.23-2.66 cm(3) g(-1)) with only a 42% reactive gluten reduction. Thus, transpeptidation during bread-making is a promising technology, although it is necessary to improve the modification process to obtain the reactive gluten reduction required in breads for the treatment of CD patients and other gluten-related disorders.

Xyloglucan-based diblock co-oligomer: synthesis, self-assembly and steric stabilization of proteins.

We propose a novel plant-based amphiphilic diblock co-oligomers (BCO) surfactant containing only carbohydrate segments and examine its potential as a biosourced stabilizer. The synthesis of an amphiphilic xyloglucan-based BCO, composed of a hydrophilic xyloglucan oligosaccharide (XGO) block "clicked" to a hydrophobic peracetylated XGO is described. Dynamic light scattering experiments correlated with transmission electron microscopy observations showed that this new class of amphiphilic BCO self-assembles in water to form spherical micelles with a hydrodynamic diameter of 22 nm. Preliminary studies indicate that the XGO-based BCO sterically stabilizes gliadin and zein nanoparticle suspensions. The stabilization results were compared to those using pluronic F-68, a commercial surfactant. For gliadin nanoparticles, both surfactants result in essentially the same morphology and polydispersity. However, for the zein nanoparticles, the XGO-based BCO stabilizer gave lower polydispersity.

A combination of two lactic acid bacteria improves the hydrolysis of gliadin during wheat dough fermentation.

The evaluation of gliadin hydrolysis during dough fermentation by using two lactic acid bacteria, Lactobacillus plantarum CRL 775 and Pediococcus pentosaceus CRL 792, as pooled cell suspension (LAB) or cell free extract (CFE) was undertaken. The CFE pool produced a greater (121%) increase in amino acid concentration than the LAB pool (70-80%). These results were correlated with the decrease (76,100 and 64,300 ppm) in the gliadin concentration of doughs supplemented with CFE and LAB, respectively, compared to control doughs. The use of LAB peptidases seemed to be a viable technologic alternative to reduce the gliadin concentration in wheat dough without using living bacteria as starter.

Dynamic rheological properties of native and cross-linked gliadin proteins.

A comparison of cross-linked and native gliadin suspensions, with respect to the state of protein globular structure was carried out using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS) and rheological analysis. Gliadin suspensions were also analyzed in the presence and absence of glycerol. DLS analysis showed that R(h) increased only with gliadin/EDC/NHS suspensions. However, Kratky plots revealed that gliadin and gliadin/L-cysteine maintained their globular shape even in absence or presence of glycerol. Rheological experiments revealed that gliadin and gliadin/L-cysteine suspension exhibited a similar profile with three main domains, and a sol-gel transition. Gliadin/EDC/NHS did not present any sol-gel transition, and this fact corroborates with DLS results and the hypothesis of lower protein-protein interaction, which are in agreement with G″ > G'.

A novel globular protein electrospun fiber mat with the addition of polysilsesquioxane.

The aim of this work has been to elaborate well defined gliadin nanofibers with incorporation of inorganic molecules, such as polyhedral oligomeric silsesquioxane (POSS). Nanofibers were obtained by electrospinning processing, controlling the relevant parameters such as tip-to-collector distance, voltage and feed rate. The fiber mats were characterized by SEM, confocal images, DSC, viscosity, FTIR and conductivimetry analysis. FTIR spectra showed characteristic absorption bands related to the presence of POSS-NH(2) within the matrices. SEM micrographs showed that gliadin fibers decreased their dimensions as the amount of POSS-NH(2) increased in the spinning solution. The electrical conductivity of gliadin solutions diminished as the concentration of POSS-NH(2) was increased. Besides, confocal micrographs revealed that POSS-NH(2) might be dispersed as nanocrystals into gliadin and gluten fibers. The dimension of gluten nanofibers was also affected by the POSS-NH(2) concentration, but conversely, this dependence was not proportional to the POSS-NH(2) amount. Somehow, the interaction between gliadin and POSS-NH(2) in aqueous TFE affected the solution viscosity and, as a consequence, higher jet instabilities and thinner fiber dimensions were obtained.

Single domain antibodies are specially suited for quantitative determination of gliadins under denaturing conditions.

Food intended for celiac patients' consumption must be analyzed for the presence of toxic prolamins using high detectability tests. Though 60% ethanol is the most commonly used solvent for prolamins extraction, 2-mercaptoethanol (2-ME) and guanidinium chloride (GuHCl) can be added to increase protein recovery. However, ethanol and denaturing agents interfere with antigen recognition when conventional antibodies are used. In the present work, a new method for gliadins quantification is shown. The method is based on the selection of llama single domain antibody fragments able to operate under denaturing conditions. Six out of 28 VHH-phages obtained retained their binding capacity in 15% ethanol. Selected clones presented a long CDR3 region containing two additional cysteines that could be responsible for the higher stability. One of the clones (named VHH26) was fully operative in the presence of 15% ethanol, 0.5% 2-ME, and 0.5 M GuHCl. Capture ELISA using VHH26 was able to detect gliadins in samples shown as negatives by conventional ELISA. Therefore, this new strategy appears as an excellent platform for quantitative determination of proteins or any other immunogenic compound, in the presence of denaturing agents, when specific recognition units with high stability are required.

Cloning, bacterial expression, purification and structural characterization of N-terminal-repetitive domain of gamma-Gliadin.

The gene encoding the repetitive domain located in the N-terminal half of gamma-Gliadin from wheat endosperm has been subcloned into a thioredoxin expression system (pET102/D-Topo). It was over-expressed as fusion protein with thioredoxin in Escherichia coli. Thioredoxin was removed by enterokinase cleavage or by acid cleavage at the respective engineered recognition sites. The soluble N-terminal half of gamma-Gliadin was purified by affinity and reverse-phase chromatography. While, the enterokinase cleavage leaded to only one species detectable by mass spectroscopy, the acid cleavage resulted in a three different length polypeptides, due to the presence of the same number of acid cleavage sites. The secondary structure of the purified protein domain was analysed by circular dichroism, showing an spectral shape common to a Poly(Pro) II conformation. The spectrum is dominated by a large negative peak centred around 201 nm and a broad shoulder centred around 225 nm. Also, the temperature denaturation process was studied. The differences observed in the spectra show two main tendencies, the increment of the shoulder intensity, and the drop of the intensity of the peak around 201. When the sample was cooled down, the change on intensity of the shoulder around 225 was completely reversible and that around the 201 nm peak reached a reversibility of 90%. Such structure and thermal behaviour are characteristic of the repetitive domains of the wheat prolamins.

Gliadin effect on fluctuation properties of phospholipid giant vesicles.

Gliadin is a fraction of wheat gluten, a protein supramolecular complex known for its remarkable and biotechnologically relevant viscoelastic properties. Very high molecular mass characterise these systems, thus hindering high-resolution structural investigations. It is known, however, that these proteins comprise rather extended, extensively interassociated structures, which respond for their peculiar mechanical behaviour. Besides these properties, some of gluten's fractions, such as gliadin, are also known to be involved in a nutritionally relevant pathology of auto-immune character, the celiac disease, supposedly related to some unusual structural features of the protein. Despite its medical relevance, however, the role played by gliadin in the etiology of the celiac disease is not sufficiently understood to date. In this work, we investigated the role of gliadin on mechanical properties of a membrane model of dioleoylphosphatidylcholine (DOPC) giant unilamellar vesicles. The technique of micropipette aspiration, coupled to videomicroscopy, was applied. The microvesicles, produced by electric field pulsing over metal-covered plates, were suctioned into the micropipettes under varying applied pressures. A significant increase in the values of the bilayer curvature constant, k(c), was observed, with a saturation effect being verified at around 0.02-0.03 gliadin/DOPC mass ratio, indicating that the membrane becomes less elastic in the presence of the protein. Possible correlations between the observed membrane fluctuation properties and the celiac disease etiology are suggested and discussed.

In vitro presentation of gliadin-derived peptides by different cell lines.

BACKGROUND: Gliadin peptide presentation and T-cell activation are critical events in the pathogenesis of celiac disease. Several studies have been performed to identify the toxic gliadin peptides but the complexity of the antigenic fraction makes this analysis difficult. In this work, an in vitro model for the analysis of gliadin peptide presentation is studied. METHODS: The human cell lines U937 and THP-1 (monocytic), DUCAF and VAVY (immortalised B cells) and HT-29 and Caco-2 (intestinal epithelial cells) were incubated with biotin-labelled gliadin (bG). FITC-labelled streptavidin was used to detect biotinylated peptides at the cell surface by flow cytometry. RESULTS: All cell lines tested showed a fluorescence signal derived from bG, that was highest when cells were stimulated with IFN-gamma for 48 h. Time course experiments performed using THP-1 cells showed that after 4-h incubation, almost a maximal signal can be reached. THP-1 cells incubated at 4 degrees C or after paraformaldehyde fixation showed a substantial signal reduction, suggesting that metabolic activity was necessary for the detection of the maximal fluorescence signal at the cell surface. The presence of HLA class II-bound biotinylated peptides was observed in cell lysates of THP-1 cells incubated with bG. CONCLUSIONS: In all cell lines tested, a specific biotin-peptide-derived signal was observed. This was increased after IFN-gamma treatment and decreased after fixation or incubation at low temperature. The signal was higher in monocytic and B-cell lines than in the epithelial cell lines. The use of this procedure could be a useful tool to study the in vitro processing and presentation of naturally gliadin-derived peptides.

Celiac disease: antibody recognition against native and selectively deamidated gliadin peptides.

BACKGROUND: Selective deamidation of glutamine residues by tissue transglutaminase (tTG) turns gliadin peptides into stronger activators of T cells from celiac disease (CD) patients. We examined the possibility that these modified peptides could be more specific epitopes for circulating antibodies than are native peptides. METHODS: Two native synthetic peptides and their respective modified sequences were used as antigens for ELISA assays: peptide-1, with residues 56-75 of alpha-type gliadin; and peptide-2, with residues 134-153 of gamma-type gliadin. We examined 40 CD patients [31 not being treated with a gluten-free diet (GFD) and 9 being treated with a GFD] and 30 non-CD patients. RESULTS: An enhanced response against deamidated peptides was observed in 4 (IgA) and 22 (IgG) of 31 untreated CD patients for peptide-1 and in 25 (IgA) and 29 (IgG) patients for peptide-2. Higher anti-gliadin antibody and anti-tTG IgA concentrations correlated with increased IgA reactivity to modified peptides. Among the nine treated CD patients, eight also displayed an improved IgG signal for the deamidated sequence. Deamidation of peptides did not increase the reactivity of non-CD sera. CONCLUSIONS: Selective deamidation specifically increases circulating antibody recognition of gliadin peptides in CD patients. This suggests that deamidated gliadin peptides are more specific CD B-cell epitopes than native peptides; this finding may be relevant for designing improved diagnostic tests.

Folding interpenetration in a gliadin model: the role of the characteristic octapeptide motif.

A model of a rheologically relevant protein, omega-gliadin, is proposed and studied in this work by means of molecular dynamics techniques. The model is based on an octapeptide repeat motif that is experimentally described as characteristic of that protein and as constituting it almost entirely. The initial molecular structure consisted of 20 such repeats. It was optimized and the dynamics developed along 980 ps, at dielectric constant epsilon = 80. Remarkable structural features were observed for the model built, such as an elongated twisted tubular overall structure with a peculiar interpenetrating folding pattern, of a very regular character, organized strand formation, topologically segregated sites on the outer surface with an alternate hydrophilic/hydrophobic character and a hydrophilic inner cavity. Dynamics produced significantly more relaxed structures, but was not able to change the main geometric features presented by the original structure. Preliminary attempts of correlating some structural/dynamic aspects observed for the model with features of gliadin rheological behavior are presented.