Digestomics of peanut allergens and characterization of digestion resistant fragments

INFOGEST metoda predstavlja standardizovani protokol za in vitro simulaciju digestije kompletne hrane, zasnovanom na fiziološki relevantnim uslovima. Predmet rada ove disertacije je ispitivanje digestibilnosti alergena kikirikija iz celog zrna primenom INFOGEST metode, kao i karakterizacija njihovih fragmenata otpornih na proteolizu. Za odstranjivanje lipida primenjena je metoda taloženja proteina, koja se pokazala kao superiornija u odnosu ekstrakciju lipida organskim rastvaračem, usled manjeg kvalitativnog i kvantitativnog gubitka proteina. U ovoj tezi je pokazano da termički tretmani kikirikija, pored matriksa hrane, dodatno otežavaju oslobađanje proteina iz zrna, što čini glavne alergene kikirikija Ara h 1, Ara h 2 Ara h 3 i Ara h 6 nedostupnijim za pepsinsku hidrolizu. Oslobađanje proteina kikirikija, kao i digestibilnost, u gastričnoj fazi se pokazala znatno izraženijom, u odnosu na intestinalnu fazu, s tim da je digestija kod pečenog kikirikija otežana u odnosu na sirovi. Nakon oralno-gastrične digestije celog zrna sirovog kikirikija, glavni alergeni kikirikija u velikoj meri ostaju intaktni, a njihovi peptidi otporni na digestiju zadržavaju alergeni kapacitet. Pokazano je da većina Ara h 2 i Ara h 6 alergena ostaje rezistentna na digestiju. Ara h 1 i Ara h 3 kaskadno podležu pepsinolizi, do fragmenata koji i dalje zadržavaju IgE vezujući potencijal. Mali peptidi koji potiču od Ara h 2 alergena, su se pokazali kao najpotentniji inhibitori vezivanja IgE iz seruma pacijenata alergičnih na kikiriki, u odnosu na male Ara h 1 i Ara h 3 peptide. U ovoj disertaciji je pokazana izuzetno važna uloga efekata matriksa hrane, kao i njene termičke obrade, na digestiju proteina hrane, koji mogu povećati stabilnost alergena hrane tokom digestije, i time omogućiti zadržavanje potencijala aktivacije alergijske reakcije nakon oralno-gastrične faze digestije.INFOGEST method is standardized protocol for in vitro simulation of complete food digestion, based on physiologicaly relevant conditions. The objective of this dissertation was to investigate digestibility of peanut allergens from whole peanut kernel by INFOGEST method, as well as to characterize their fragments resistant to proteolysis. For delipidation, protein precipitation approach was applied, showing to be superior in comparison to delipidation by organic solevent, due to lower qualitative and quantitative protein loss. In this thesis it was shown that peanut thermal processing, in addition to effect of food matix, further complicates the extractability and digestibility of proteins from the grain, making peanut allergens Ara h 1, 2, 3 and 6, less accessible for pepsin hydrolysis. Extractability and digestibility of peanut proteins in the gastric phase have shown to be significantly more pronounced, in comparison to intestinal phase, and roasted peanut digestion was impaired compared to the raw. It was shown that after oral and gastric digestion of whole raw peanut grains peanut allergens largely remain intact, and their digestion resistant peptides retain allergenic capacity. The most Ara h 2 and Ara h 6 allergens have been shown to remain resistant to digestion. Ara h 1 and Ara h 3 undergo pepsinolysis with cascade pattern to consequently smaller peptide fragments with retained IgE binding capacity. Small peptides from Ara h 2 allergens were the most potent inhibitors of IgE binding from sera of peanut allergic patients, compared to small peptides from Ara h 1 and Ara h 3. This thesis points to the great importance of the effects of food matrix, as well as food thermal processing, on protein digestibility, which can create additional stability of food allergens during digestion, and thus enable retaining of their potential for the sensitization or triggering of allergic reactions

Food allergies on the rise: the role of anthropogenic chemicals

Food allergies have increased dramatically in the last decade, especially in developedcountries. Food tolerance requires strict maintenance of a specific microbial portfolio inthe gastrointestinal tract, as changes in the gut microbiome can lead to its disruption,which in turn causes inflammation and pathogenic gut conditions leading to thedevelopment of food allergies. Any environmental factors that lead to a disturbanceand/or malfunction of the gastrointestinal tract and digestive performance favor thedevelopment of food allergies.Based on that, what do we know about the role of increasing anthropogenic chemicals,including emerging ones, resulting from the new global situation?There is awareness that their effects are multifaceted, e.g., chemicals affect the growth ofplants and animals and thus the quality of the food produced. In addition, chemicals affectour food during its production and processing, but also affect our body andgastrointestinal tract. It is time to fill the knowledge gaps and understand how theseinteractions between environmental triggers such as industrial and traffic pollution,transition and heavy metals, pesticides, chemtrails, etc., affect food allergens and theirallergenicity, adjuvant effects, and the increasing prevalence of food allergies.Some improvements in this area are already being made through advances in ‘omics’technologies (i.e., proteomics, genomics, metabolomics) and systems biology approachesthat will hopefully provide a scientific understanding of the relationship betweenincreasing food allergies and the increasingly present wide variety of anthropogenicchemicals in our environment

Food allergies on the rise: the role of anthropogenic chemicals

Food allergies have increased dramatically in the last decade, especially in developed countries. Food tolerance requires strict maintenance of a specific microbial portfolio in the gastrointestinal tract, as changes in the gut microbiome can lead to its disruption, which in turn causes inflammation and pathogenic gut conditions leading to the development of food allergies. Any environmental factors that lead to a disturbance and/or malfunction of the gastrointestinal tract and digestive performance favor the development of food allergies. Based on that, what do we know about the role of increasing anthropogenic chemicals, including emerging ones, resulting from the new global situation? There is awareness that their effects are multifaceted, e.g., chemicals affect the growth of plants and animals and thus the quality of the food produced. In addition, chemicals affect our food during its production and processing, but also affect our body and gastrointestinal tract. It is time to fill the knowledge gaps and understand how these interactions between environmental triggers such as industrial and traffic pollution, transition and heavy metals, pesticides, chemtrails, etc., affect food allergens and their allergenicity, adjuvant effects, and the increasing prevalence of food allergies. Some improvements in this area are already being made through advances in ‘omics’ technologies (i.e., proteomics, genomics, metabolomics) and systems biology approaches that will hopefully provide a scientific understanding of the relationship between increasing food allergies and the increasingly present wide variety of anthropogenic chemicals in our environment

Supplementary data for article: Stanić-Vučinić, D.; Prodić, I.; Apostolović, D.; Nikolić, M.; Ćirković-Veličković, T. Structure and Antioxidant Activity of Beta-Lactoglobulin-Glycoconjugates Obtained by High-Intensity-Ultrasound-Induced Maillard Reaction in Aqueous Model Systems under Neutral Conditions. Food Chemistry 2013, 138 (1), 590–599. https://doi.org/10.1016/j.foodchem.2012.10.087

Supplementary material for: [https://doi.org/10.1016/j.foodchem.2012.10.087]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1583

LEA4 proteins: How disordered are they?

Abstract book: 1st ML4NGP meeting on machine learning and non-globular proteins, July 5-7, 2023, Bratislava, Slovaki

The use of tryptic food protein digests data in public proteomic repositories to assess the effects of chemical and post-translational modifications on digestion outcomes

Porcine-derived trypsin generated proteomic data of the major peanut allergen Ara h 1 from the peanut was reassessed to search for possible facilitating/hindrance effects on trypsin digestion efficacy caused by post-translational and chemical modifications (PTMs) positioned on arginine or lysine (K/R) residues. If the potential effects caused by PTMs are observed with porcine trypsin, they can be just augmented and more pronounced within human intestinal digestion. The reasoning is in inferior performance of human trypsin compared to porcine-derived used in proteomic digestion protocols, also in the lower trypsin-to-sample ratio and much shorter digestion times, even though gastric digestion precedes and trypsin is not the sole digestive enzyme. A novel method was developed to decipher cleavage or miscleavage outcomes at scissile bonds in each, modified and unmodified sequence counterparts, using PEAKS Studio-X+ (Bioinformatics Solutions Inc., Ontario, Canada) in the reassessment of high-resolution tandem mass spectrometry data, from 18-hour long trypsin digestion proteomic protocols. In general, eight site-specific and modified K/R residues with methylation, dihydroxy and formylation showed significantly higher content of miscleaved bonds (at least >10%) compared to their unmodified counterpart peptides. Specifically, dihydroxylation and formylation hindered trypsin efficacy, while methylation on several K/R showed opposite effects. It is essential to elucidate the specific impacts of modifications on trypsin digestion performance and if there are additional effects generated by food processing, which could influence digestion outcomes and allergenicity of food proteins/peptides.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Dehydrins in the service of protecting the DNA helix from the aspect of molecular dynamics (MD)

Drought stress is one of the greatest threats to global food security, posing a major challenge to agriculture. Understanding the molecular mechanisms underlying desiccation tolerance in resurrection plants like Ramonda serbica Panc., can provide valuable insights for improving crop resilience. Dehydrins are intrinsically disordered proteins known to accumulate in these plants in response to desiccation. Among several proposed physiological roles, it has been suggested that dehydrins can protect DNA from damage during water shortage. Here, we have characterised dehydrins from R. serbica, selected a representative one and evaluated its potential to interact with DNA. Most of the R. serbica dehydrins were designated as hydrophilins (glycine content >6%; GRAVY index <1). They exhibit a high disorder propensity, making them quite dynamic in solution. Furthermore, they were predicted to localize in the nucleus. To examine the potential interactions with DNA in silico, we have selected a representative, highly hydrophilic dehydrin (Gravy index: –1.29) containing a high percentage of glycine (22.6%) and charged amino acids (lysine, glutamate and aspartate). Its 3D structures were determined using the Phyre 2 intensive modelling and AlphaFold. The dehydrin-DNA complex was manually adjusted, following molecular dynamic simulation (MDS) in both cases of hydration and desiccation. To simulate complete hydration, the DNAdehydrin complex was solvated in a water box, with final dimensions of 100×69×82 Å, neutralised with 0.15 M NaCl. The system underwent a 10,000-step energy minimization, consecutive 1250 ps equilibration NVE (constant number of atoms, volume and energy) heating from 10 K to 298 K and 100 ns NPT (constant number of atoms, pressure and temperature) MD production at 1 bar, and 1 fs integration step. In all simulations, periodic boundary conditions (PBC) were implemented and the CHARMM36 force field was used. The obtained results revealed that selected dehydrin can interact with both minor and major DNA grooves. The phosphate groups from the DNA molecule form salt bridges with the positively charged lysines from polylysine, K-segment, contributing to the complex stability. Overall, we have provided evidence for possible dehydrin-DNA interactions. However, the exact nature and significance of these interactions is still an area of active research in vitro.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

INFOGEST Digestion Assay of Raw and Roasted Hazelnuts and Its Impact on Allergens and Their IgE Binding Activity

Most of the food allergens sensitized via the gastrointestinal tract resist thermal treatments and digestion, particularly digestion by pepsin. Roasted hazelnuts are more commonly consumed than raw ones. Since no studies have characterized gastric digestion protein fragments of raw and roasted hazelnuts nor their IgE binding properties, we compared these aspects of raw and roasted hazelnuts’ gastric digesta obtained by INFOGEST protocol. Their electrophoretically resolved profiles were probed with hazelnut allergic patients’ sera in 1D and 2D immunoblots. Electrophoretic profiles demonstrated pepsin digestion of all hazelnut allergens to varying extents. While 2D immunoblots indicated that roasting slightly reduced allergenicity, IgE ELISA with the pool of sera showed a slight significant (10%) increase in IgE binding in both gastric digesta. Cor a 9 isolated from the raw and roasted hazelnuts, characterized by far and near CD, remained stable after roasting, with preserved IgE reactivity. Its immunoreactivity contribution by inhibitory ELISA was noticeable in raw and roasted hazelnut digesta; its activity was slightly stronger in the roasted preparations. Roasting has a visible impact on proteins; however, it did not affect overall IgE reactivity. Gastric digestion slightly increases the overall IgE reactivity in raw and roasted hazelnuts, and may therefore impact the profiles of allergens and their fragments available to interact with the immune system in the small intestine

Comparative study of raw and thermally treated peanut major allergen post- translational modifications (PTMs)

Introduction. Peanut allergy affects a large portion of world population causing reactions ranging from mild to severe. Major peanut allergen IgE epitopes are well characterized but little is known about their post-translational modifications (PTM) and how they are affected by thermal treatment. PTM profile may differ between raw and thermally treated peanut, which could affect its allergic potential depending on type, size and position of modifications. Objective. Our aim was to analyse and compare PTM profiles of 4 major peanut allergens - Ara h 1, Ara h 2, Ara h 3 and Ara h 6, as well as their amounts in raw and roasted samples using bottom-up proteomics methods. Methodology. Full peanut protein extracts (both thermally treated and non-treated) were digested in gel and in solution, and analysed by a Top10 nLC-MS/MS method by LTQ Orbitrap XL (Thermo Fisher Scientific Inc., Germany). Within the extracts major allergens - Ara h 1, Ara h 2, Ara h 3 and Ara h 6 were identified, label free quantified (LFQ) and searched for PTMs by Peaks X software (Bioinformatics solutions Inc.I, Canada). Epitope sequences were acquired from the Immune Epitope Database (IEDB www.iedb.org). Main findings. LFQ results show that there is no significant change in the amountsof any of the studied allergens between raw and roasted extracts.Out of the 4 allergens Ara h 6 is modified in the highest portion, with respect to the protein size: 15% and 12% of its positions are modified in raw and roasted sample, respectively. Total of 21 modifications were quantified between the two preparations, with oxidation (M), methylation (K,R) and dethiomethylation affecting the largest number of peptides. Conclusions. Peanut allergen epitopes are indeed carriers of PTMs that differ in pattern and quantity between treated and non-treated extracts. The in silico discovered PTMs could affect protein digestibility and allergenicity. Further investigation is necessary in order to fully understand the impact protein modifications could have on their allergenic potential

Investigation of raw and thermally treated peanut major allergen post- translational modifications (PTMs)

Introduction. Peanut allergy affects a large portion of world population causing reactions ranging from mild to severe. Major peanut allergen IgE epitopes are well characterized but little is known about their post-translational modifications (PTM) and how they are affected by thermal treatment. PTM profile may differ between raw and thermally treated peanut, which could affect its allergic potential depending on type, size and position of modifications. Objective. Our aim was to analyse and compare PTM profiles of 4 major peanut allergens - Ara h 1, Ara h 2, Ara h 3 and Ara h 6, as well as their amounts in raw and roasted samples using bottom-up proteomics methods. Methodology. Full peanut protein extracts (both thermally treated and non-treated) were digested in gel and in solution, and analysed by a Top10 nLC-MS/MS method by LTQ Orbitrap XL (Thermo Fisher Scientific Inc., Germany). Within the extracts major allergens - Ara h 1, Ara h 2, Ara h 3 and Ara h 6 were identified, label free quantified (LFQ) and searched for PTMs by Peaks X software (Bioinformatics solutions Inc.I, Canada). Epitope sequences were acquired from the Immune Epitope Database (IEDB www.iedb.org). Main findings. LFQ results show that there is no significant change in the amounts of any of the studied allergens between raw and roasted extracts. Out of the 4 allergens Ara h 6 is modified in the highest portion, with respect to the protein size: 15% and 12% of its positions are modified in raw and roasted sample, respectively. Total of 21 modifications were quantified between the two preparations, with oxidation (M), methylation (K,R) and dethiomethylation affecting the largest number of peptides. Conclusions. Peanut allergen epitopes are indeed carriers of PTMs that differ in pattern and quantity between treated and non-treated extracts. The in silico discovered PTMs could affect protein digestibility and allergenicity. Further investigation is necessary in order to fully understand the impact protein modifications could have on their allergenic potential