Crosstalk Between the Immune System and Plant-Derived Nanovesicles: A Study of Allergen Transporting

Background: Nanometer-sized membrane-surrounded vesicles from different parts of plants including fruits are gaining increasing attention due to their anti-inflammatory and anticancer effects demonstrated by in vitro and in vivo studies, and as nanovectors for molecular delivery of exogenous substances. These nanomaterials are very complex and contain a diverse arsenal of bioactive molecules, such as nucleic acids, proteins, and lipids. Our knowledge about the transport of allergens in vesicles isolated from plant food is limited today.Methods: Here, to investigate the allergenicity of strawberry-derived microvesicles (MVs), nanovesicles (NVs), and subpopulations of NV, we have set up a multidisciplinary approach. The strategy combines proteomics-based protein identification, immunological investigations, bioinformatics, and data mining to gain biological insights useful to evaluate the presence of potential allergens and the immunoglobulin E (IgE) inhibitory activity of vesicle preparations.Results: Immunological test showed that several proteins of strawberry-derived vesicles compete for IgE binding with allergens spotted on the FABER biochip. This includes the known strawberry allergens Fra a 1, Fra a 3, and Fra a 4, and also other IgE-binding proteins not yet described as allergens in this food, such as gibberellin-regulated proteins, 2S albumin, pectate lyase, and trypsin inhibitors. Proteomics identified homologous sequences of the three strawberry allergens and their isoforms in total protein extract (TPE) but only Fra a 1 and Fra a 4 in the vesicle samples. Label-free quantitative proteomic analysis revealed no significant enrichment of these proteins in strawberry vesicles with respect to TPE.Conclusion: Immunological tests and bioinformatics analysis of proteomics data sets revealed that MVs and NVs isolated from strawberries can carry functional allergens their isoforms as well as proteins potentially allergenic based on their structural features. This should be considered when these new nanomaterials are used for human nutraceutical or biomedical applications.

The usefulness of molecular diagnosis in the assessment of the aetiology, clinical phenotypes and risk of food-induced anaphylaxis in children

Aim: Food-induced anaphylaxis is most common in the paediatric population and has an unpredictable course. The aim of this paper was to perform a demographic and clinical assessment of food-induced anaphylaxis in children using molecular diagnosis. Materials and methods: The study included 541 children aged 0–18 years who developed 893 sudden reactions to food. Levels of IgEs against 112 allergen molecules were measured in each child. We analysed demographic and clinical data in two age groups. The aetiology of anaphylaxis was determined at the level of source allergens and at the level of allergen molecules. We also determined the risk factors for severe clinical course of reactions. Results: A total of 631 food-induced anaphylactic reactions developed by 421 children were included in the analysis. The group of children aged 0–6 years was mostly composed of boys (p = 0.0023) and children with atopic dermatitis (p = 0.0001). Also, cutaneous and mucosal symptoms were more common (p < 0.0001), and milk casein, Bos d 8, was the most common cause of anaphylaxis in this group (p < 0.0001). In the group of 7–18-year-olds, anaphylaxis was more common in children with no asthma or atopic dermatitis (p = 0.0001); hazelnuts (p = 0.0005) and, in terms of allergen molecules, walnut 2S albumin, Jug r 1 (p = 0.0011), were a more common cause of reaction; as well as exercise-induced anaphylaxis (p < 0.0001) and cardiovascular symptoms (p = 0.0247) were more common. In the study population, more severe anaphylaxis was more common in children without asthma or atopic dermatitis (p = 0.0428) and in the case of anaphylaxis induced by cashew nut 2S albumin, Ana o 3 (p < 0.0001) and wheat allergen, Tri a 14 (p = 0.0143). Conclusions: Molecular diagnostics allows for a detailed assessment of the aetiology and the risk of severe food-induced anaphylaxis.

Seed Storage Proteins, 2S Albumin And 11S Globulin, Associated to Severe Allergic Reactions after Flaxseed Intake

Background: Given the increased popularity of flaxseed in meals, several cases of allergy to these seeds have been reported. Little is known about allergens implicated in hypersensitivity reactions to these seeds. The present work aimed to identify the allergens involved in IgE-mediated reactions in five patients with a clinical history of severe systemic symptoms after flaxseed consumption. Methods: Proteins susceptible to be allergens with IgE-binding capacity were purified from flaxseed extract by chromatographic techniques. Their identification was achieved via MALDI-TOF mass spectrometry. Immunoassays were performed using the five allergic patient’s era either by testing them individually or as a pool. Results: Four out of five patients recognized a low-molecular-mass protein (around 13kDa) by immunoblotting of the flaxseed extract, while two patients recognized a protein of approximately 55 kDa. They were identified by mass spectrometry as flaxseed 2S albumin, included into WHO/IUIS allergen nomenclature as Lin u 1,and 11S globulin, respectively. Inhibition assays revealed in vitro IgE-cross-reactivity of Lin u 1 with peanut and cashew nut proteins, while IgE recognition of 11S globulin by patients’ sera was partially inhibited by several plant-derived sources. Conclusions: Seed storage proteins from flaxseed were involved in the development of severe symptoms in five individuals and exhibited cross-reactivity with other allergenic sources. Besides the severity of flaxseed allergy in patients sensitized to 2S albumin, it is the first time that the 11S globulin is identified as a potential allergen. We consider that these data should be taken into account for a more accurate diagnosis of patients.

Characterization of Relevant Biomarkers for the Diagnosis of Food Allergies: An Overview of the 2S Albumin Family

2S albumins are relevant and often major allergens from several tree nuts and seeds, affecting mainly children and young people. The present study aims to assess how the structural features of 2S albumins could affect their immunogenic capacity, which is essential to comprehend the role of these proteins in food allergy. For this purpose, twelve 2S albumins were isolated from their respective extracts by chromatographic methods and identified by MALDI-TOF mass-spectrometry. Their molecular and structural characterization was conducted by electrophoretic, spectroscopic and in silico methods, showing that these are small proteins that comprise a wide range of isoelectric points, displaying a general high structure stability to thermal treatment. Despite low amino acid sequence identity, these proteins share structural features, pointing conformational epitopes to explain cross-reactivity between them. Immunoblotting with allergic patients’ sera revealed those possible correlations between evolutionarily distant 2S albumins from different sources. The availability of a well-characterized panel of 2S albumins from plant-derived sources allowed establishing correlations between their structural features and their allergenic potential, including their role in cross-reactivity processes.

Effect of O-linked glycosylation on the antigenicity, cellular uptake and trafficking in dendritic cells of recombinant Ber e 1

Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy.

Vicilin and legumin storage proteins are abundant in water and alkali soluble protein fractions of glandless cottonseed

In this work, we sequentially extracted water (CSPw)- and alkali (CSPa)-soluble protein fractions from glandless cottonseed. SDS-Gel electrophoresis separated CSPw and CSPa to 8 and 14 dominant polypeptide bands (110–10 kDa), respectively. Liquid chromatography-electrospray ionization-tandem mass spectrometry identified peptide fragments from 336 proteins. While the majority of peptides were identified as belonging to vicilin and legumin storage proteins, peptides from other functional and uncharacterized proteins were also detected. Based on the types (unique peptide count) and relative abundance (normalized total ion current) of the polypeptides detected by mass spectrometry, we found lower levels (abundance) and types of legumin isoforms, but higher levels and more fragments of vicilin-like antimicrobial peptides in glandless samples, compared to glanded samples. Differences in peptide fragment patterns of 2S albumin and oleosin were also observed between glandless and glanded protein samples. These differences might be due to the higher extraction recovery of proteins from glandless cottonseed as proteins from glanded cottonseed tend to be associated with gossypol, reducing extraction efficiency. This work enriches the fundamental knowledge of glandless cottonseed protein composition. For practical considerations, this peptide information will be helpful to allow better understanding of the functional and physicochemical properties of glandless cottonseed protein, and improving the potential for food or feed applications.

Inhibition of SARS-CoV-2 3CL Mpro by Natural and Synthetic Inhibitors: Potential Implication for Vaccine Production Against COVID-19

COVID-19 has created a pandemic situation all over the world. It has spread in nearly every continent. Researchers all over the world are trying to produce an effective vaccine against this virus, however; no specific treatment for COVID-19 has been discovered -so far. The current work describes the inhibition study of the SARS-CoV-2 main proteinase or 3CL Mpro by natural and synthetic inhibitors, which include 2S albumin and flocculating protein from Moringa oleifera (M. oleifera) and Suramin. Molecular Docking study was carried out using the programs like AutoDock 4.0, HADDOCK2.4, patchdock, pardock, and firedock. The global binding energy of Suramin, 2S albumin, and flocculating proteins were −41.96, −9.12, and −14.78 kJ/mol, respectively. The docking analysis indicates that all three inhibitors bind at the junction of domains II and III. The catalytic function of 3CL Mpro is dependent on its dimeric form, and the flexibility of domain III is considered important for this dimerization. Our study showed that all three inhibitors reduce this flexibility and restrict their motion. The decrease in flexibility of domain III was further confirmed by analysis coming from Molecular dynamic simulation. The analysis results indicate that the temperature B-factor of the enzyme decreases tremendously when the inhibitors bind to it. This study will further explore the possibility of producing an effective treatment against COVID-19.

IgE-Binding Epitopes of Pis v 1, Pis v 2 and Pis v 3, the Pistachio (Pistacia vera) Seed Allergens

Sequential IgE-binding epitopes were identified on the molecular surface of the Pis v 1 (2S albumin), Pis v 2 (11S globulin/legumin) and Pis v 3 (7S globulin/vicilin)—major allergens from pistachio (Pistacia vera) seeds—using the Spot technique. They essentially consist of hydrophilic and electropositively charged residues well exposed on the surface of the allergens. Most of the epitopic regions identified on Pis v 1 and Pis v 3 do not coincide with the putative N-glycosylation sites and thus are not considered as glycotopes. Surface analysis of these epitopic regions indicates a high degree of conformational similarity with the previously identified epitopic regions of the corresponding allergens Ana o 1 (vicilin), Ana o 2 (legumin) and Ana o 3 (2S albumin) from the cashew (Anacardium occidentale) nut. These results offer a molecular basis for the IgE-binding cross-reactivity often observed between pistachio and cashew nut. They support the recommendation for prescribing pistachio avoidance in cashew allergic patients. Other conformational similarities were identified with the corresponding allergens Ses i 1 (2S albumin), Ses i 3 (vicilin) and Ses i 6 (legumin) from sesame (Sesamum indicum), and Jug r 1 (2S albumin), Jug r 2 (vicilin) and Jug r 4 (legumin) from walnut (Juglans regia). Conversely, conformation of most of the epitopic regions of the pistachio allergens often differs from that of epitopes occurring on the molecular surface of the corresponding Ara h 1 (vicilin), Ara h 2 (2S albumin) and Ara h 3 (legumin) allergens from peanut (Arachis hypogaea).