Allergenomic discovery pipeline for allergenic proteins complemented by large-scale genomic and proteomic resources
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Elite
ORGANISM(S): Crassostrea Gigas (pacific Oyster) (crassostrea Angulata)
TISSUE(S): Whole Body
SUBMITTER: 
Roni Nugraha
LAB HEAD: Andreas Lopata
PROVIDER: PXD006624 | Pride | 2022-02-23
REPOSITORIES: Pride
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Project description:Current diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive, high-throughput screening of patients. Additionally, extracts used in these methods are made from washed pollen that lacks pollen surface materials that may contain allergens. We sought to develop a high-throughput assay to rapidly measure allergen-specific IgE levels in sera and to explore the relative allergenicity of different pollen fractions (i.e. surface, cytoplasmic, commercial extracts). To do this, we generated a protein microarray containing surface, cytoplasmic, and commercial extracts from 22 pollen species, commercial extracts from nine non-pollen allergens, and five recombinant allergenic proteins. Arrays were incubated with <25uL of serum from 176 individuals and bound IgE was detected by indirect immunofluorescence, providing a high-throughput measurement of IgE levels.
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Project description:The frequent use of precautionary food allergen labeling statements such as “may contain” poses challenges to allergic individuals relying on such labeling to determine whether a food is safe to consume. To survey the frequency at which these precautionary statements indicate allergen contamination in commercial foods we developed a multiplexed liquid chromatography-mass spectrometry assay targeting 14 common allergens. For each allergen, we selected two or more tryptic peptides belonging to two or more clinically-recognized allergenic proteins by following one of two approaches. For certain allergenic proteins, a wealth of existing mass spectrometry literature suggests ideal tryptic peptide targets. In these cases, such for milk proteins Bos d 5 and Bos d 9, as well as peanut protein Ara h 3, we chose peptides using the Allergen Peptide Browser (APB), a freely available online database we developed. To choose peptides for the remaining allergenic proteins, as well as validate our choices, we applied shotgun proteomics to pure allergen protein extracts of the aforementioned allergens.
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Project description:Aspergillus terreus has been described as an allergenic fungus in addition to causing infection to both human beings and plants. However, the allergens in this fungus are still unknown. The identification of allergens is essential to develop diagnostic and therapeutic avenues. Employing a proteomic approach, sixteen allergens were identified based on two-dimensional immuno-blotting with A. terreus susceptible patient sera. Amongst them, triosephosphate isomerase (Asp t 36), one of the dominant immunoglobulin E (IgE)-reactive proteins, was purified under native conditions. To characterize Asp t 36, the gene was cloned and expressed in E. coli. Phylogenetic analysis showed it to be highly conserved with close similarity to Dermatophagoides farinae. Four immuno-dominant epitopes were identified using synthetic peptides and mapped on the tertiary structure of Asp t 36. Among these, two were found to create a continuous surface patch on the 3D structure rendering it to be an IgE binding hotspot. Asp t 36 showed similar secondary structure and temperature sensitivity with other reported triosephosphate isomerase allergens. In-vivo studies using the murine model demonstrated rAsp t 36 able to stimulate airway inflammation, such as an influx of eosinophils, goblet cell hyperplasia, elevated serum immunoglobulins and induction of Th2 cytokines. Collectively, our results reveal the immunogenic property of Asp t 36, a major allergen from A. terreus for the first time in any fungi. This allergen could serve as a potent candidate for investigating component resolved diagnosis and immunotherapy.
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Project description:Background: Respiratory allergy triggered by pollen allergens is increasing at an alarming rate worldwide. Sunflower pollen is thought to be an important source of inhalant allergens. Present study aims to identify the prevalence of sunflower pollinosis among the Indian allergic population and characterizes the pollen allergens using immuno-proteomic tools. Methodology: Clinico-immunological tests were performed to understand the prevalence of sensitivity towards sunflower pollen among the atopic population. Sera from selected sunflower positive patients were used as probe to detect the IgE-reactive proteins from the one and two dimensionally separated proteome of sunflower pollen. The antigenic nature of the sugar moiety of the glycoprotein allergens was studied by meta-periodate modification of IgE-immunoblot. Finally, these allergens were identified by mass-spectrometry (MALDI TOF/TOF and LC ESI qTOF). MASCOT searching was performed against NCBInr database. However, Helianthus annuus genome is not fully sequenced and partially annotated. So in case of low confidence (p> 0.05) protein identification, searching was performed against EST library of Helianthus annuus. Results: Prevalence of sunflower pollen allergy was observed among 21% of the atopic population and associated with elevated level of specific IgE and histamine in the sera of these patients. Immunoscreening of sunflower pollen proteome with patient serum detected seven IgE-reactive proteins with varying molecular weight and pI. Hierarchical clustering of 2D-immunoblot data highlighted three allergens characterized by a more frequent immuno-reactivity and increased levels of IgE antibodies in the sera of susceptible patients. These allergens were considered as the major allergens of sunflower pollen and were found to have their glycan moiety critical for inducing IgE response. Homology driven search of MS/MS data of these IgE-reactive proteins identified seven previously unreported allergens from sunflower pollen. Three major allergenic proteins were identified as two non-isoformic pectate lyases and a cystein protease. Conclusion: Novelty of the present report is the identification of a panel of seven sunflower pollen allergens for the first time at immuno-biochemical and proteomic level, which substantiated the clinical evidence of sunflower allergy. Further purification and recombinant expression of these allergens will improve component-resolved diagnosis and therapy of pollen allergy.
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Project description:Thermal processing of allergenic foods has the potential to induce a number of changes in the constituent allergens, but many of the effects of heat treatment are poorly defined. Like numerous other commonly allergenic tree nuts, walnuts often undergo heat treatment, such as roasting, prior to consumption. This study evaluated the changes in solubility and detectability of allergens from roasted walnuts using tandem mass spectrometry methods. Walnuts were roasted at 132 °C or 180 °C for 5, 10, or 20 minutes prior to preparation for LC-MS/MS using sequential or simultaneous protocols for extraction and trypsin digestion. LC-MS/MS data analysis incorporated label-free quantification and Maillard adduct screening. Relatively minor changes in detection were demonstrated following roasting in some allergenic proteins (2S albumin, LTP, and the 7S vicilin N-terminal region) with the LC-MS/MS method. The mature 7S vicilin and 11S legumin, however, showed significantly increased detection in the highest heat treatment sample when using the simultaneous extraction/digestion protocol, an effect likely due to increased digestibility of the proteins following heating. The results of this study indicate that individual walnut allergens respond differently to thermal processing, and the detection of these proteins by LC-MS/MS is dependent on the protein in question, its susceptibility to proteolytic digestion, the degree of thermal processing, and the sample preparation methodology.
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Project description:The recently published genome of peanut enables detailed molecular description of seed storage proteins, particularly allergens, which make up the peanut seed proteome. A detailed description of the allergenic proteome was produced using peanuts of different genetic backgrounds to increase the coverage of potentially expressed proteins and additionally allows for analysis of protein variability. Using relative quantification by mass spectrometry, minor variation of some allergenic proteins was observed but total levels of Ara h 1, 2, 3 and 6 were relatively consistent among genotypes. The isoform compositions of Ara h 1, 2 and 6 were similar. Three Ara h 3 isoforms were variable among genotypes and contributed to a large proportion of total Ara h 3 when present. The immunologically relevant amino acid modification hydroxyproline, previously thought to be present only on Ara h 2, was detected on Ara h 1 and 3 in consistent proportions given when the associated isoforms were present. The relevance of the data was evaluated in terms of food allergy regarding applications to reference materials, diagnostic and immunotherapeutic use, detection of peanuts in food, and risk assessment. The data can be used to design targeted mass spectrometry methods to permit analytical control of such materials.
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Project description:BEAS-2B cells, at air liquid interface, were exposed to birch pollen extract or house dust mite extract in a cloud chamber and, later, to UFP rich combustion aerosols in an in vitro exposure system. As control the same exposure was performed without allergen containing extracts. The goal was to understand the effect of allergenic pre-exposure to a UFP rich combustion aerosol exposed cells and their effect on allergic sensitization, using an established model that mimics more closely real life exposures.
2023-03-01 | GSE216533 | GEO
Project description:Why and when the immune system skews to Th2 mediated allergic immune responses is still poorly characterized. With two homologous lipocalins, the major respiratory dog allergen Can f 1 and the human endogenous, non-allergenic Lipocalin-1, we investigated their impact on human monocyte derived dendritic cells (DC). The two lipocalins had differential effects on DC according to their allergenic potential. Compared to Lipocalin-1, Can f 1 persistently induced lower levels of the Th1 skewing maturation marker expression, tryptophan breakdown and IL-12 production in DC. As a consequence, T cells stimulated by DC treated with Can f 1 produced more of the Th2 signature cytokine IL-13 and lower levels of the Th1 signature cytokine IFN-γ than T cells stimulated by Lipocalin-1 treated DC. These data were partially verified by a second pair of homologous lipocalins, the cat allergen Fel d 4 and its putative human homologue MUP. Our data indicate that the crosstalk of DC with lipocalins alone has the potential to direct the type of immune response to these particular antigens. A global gene expression analysis further supported these results and indicated significant differences in intracellular trafficking, sorting and antigen presentation pathways when comparing Can f 1 and Lipocalin-1 stimulated DC. With this study we contribute to a better understanding of the induction phase of a Th2 immune response.
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Project description:Purpose: Delonix regia or Gulmohor commonly grows here and there in Indian villages as well as it is used for megacity beautification and environmental management due to its evergreen nature and vibrant flower colour. However, an increasing incidence of seasonal pollinosis was observed among the inhabitants living in close vicinity to this tree suggesting a possible link between the airborne pollen load and the concomitant respiratory hazards. This prompted us to investigate the allergens in the pollen of this dominant avenue tree. Methodology: Allergenicity of D. regia pollen grains was first checked by Skin Prick Test (SPT) and further confirmed by in vitro tests, such as, ELISA, Dot Blot and histamine release assay. The total proteome profiling was done by 2D PAGE and it was confronted with the pooled sera of 10 patients. The IgE reactive proteins were identified by MALDI TOF/TOF in Autoflex speed (Bruker,Germany). The raw spectra were searched against NCBInr database using MASCOT search engine. Result: Delonix regia, pollen grains have been found in considerable amount in the air during its flowering season (May to July). Approximately 31% of atopic individuals were found allergic to D. regia, pollen with elevated level of specific IgE and histamine in the serum. Total 8 IgE reactive proteins have been identified by homology driven proteomics. These proteins are ATP synthase beta subunit (spot no 5), Actin (spot no 6), Hypothetical actin like protein (spot no 7), Recombinant S-adenosylmethionine synthase 2 (spot no 9), UDP-arabinopyranose mutase (spot no 13), Luminal-binding 5 (spot no 2), ELF3-like protein (spot no 8) and hypothetical protein OsJ_04810 (spot no 11). Among these eight identified allergenic proteins, five were previously reported as allergen from different sources whereas the rest three have been reported for the first time as novel allergens. Conclusion: Novelty of this study was to identify 8 allergens from D.regia for the first time using immune-biochemical and proteomic techniques. Further studies will open up new avenues in component resolved diagnosis of pollen allergy.
2021-09-08 | PXD015697 | Pride
