Dataset Information

Gastrointestinal digestion of hazelnut allergens on molecular level: Elucidation of degradation kinetics and resistant immunoactive peptides using mass spectrometry

ABSTRACT: Allergy to hazelnut seeds ranks among the most prevalent food allergies in Europe. The aim of this study was to elucidate the gastrointestinal digestion of hazelnut allergens on molecular level. Hazelnut flour was digested in vitro following the Infogest consensus model. For six allergenic proteins, the time-dependent course of digestion was monitored by SDS-PAGE and HPLC−MS/MS, and degradation products were characterized by a bottom-up proteomics approach. Depending on the molecular structure, a specific biochemical fate was observed for each allergen, and degradation kinetics were traced back to the peptide level. 1183 peptides were characterized, including 130 peptides that carry known IgE-binding epitopes and may represent sensitizers for hazelnut allergy. The kinetics of peptide formation and degradation were determined by label free quantification and follow a complex multi-stage mechanism.

INSTRUMENT(S): LTQ Orbitrap XL

ORGANISM(S): Corylus Avellana

TISSUE(S): Seed

SUBMITTER: Robin Korte

LAB HEAD: Jens Brockmeyer

PROVIDER: PXD006412 | Pride | 2017-07-11

REPOSITORIES: Pride

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Gastrointestinal digestion of hazelnut allergens on molecular level: Elucidation of degradation kinetics and resistant immunoactive peptides using mass spectrometry.

Korte Robin R Bräcker Julia J Brockmeyer Jens J

Molecular nutrition & food research 20170814 10

<h4>Scope</h4>Allergy to hazelnut seeds ranks among the most prevalent food allergies in Europe. The aim of this study was to elucidate the gastrointestinal digestion of hazelnut allergens on molecular level.<h4>Methods and results</h4>Hazelnut flour was digested in vitro following the Infogest consensus model. For six allergenic proteins, the time-dependent course of digestion was monitored by SDS-PAGE and HPLC-MS/MS, and degradation products were characterized by a bottom-up proteomics approac ...[more]

PMID: 28677900

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Project description:Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that amylase binding to S. gordonii modulates expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect differentially expressed genes in S. gordonii strain CH1 in response to the binding of purified human salivary amylase as compared to exposure to heat-denatured amylase. Selected genes found to be differentially expressed were validated by qRT-PCR. Five genes from the fatty acid synthesis (FAS) cluster were highly (10-35 fold) up-regulated in amylase treated S. gordonii CH1 cells compared to the denatured-amylase treated cells. An abpA-deficient strain of S. gordonii exposed to amylase did not show a similar response in FAS gene expression as observed in the parental strain. Predicted phenotypic effects of amylase binding to S. gordonii strain CH1 associated with increased expression of FAS genes leading to changes in fatty acid synthesis were noted, as evidenced by increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase exposed abpA-deficient strain, suggesting for the role of AbpA in amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response in S. gordonii, resulting in a phenotype adjustment that is potentially advantageous for bacterial survival in the oral environment. In order to identify amylase-regulated genes, S. gordonii CH1 was grown statically in 40 ml CDM at 37°C in a candle jar to mid-log phase corresponding to an optical density at 600 nm of 0.5 to 0.6.The mid-log phase bacterial culture was divided into two aliquots of equal volume. Bacterial cells from all aliquots were pelleted by centrifugation at 6,000 x g in a Sorvall RC6 centrifuge at 20°C, and washed once with simulated salivary buffer preconditioned to 37°C. Simulated salivary buffer containing 0.4 mg/ml purified, non-glycosylated salivary amylase (native amylase) and preconditioned to 37°C was added to the cells of the fist aliquot; to the cells of the second aliquot simulated salivary buffer containing 0.4 mg/ml of the same salivary amylase denatured by heating to 100°C {Heinen, 1976} and cooled to 37°C was added, as a negative control. Each aliquot, amylase treated and control, was incubated statically for 15 min at 37°C in a candle jar. Total RNA was immediately isolated by the hot acid phenol method as described previously {Vickerman, 2007}, followed by treatment with TurboDNase (Applied Biosystems/Ambion, Austin, TX) according to manufacturer’s protocol. Remaining contaminants were removed using the RNeasy minikit column (Qiagen, Valencia, CA) with the cleanup protocol. Total RNA was quantified using the Nanodrop 2000 spectrophotometer and RNA integrity determined by agarose gel electrophoresis. Total RNA was used immediately for cDNA synthesis. The Cy dye-labeled cDNA from the amylase-treated aliquot of the culture was mixed with Cy dye-labeled cDNA from the denatured amylase-treated control aliquot, and used to probe the S. gordonii microarray slides. Each amylase-exposure experiment was repeated from four biological replicates. To confirm microarray results the cDNA from each strain was labeled with the opposite Cy dye and hybridized to similar arrays for the flip-dye comparison. Overall design 4 samples were analyzed. The quality controls were 4 biological replicates and dye-swap technical replicates for each biological replicate.

Dataset Information

Gastrointestinal digestion of hazelnut allergens on molecular level: Elucidation of degradation kinetics and resistant immunoactive peptides using mass spectrometry

Publications

Gastrointestinal digestion of hazelnut allergens on molecular level: Elucidation of degradation kinetics and resistant immunoactive peptides using mass spectrometry.

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