Influence of peanut matrix on stability of allergens in gastric-simulated digesta: 2S albumins are main contributors to the IgE reactivity of short digestion-resistant peptides.

BACKGROUND: Most food allergens sensitizing via the gastrointestinal tract are stable proteins that are resistant to pepsin digestion, in particular major peanut allergens, Ara h 2 and Ara h 6. Survival of their large fragments is essential for sensitizing capacity. However, the immunoreactive proteins/peptides to which the immune system of the gastrointestinal tract is exposed during digestion of peanut proteins are unknown. Particularly, the IgE reactivity of short digestion-resistant peptides (SDRPs; <10 kDa) released by gastric digestion under standardized and physiologically relevant in vitro conditions has not been investigated. OBJECTIVE: The aim of this study was to investigate and identify digestion products of major peanut allergens and in particular to examine IgE reactivity of SDRPs released by pepsin digestion of whole peanut grains. METHODS: Two-dimensional gel-based proteomics and shotgun peptidomics, immunoblotting with allergen-specific antibodies from peanut-sensitized patients, enzyme-linked immunosorbent inhibition assay and ImmunoCAP tests, including far ultraviolet-circular dichroism spectroscopy were used to identify and characterize peanut digesta. RESULTS: Ara h 2 and Ara h 6 remained mostly intact, and SDRPs from Ara h 2 were more potent in inhibiting IgE binding than Ara h 1 and Ara 3. Ara h 1 and Ara h 3 exhibited sequential digestion into a series of digestion-resistant peptides with preserved allergenic capacity. A high number of identified SDRPs from Ara h 1, Ara h 2 and Ara h 3 were part of short continuous epitope sequences and possessed substantial allergenic potential. CONCLUSION AND CLINICAL RELEVANCE: Peanut grain digestion by oral and gastric phase enzymes generates mixture of products, where the major peanut allergens remain intact and their digested peptides have preserved allergenic capacity highlighting their important roles in allergic reactions to peanut.

Peanut protein structure, polyphenol content and immune response to peanut proteins in vivo are modulated by laccase.

Food texture can be improved by enzyme-mediated covalent cross-linking of different food components, such as proteins and carbohydrates. Cross-linking changes the biological and immunological properties of proteins and may change the sensitizing potential of food allergens. In this study we applied a microbial polyphenol oxidase, laccase, to cross-link peanut proteins. The size and morphology of the obtained cross-linked proteins were analyzed by electrophoresis and electron microscopy. Structural changes in proteins were analyzed by CD spectroscopy and by using specific antibodies to major peanut allergens. The bioavailability of peanut proteins was analyzed using a Caco-2 epithelial cell model. The in vivo sensitizing potential of laccase-treated peanut proteins was analyzed using a mouse model of food allergy. Finally, peanut polyphenols were analyzed by UHPLC-MS/MS, before and after the enzymatic reaction with laccase. Laccase treatment of peanut proteins yielded a covalently cross-linked material, with the modified tertiary structure of peanut proteins, improved bioavailability of Ara h 2 (by 70 fold, p < 0.05) and modulated allergic immune response in vivo. The modulation of the immune response was related to the increased production of IgG2a antibodies 11 fold (p < 0.05) and reduced IL-13 secretion in in vitro cultured splenocytes 7 fold (p < 0.05). Analysis of the peanut polyphenol content and profile by HPLC-MS/MS revealed that laccase treatment depleted the peanut extract of polyphenol compounds leaving mostly isorhamnetin derivatives and procyanidin dimer B-type in detectable amounts. Treatment of complex food extracts rich in polyphenols with laccase results in both protein cross-linking and modification of polyphenol compounds. These extensively cross-linked proteins have unchanged potency to induce allergic sensitization in vivo, but certain immunomodulatory changes were observed.

Xylem parenchyma cell walls lack a gravitropic response in conifer compression wood.

MAIN CONCLUSION: Cell wall fluorescence and immunocytochemistry demonstrate that xylem parenchyma cell walls do not show changes in structure and composition related to gravitropic response comparable to those of tracheids, even when they have lignified secondary cell walls. Tracheid cell walls in compression wood have altered composition and structure which generates the strain responsible for correction of stem lean as part of the gravitropic response of woody plants. Xylem parenchyma cell walls vary among conifer species and can be lignified secondary walls (spruce) or unlignified primary walls (pine). It can be expected that xylem parenchyma with lignified secondary cell walls might show features of compression wood comparable to those of tracheids that have a similar type of cell wall. A comparison of xylem parenchyma cell walls in normal and compression wood in species with lignified and non-lignified parenchyma cell walls provides a unique opportunity to understand the process of reaction wood formation in conifers. Using both UV/visible fluorescence microscopy of cell wall fluorophores and immunocytochemistry of galactan and mannan epitopes, we demonstrate that xylem parenchyma cell walls do not show the changes in composition and structure typical of compression wood tracheids. Adjacent cells of different types but with similar cell wall structure can undergo cell wall developmental changes related to support or defence functions independent of their neighbours. Tracheids are sensitive to gravitropic signals while xylem parenchyma cells are not.