RESUMEN
BACKGROUND: Protein oxidation can occur as a consequence of lipid peroxidation during food processing. The aim of this work was to explore the effect of malondialdehyde (MDA) modification of ovalbumin (OVA) on its interaction with T84 intestinal cells. METHODS: Molecular dynamics simulation was employed for the prediction of MDA modification in the OVA, while introduced structural changes were evaluated by measurement of carbonyl group content, fluorescence spectra, MS/MS analysis, and IgE reactivity. Effects of MDA modified OVA on T84 epithelial cells were analyzed by gene expression for pro-inflammatory cytokines and protein secretion. RESULTS: Out of 9 predicted, five modified Lys residues were confirmed by MS/MS analysis: 51TQINKVVR58, 85DILNQITKPNDVYSFSLASR104, 111YPILPEYLQCVKELYR126, 187AFKDEDTQAMPFR199, 277KIKVYLPR284, and 278IKVYLPR284. The introduced MDA modifications influenced profile of IgE reactivity to OVA. Treatment of T84 epithelial cells with OVA and OVA modified with 1mM MDA, induced up-regulation of pro-inflammatory cytokines (IL-1ß, IL-25, IL-33, TSLP and TNFα), while OVA modification with 10mM MDA induced down regulation of the cytokine expression profile, except for IL-1ß. OVA and OVA modified with 1mM MDA induced secretion of epithelial cells specific cytokine IL-33. CONCLUSIONS: This finding indicated that OVA and its MDA modified form have the potential to trigger the innate immunity by inducing up-regulation and secretion of pro-allergenic IL-33 in T84 intestinal epithelial cells. GENERAL SIGNIFICANCE: Interactions of ovalbumin and its MDA modified form with intestinal epithelial cells can induce a specific immunological priming necessary for the downstream activation of innate immunity.
Asunto(s)
Células Epiteliales/metabolismo , Malondialdehído/farmacología , Ovalbúmina/metabolismo , Línea Celular , Citocinas/metabolismo , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunoglobulina E/metabolismo , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Interleucina-33/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Simulación de Dinámica Molecular , Espectrometría de Masas en Tándem/métodos , Regulación hacia Arriba/efectos de los fármacos , Linfopoyetina del Estroma TímicoRESUMEN
A conjugate of lysozyme with avarone, a bioactive sesquiterpene quinone of marine origin, and its three derivatives were synthesized. MALDI TOF mass spectral analysis and tryptic digestion showed that the only residue in lysozyme that was modified by all derivatives was lysine 97. The identity of the residue was in full correlation with the prediction obtained by molecular modeling. All bioconjugates preserved most of the enzymatic activity of lysozyme. The melting point of the conjugates was slightly increased in comparison to lysozyme, indicating a slight stabilization of structure. The antibacterial activity of all the conjugates to both Gram positive and Gram negative bacteria was stronger than the activity of either lysozyme or the quinones, the MIC values being in low micromolar range for some conjugates.