Open AccessKiwifruit protease Act d 1 compromises the intestinal barrier by disrupting tight junctions
Author(s) -
Grozdanovic Milica,
Cavic Milena,
Andjelkovic Uros,
Petersen Arnd,
Smit Joost,
GavrovicJankulovic Marija
Publication year - 2014
Publication title -
clinical and translational allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.979
H-Index - 37
ISSN - 2045-7022
DOI - 10.1186/2045-7022-4-s2-o10
Subject(s) - tight junction , occludin , barrier function , in vivo , dextran , caco 2 , intestinal permeability , protease , in vitro , microbiology and biotechnology , medicine , biophysics , chemistry , biochemistry , biology , immunology , enzyme
Method In vitro analysis of Act d 1 digestion stability in simulated conditions of the gastrointestinal tract was performed by means of SDS-PAGE, zymography, ESI-TOF and immunoelectrophoresis. In addition, the influence of Act d 1 on tight junctions of Caco-2 cells was assessed by immunofluorescence and by measuring changes in transepithelial resistance and FITC-dextran leakage across cell monolayers. In vivo studies were performed to determine the effect of Act d 1 on intestinal permeability in mice. Results Act d 1 isolated from kiwifruit under native conditions retained its primary structure, immunological reactivity and proteolytic activity after 2 h of simulated gastric digestion, followed by 2 h of simulated intestinal digestion. Exposure of confluent Caco-2 cells to Act d 1 reduced the transepithelial resistance of cell monolayers by 18.1% after 1h (P < 0.01) and 25.6% after 4 h (P < 0.001). The loss of barrier function was associated with leakage of FITC-dextran across the monolayers. Confocal microscopy revealed that Act d 1 treatment lead to disruption of tight junction proteins occludin and ZO-1. None of these effects were observed with heat inactivated Act d 1. In vivo measurements of intestinal permeability in mice showed that following administration of 40 kDa FITC-dextran by oral gavage, significantly higher concentrations of FITCdextran (2.33 µg/mL) were later detected in the sera of Act d 1 treated mice in comparison to the control group (0.5 µg/mL, P < 0.05). Conclusion