title: Kiwifruit cysteine protease actinidin compromises the intestinal barrier by disrupting tight junctions.
abstract: BACKGROUND
  The intestinal epithelium forms a barrier that food allergens must cross in order to induce sensitization. The aim of this study was to evaluate the impact of the plant-derived food cysteine protease--actinidin (Act d1) on the integrity of intestinal epithelium tight junctions (TJs).
  
  
  METHODS
  Effects of Act d1 on the intestinal epithelium were evaluated in Caco-2 monolayers and in a mouse model by measuring transepithelial resistance and in vivo permeability. Integrity of the tight junctions was analyzed by confocal microscopy. Proteolysis of TJ protein occludin was evaluated by mass spectrometry.
  
  
  RESULTS
  Actinidin (1 mg/mL) reduced the transepithelial resistance of the cell monolayer by 18.1% (after 1 h) and 25.6% (after 4 h). This loss of barrier function was associated with Act d 1 disruption of the occludin and zonula occludens (ZO)-1 network. The effect on intestinal permeability in vivo was demonstrated by the significantly higher concentration of 40 kDa FITC-dextran (2.33 μg/mL) that passed from the intestine into the serum of Act d1 treated mice in comparison to the control group (0.5 μg/mL). Human occludin was fragmented, and putative Act d1 cleavage sites were identified in extracellular loops of human occludin.
  
  
  CONCLUSIONS
  Act d1 caused protease-dependent disruption of tight junctions in confluent Caco-2 cells and increased intestinal permeability in mice.
  
  
  CONCLUSIONS
  In line with the observed effects of food cysteine proteases in occupational allergy, these results suggest that disruption of tight junctions by food cysteine proteases may contribute to the process of sensitization in food allergy.
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia; Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, 900 S. Ashland Ave., MBRB 1354 (M/C 669), Chicago, IL 60607, USA.
authors.forename: Milica M
authors.initials: MM
authors.lastname: Grozdanovic
authors.affiliation: Department of Experimental Oncology, Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia.
authors.forename: Milena
authors.initials: M
authors.lastname: Čavić
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia.
authors.forename: Andrijana
authors.initials: A
authors.lastname: Nešić
authors.affiliation: Department of Biotechnology, University of Rijeka, Radmile Matejcic 2, 51000 Rijeka, Croatia; Department of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Studentskitrg 12-16, 11000 Belgrade, Serbia.
authors.forename: Uroš
authors.initials: U
authors.lastname: Andjelković
authors.affiliation: Institute for Risk Assessment Sciences, Utrecht University, Yalelaan 104, 3584 CM Utrecht, The Netherlands.
authors.forename: Peyman
authors.initials: P
authors.lastname: Akbari
authors.affiliation: Institute for Risk Assessment Sciences, Utrecht University, Yalelaan 104, 3584 CM Utrecht, The Netherlands.
authors.forename: Joost J
authors.initials: JJ
authors.lastname: Smit
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia. Electronic address: mgavrov@chem.bg.ac.rs.
authors.forename: Marija
authors.initials: M
authors.lastname: Gavrović-Jankulović
mesh_terms: D029042:Actinidia / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D018938:Caco-2 Cells; D003546:Cysteine Endopeptidases / Q000494:pharmacology*; D005512:Food Hypersensitivity / Q000209:etiology; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000187:drug effects*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D062793:Occludin / Q000378:metabolism; D010539:Permeability; D019108:Tight Junctions / Q000187:drug effects*
chemical_list: C568985:OCLN protein, human; D062793:Occludin; D003546:Cysteine Endopeptidases; C018745:actinidain
keywords: Actinidin; Cysteine protease; Intestinal permeability; Occludin; Tight junctions