title: Mechanisms of epithelial translocation of the alpha(2)-gliadin-33mer in coeliac sprue.
abstract: OBJECTIVE
  The alpha(2)-gliadin-33mer has been shown to be important in the pathogenesis of coeliac disease. We aimed to study mechanisms of its epithelial translocation and processing in respect to transcytotic and paracellular pathways.
  
  
  METHODS
  Transepithelial passage of a fluorescence-labelled alpha(2)-gliadin-33mer was studied in Caco-2 cells by using reverse-phase high-performance liquid chromatography, mass spectrometry, confocal laser scanning microscopy (LSM) and fluorescence activated cell sorting (FACS). Endocytosis mechanisms were characterised with rab-GFP constructs transiently transfected into Caco-2 cells and in human duodenal biopsy specimens.
  
  
  RESULTS
  The alpha(2)-gliadin-33mer dose-dependently crossed the epithelial barrier in the apical-to-basal direction. Degradation analysis revealed translocation of the 33mer polypeptide in the uncleaved as well as in the degraded form. Transcellular passage was identified by confocal LSM, inhibitor experiments and FACS. Rab5 but not rab4 or rab7 vesicles were shown to be part of the transcytotic pathway. After pre-incubation with interferon-gamma, translocation of the 33mer was increased by 40%. In mucosal biopsies of the duodenum, epithelial 33mer uptake was significantly higher in untreated coeliac disease patients than in healthy controls or coeliac disease patients on a gluten-free diet.
  
  
  CONCLUSIONS
  Epithelial translocation of the alpha(2)-gliadin-33mer occurs by transcytosis after partial degradation through a rab5 endocytosis compartment and is regulated by interferon-gamma. Uptake of the 33mer is higher in untreated coeliac disease than in controls and coeliac disease patients on a gluten-free diet.
authors.affiliation: Klinik für Gastroenterologie, Charité, Campus Benjamin Franklin, 12200 Berlin, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Schumann
authors.forename: J F
authors.initials: JF
authors.lastname: Richter
authors.forename: I
authors.initials: I
authors.lastname: Wedell
authors.forename: V
authors.initials: V
authors.lastname: Moos
authors.forename: M
authors.initials: M
authors.lastname: Zimmermann-Kordmann
authors.forename: T
authors.initials: T
authors.lastname: Schneider
authors.forename: S
authors.initials: S
authors.lastname: Daum
authors.forename: M
authors.initials: M
authors.lastname: Zeitz
authors.forename: M
authors.initials: M
authors.lastname: Fromm
authors.forename: J D
authors.initials: JD
authors.lastname: Schulzke
mesh_terms: D018938:Caco-2 Cells; D002446:Celiac Disease / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004386:Duodenum / Q000378:metabolism; D005903:Gliadin / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007408:Intestinal Absorption; D007413:Intestinal Mucosa / Q000378:metabolism*; D018613:Microscopy, Confocal; D010446:Peptide Fragments / Q000378:metabolism*; D015398:Signal Transduction; D014178:Translocation, Genetic / Q000187:drug effects; D020691:rab GTP-Binding Proteins / Q000502:physiology
chemical_list: D010446:Peptide Fragments; C489632:alpha2-gliadin (56-88); D007371:Interferon-gamma; D005903:Gliadin; D020691:rab GTP-Binding Proteins