title: Targeted disruption of core 1 β1,3-galactosyltransferase (C1galt1) induces apical endocytic trafficking in human corneal keratinocytes.
abstract: BACKGROUND
  Exposed mucosal surfaces limit constitutive endocytosis under physiological conditions to prevent uptake of macromolecules and pathogens and, therefore, cellular damage. It is now accepted that cell surface mucins, a group of high molecular weight glycoproteins on the epithelial glycocalyx, defined by their extensive O-glycosylation, play a major role in maintaining barrier function in these surfaces, but the precise mechanisms are unclear.
  
  
  RESULTS
  In this work, we utilized a stable tetracycline-inducible RNA interfering system targeting the core 1 ß1,3-galactosyltransferase (C1galt1 or T-synthase), a critical galactosyltransferase required for the synthesis of core 1 O-glycans, to explore the role of mucin-type carbohydrates in apical endocytic trafficking in human corneal keratinocytes. Using cell surface biotinylation and subcellular fractionation, we found increased accumulation of plasma membrane protein in endosomes after C1galt1 depletion. Confocal laser scanning microscopy and fluorometry revealed increased translocation of negatively charged fluorescent nanospheres after C1galt1 knockdown sustained by an active transport process and largely independent of apical intercellular junctions. Internalization of nanospheres could be blocked by dynasore, nocodazole, chlorpromazine, and hyperosmotic sucrose, suggesting a mechanism for clathrin-coated pit budding and vesicular trafficking. This possibility was supported by experiments showing nanosphere colocalization with clathrin heavy chain in the cytoplasm.
  
  
  CONCLUSIONS
  Together, the data suggest that core 1 O-glycans contribute to maintenance of apical barrier function on exposed mucosal surfaces by preventing clathrin-mediated endocytosis.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Escuela Universitaria de Óptica, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Guzman-Aranguez
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Woodward
authors.forename: Jesús
authors.initials: J
authors.lastname: Pintor
authors.forename: Pablo
authors.initials: P
authors.lastname: Argüeso
mesh_terms: D002966:Clathrin / Q000378:metabolism; D003315:Cornea / Q000166:cytology*; D004705:Endocytosis / Q000235:genetics*; D005700:Galactosyltransferases / Q000172:deficiency* / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D053758:Nanoparticles; D021381:Protein Transport / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D002966:Clathrin; D034741:RNA, Small Interfering; C505293:C1GALT1 protein, human; D005700:Galactosyltransferases