title: Epithelial sorting of a glycosylphosphatidylinositol-anchored bacterial protein expressed in polarized renal MDCK and intestinal Caco-2 cells.
abstract: To evaluate whether a glycosylphosphatidylinositol (GPI) anchor can function as a protein sorting signal in polarized intestinal epithelial cells, the GPI-attachment sequence from Thy-1 was fused to bacterial endoglucanase E' (EGE') from Clostridium thermocellum and polarity of secretion of the chimeric EGE'-GPI protein was evaluated. The chimeric EGE'-GPI protein was shown to be associated with a GPI anchor by TX-114 phase-partitioning and susceptibility to phosphoinositol-specific phospholipase C. In polarized MDCK cells, EGE' was localized almost exclusively to the apical cell surface, while in polarized intestinal Caco-2 cells, although 80% of the extracellular form of the enzyme was routed through the apical membrane over a 24 hour period, EGE' was also detected at the basolateral membrane. Rates of delivery of EGE'-GPI to the two membrane domains in Caco-2 cells, as determined with a biotinylation protocol, revealed apical delivery was approximately 2.5 times that of basolateral. EGE' delivered to the basolateral cell surface was transcytosed to the apical surface. These data indicate that a GPI anchor does represent a dominant apical sorting signal in intestinal epithelial cells. However, the mis-sorting of a proportion of EGE'GPI to the basolateral surface of Caco-2 cells provides an explanation for additional sorting signals in the ectodomain of some endogenous GPI-anchored proteins.
authors.affiliation: Department of Physiological Sciences, University of Newcastle upon Tyne, Medical School, UK.
authors.forename: K L
authors.initials: KL
authors.lastname: Soole
authors.forename: M A
authors.initials: MA
authors.lastname: Jepson
authors.forename: G P
authors.initials: GP
authors.lastname: Hazlewood
authors.forename: H J
authors.initials: HJ
authors.lastname: Gilbert
authors.forename: B H
authors.initials: BH
authors.lastname: Hirst
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002480:Cellulase / Q000032:analysis / Q000096:biosynthesis*; D003013:Clostridium / Q000201:enzymology*; D003110:Colonic Neoplasms; D004285:Dogs; D004848:Epithelium / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D007668:Kidney; D007700:Kinetics; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D018800:Thy-1 Antigens / Q000096:biosynthesis; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D017261:Glycosylphosphatidylinositols; D011994:Recombinant Proteins; D018800:Thy-1 Antigens; D002480:Cellulase