Targeted disruption of core 1 β1,3-galactosyltransferase (C1galt1) induces apical endocytic trafficking in human corneal keratinocytes.

PloS One
Ana Guzman-AranguezPablo Argüeso

Abstract

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. 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 ...Continue Reading

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Citations

Aug 8, 2014·Journal of Ocular Pharmacology and Therapeutics : the Official Journal of the Association for Ocular Pharmacology and Therapeutics·Ana Guzman-AranguezJesús Pintor
Dec 3, 2014·Progress in Retinal and Eye Research·A J BronF V Bright
Jan 18, 2013·Japanese Journal of Ophthalmology·Pablo Argüeso
Feb 1, 2013·Contact Lens & Anterior Eye : the Journal of the British Contact Lens Association·Joseph P ShovlinFiona Stapleton
Mar 11, 2018·Biochemical Society Transactions·Maria C Rodriguez Benavente, Pablo Argüeso
Feb 3, 2018·Journal of Inherited Metabolic Disease·R FranciscoVanessa Dos Reis Ferreira
Feb 12, 2019·Journal of Inherited Metabolic Disease·Rita FranciscoVanessa Dos Reis Ferreira
Oct 12, 2018·PloS One·Andrew WebsterM Elizabeth Fini

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Methods Mentioned

BETA
transmission electron microscopy
electrophoresis
confocal microscopy
Protein Assay

Software Mentioned

GraphPad
JACoP
ImageJ
InStat3

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