Abstract
BackgroundExposed 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.Methodology/Principal FindingsIn 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/SignificanceTogether, the data suggest that core 1 O-glycans contribute to maintenance of apical barrier function on exposed mucosal surfaces by preventing clathrin-mediated endocytosis.
Highlights
Rapid endocytosis and recycling of plasma membrane in mammalian cells contribute to the internalization of important nutrients, effector molecules, and macromolecules or particles that either or non bind to the cell surface [1]
Results from our previous studies indicated that tetracycline-inducible human corneal keratinocytes stably transfected with C1galt1 shRNA are characterized by reduced core 1 and galectin-3 on the cell surface, concomitant with impaired barrier function [19]
A second barrier on mucosal surfaces is composed of membrane-associated mucins on the epithelial glycocalyx that can extend up to 500 nm above the plasma membrane and, provide steric hindrance [12]
Summary
Rapid endocytosis and recycling of plasma membrane in mammalian cells contribute to the internalization of important nutrients, effector molecules (e.g., growth factors, hormones, antibodies), and macromolecules or particles that either or non bind to the cell surface [1]. Endocytic activity in corneal keratinocytes leads to internalization of viruses and bacteria [4,5] and, to infection, a leading cause of blindness worldwide. This resistance to apical internalization impairs the delivery of therapeutic components into the eye, as it is generally accepted that topical administration of macromolecules, such as genes or nanoparticles sized between 1 and 100 nanometers, cannot effectively cross the corneal epithelium barrier [6,7]. The molecular mechanisms limiting apical plasma membrane plasticity in these exposed mucosal surfaces remains largely unexplored. It is 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
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