Antiadhesive character of mucin O-glycans at the apical surface of corneal epithelial cells.

Cell surface–associated mucins, a group of high-molecular-weight glycoproteins lining the apical cell surface on mucosal tissues, constitute a major component of the glycocalyx on epithelial cells.1,2 To date, 10 members of this class of molecules, designated MUC, have been described, all being type I membrane proteins with single transmembrane domains.3 The human ocular surface epithelia express at least three membrane-associated mucins, MUC1, -4, and -16.4 Structurally, they are defined by the presence of central tandem repeats of amino acids rich in serine, threonine, and proline residues, and by their extensive O-glycosylation. The amount of O-glycans varies depending on the tissue in which they are expressed (e.g., representing up to 50% of the mucin mass in the mammary gland and up to 80% in the pancreas).5 Alteration in mucin-type O-glycosylation has been demonstrated in patients with drying diseases such as dry eye6,7 and dry mouth.8,9 In these diseases—frequently chronic and characterized by adhesiveness of the apical surface epithelia—lubricants are usually applied to reduce epithelial damage to the cell surface by abrasive stress. Because of their large extracellular domain, which extends up to 500 nm above the plasma membrane, cell surface mucins have been shown to confer an antiadhesive character to cell membranes.10,11 Preclusion of adhesion has been shown to depend on the level of mucin expression, and the length and number of tandem repeats that extend into the pericellular space. In this regard, overproduction of cell surface mucin by transfecting HBL-100 human mammary epithelial cells and A375 human melanoma cells with full-length complementary DNA encoding MUC1 has been shown to strongly reduce cellular aggregation.12 Similarly, transfection of A375 human melanoma cells or murine L929 cells with a series of recombinant cDNA containing increasing lengths of cell surface-associated mucins shows a decreasing number of cell–cell interactions.13,14 Because of the extensive glycosylation of mucins, it has been proposed that O-glycans may also contribute to the antiadhesive character of mucins by either preserving the extended, rigid structure of the mucin molecule or by charge repulsion, due to the abundance of negatively charged sialic acids.11 Removal of sialic acid by neuraminidase, however, has yielded controversial results in aggregation assays, showing that sialic acid residues are either not required for the antiadhesive effect of the mucin or partially contribute to modulating cell adhesion.12,13 To test whether mucin O-glycans and negative charges prevent apical cell–cell interactions, we have disrupted the biosynthesis of cell surface O-glycans in vitro by metabolic interference using benzyl-α-GalNAc.15,16 This chemical primer competes with endogenous substrates for elaboration of the core GalNAc residue; therefore, cells treated with this compound express truncated mucin-type O-linked glycans.17 Using static and dynamic flow assays, we showed that cell surface O-glycans contribute to the prevention of adhesion on the apical surface of normal, differentiated corneal epithelial cells and that this effect is not mediated by negative charges.