N-Glycosylation affects the stability and barrier function of the MUC16 mucin

Abstract

Transmembrane mucins are highly <i>O</i>-glycosylated glycoproteins that coat the apical glycocalyx on mucosal surfaces and represent the first line of cellular defense against infection and injury. Relatively low levels of <i>N</i>-glycans are found on transmembrane mucins, and their structure and function remain poorly characterized. We previously reported that carbohydrate-dependent interactions of transmembrane mucins with galectin-3 contribute to maintenance of the epithelial barrier at the ocular surface. Now, using MALDI-TOF mass spectrometry, we report that transmembrane mucin <i>N</i>-glycans in differentiated human corneal epithelial cells contain primarily complex-type structures with <i>N</i>-acetyllactosamine, a preferred galectin ligand. In <i>N</i>-glycosylation inhibition experiments, we find that treatment with tunicamycin and siRNA-mediated knockdown of the Golgi <i>N</i>-acetylglucosaminyltransferase I gene (<i>MGAT1</i>) induce partial loss of both total and cell-surface levels of the largest mucin, MUC16, and a concomitant reduction in glycocalyx barrier function. Moreover, we identified a distinct role for <i>N</i>-glycans in promoting MUC16's binding affinity toward galectin-3 and in causing retention of the lectin on the epithelial cell surface. Taken together, these studies define a role for <i>N</i>-linked oligosaccharides in supporting the stability and function of transmembrane mucins on mucosal surfaces.