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Takazumi Taniguchi, Ashley M Woodward, Paula Magnelli, Pablo Argueso; Structure and Biosynthesis of Transmembrane Mucin N-glycans in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5652.
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© ARVO (1962-2015); The Authors (2016-present)
Transmembrane mucins are critical to the maintenance of the ocular surface epithelial barrier through their interaction with the carbohydrate-binding protein galectin-3. Although mucin N-glycans have been implicated in crucial recognition events, little is known about their overall structure or role at the ocular surface. The goal of this study was to determine the structure of transmembrane mucin N-glycans, and to evaluate the gene expression of a series of enzymes responsible for the maturation of N-glycans in immortalized human corneal epithelial (HCLE) cells.
HCLE cell cultures were grown in conditions that promote differentiation and stratification as described previously. HCLE cell-associated transmembrane mucins were isolated using size-exclusion chromatography followed by isopycnic density gradient centrifugation in cesium chloride. The presence of mucins MUC1, MUC4, MUC16 and MUC20 was determined using Western blot during the purification process. Purified mucins were digested with PNGase-F, and the N-glycan structures were then analyzed using liquid chromatography-mass spectrometry (LC-MS). Gene expression was measured by quantitative PCR.
MUC1, MUC4 and MUC16 were eluted within the void volume following size-exclusion chromatography, whereas the small-molecular-weight MUC20 was not. Following density gradient centrifugation, LC-MS showed the presence of high-mannose and complex-type N-glycans in the high-molecular-weight mucin fraction. Lactosamine sequences, a preferential binding partner for galectin-3, were observed in complex-type N-glycans. Glycosidases (MAN2A1 and MAN2A2) and glycosyltransferases (MGAT1, MGAT2, MGAT4A, MGAT4B and MGAT5), which are essential for the generation of complex-type N-glycans with lactosamine residues, were expressed in HCLE cells, with MAN2A1 being the most highly expressed.
High-molecular-weight transmembrane mucins contain complex-type N-glycans with lactosamine residues in human corneal epithelial cells. These data suggest that N-glycans from transmembrane mucins may associate with galectin-3 at the ocular surface.
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