Role of the Lewis^x Glycan Determinant in Corneal Epithelial Cell Adhesion and Differentiation

In this study we demonstrate that in corneal epithelium there is cell-cell contact-regulated expression of a 145-kDa glycoprotein (GP) bearing the glycan determinant Lewis^x (Le^x) (Galβ(1,4)[Fucα (1,3)]GlcNAc). This glycoprotein (Le^x-GP) was expressed in confluent/contact-inhibited cultures but not in sparse cultures of corneal epithelium. In contrast, a 135-kDa glycoprotein bearing precursor, unfucosylated, lactosamine-containing glycans (Galβ1-4GlcNAcβ1-R) was expressed in sparse cultures. Immunofluorescence staining and confocal microscopy of confluent cultures revealed that in corneal epithelium, Le ^x antigen is located in high density at sites of cell-cell adhesion. In in vitro cell-cell adhesion assays, anti-Le^x, but not anti-sialyl-Le^x monoclonal antibodies, inhibited the formation of corneal epithelial cell-cell adhesion. Also, when added to confluent cultures, antibodies to Le^x disrupted the monolayer and caused tightly packed polygonal cells to round up. Analysis of the expression of Fut genes that encode α-1,3-fucosyltransferases, the enzymes that generate the Le ^x determinant, revealed that confluent/contact-inhibited cultures of rabbit corneal epithelium contain markedly elevated levels of Fut4 and Fut3/5/6 gene transcripts compared with sparse cultures. These data suggest that the Fut4 and Fut3/5/6 genes are targets of cell-cell contact-regulated signals and that Fut gene products direct cell-cell contact-associated expression of Le ^x on the Le^x-GP in corneal epithelium. Immunohistochemical analysis revealed that the expression of Le^x antigen in the epithelium of adult and developing corneas is related to the stage of differentiation of the cells. Although early differentiated cells robustly expressed Le^x, relatively undifferentiated cells did not, and the expression level was relatively low in terminally differentiated cells. Overall, these data provide evidence that a Le^x-bearing glycoprotein plays a role through the Le^x determinant in corneal epithelial cell-cell adhesion, and these data suggest that Le^x-mediated cell-cell interactions contribute to mechanisms that mediate corneal epithelial cell differentiation.