α-2,3 sialylation differentiate the limbal and corneal epithelial cell phenotypes

Purpose. The initial differentiation event for the corneal epithelial cell lineage occurs as the limbally localized stem cells yield, through mitosis, the highly proliferative, transiently amplifying corneal peripheral cells. This differentiation is characterized by the expression of tissue-specific cytokeratins, as well as the loss of α-enolase and pigmentation. All these are intracellular events. The aim of this study was to identify and characterize, through lectin analysis, changes in cell surface properties associated with differentiation. Methods. Cryostat sections of the limbo- corneal area from freshly dissected pigmented rabbit corneas were stained with fluorescent lectins. Results. Peanut lectin (PNA; binds to Ser/Threo- GalNAc-β-1,3-Gal, if the Gal residue is not sialylated) stained the plasma membrane of all layers of the conjunctiva and limbus but was excluded from corneal cell membranes. Maakia amurensis agglutinin (MAA; binds to sialic acid attached to galactose through α-2,3 bonds in either N-glycans or O- glycans) stained exclusively corneal cell plasma membrane. After complete tissue desialylation, all corneal plasma membranes became PNA positive with equal stain intensity across both sides of the limbo-corneal margin. The binding of the agglutinins from Limax flavus (binds unselectively to sialic acid) and Sambucus nigra (binds to sialic acid attached through α-2,6 bonds) to the basement membrane displayed a large increase at the corneal side of limbo-corneal demarcation. Conclusions. Limbal (stem) cells express on the cell surface unsialylated galactose residues that are recognized by PNA and that lack any sialic acid bound through α-2,3 bonds. The initial differentiation involves sialylation of these residues and the concurrent appearance of α-2,3 sialic acid residues, suggesting expression or activation of α-2-3 sialytransferase. Changes in basement membrane composition, charge, or both may underpin this expression.