Synthesis of Rabbit Corneal Epithelial Glycocalyx in vitro

Abstract

A method to study the synthesis and cellular processing of epithelial apical membrane glycoproteins in the rabbit cornea was developed. Fluorescent derivatives of wheat germ agglutinin (WGA; α-N-acetylglucosamine and sialic acid hapten affinities), succinylated WGA (α-N-acetylglucosamine hapten affinity) and concavalin A (Con A; d-mannose and d-glucose hapten affinity) were reacted with the corneal surface and the extent of binding attained was measured by en face, microscope-aided fluorophotometry. Minimal binding of succinylated WGA and a large reduction in WGA binding following neuraminidase treatment demonstrated that the attachment of WGA to the corneal surface occurred via sialic acid residues, i.e. via structures associated with terminal glycosylation. Corneas were treated with digitonin to induce the exfoliation of the outer squamous-like cell layers. The time-dependent changes in lectin binding density at the apical surface of the newly exposed intrastratal cells were then determined. Binding densities for WGA and Con A at the time of exfoliation of the digitonin-devitalized squamous cell layers (< 2 hr post-devitalization) were similar to the densities measured at the surface of untreated corneas. Over the subsequent 18-20 hr, the WGA and Con A binding increased by 2·63 ± 0·24 and 3·0 ± 0·68 (± S.D., n = 4) fold, respectively. The effect of inhibitors of transcription (actinomycin D, α-amanitin), translation (cycloheximide), core glycosylation of polypeptides (tunicamycin), endoplasmic reticulum glucosidases (deoxinojirimycin) and Golgi mannosidase (swainsonine) indicated that the increases were underpinned by new glycoprotein synthesis driven by a stable, pre-existing mRNA pool. Retinoic acid (2 μM) inhibited the increase in WGA binding by 55 ± 6% (n = 4) but did not affect the Con A density increase suggesting that this agent either, modifies the terminal glycosylation pattern of apical membrane proteins and/or inhibits the synthesis of proteins bearing sialic acid. Actinomycin D or α-aminitin reverted the retinoic acid action, indicating that the retinoid effect is mediated by induced gene expression.