OCULAR SURFACE MUCIN ppGaNTase GLYCOSYLTRANFERASES

University Of Louisville, Louisville KY

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Abstract

DESCRIPTION (provided by applicant): The mucus layer, secreted primarily by the conjunctival goblet cells but also by the corneal and conjunctival epithelia, is a critical protective layer for the ocular surface; shielding it from pathogenic and environmental challenges. Mucins are a heterogeneous group of highly glycosylated proteins that are present both on the ocular surface cells and as major soluble components of tear fluid. The first and rate-limiting step of O-glycosylation of mucins requires the action of at least one isoform of the UDPGalNAc: polypeptide N-Acetylgalactosaminyltransferase (ppGaNTase) family of glycosyltransferases. Without glycosylation, mucins would lack the viscoelastic properties required for proper function. The hypothesis of this project is that one cause of dry eye disease is abnormal mucin glycosylation due to altered expression of ppGaNTase isoforms. In the first aim we will compare the levels of mRNA expression of ppGaNTase isoforms in normal and dry eye patients by real-time PCR using the Taqman technique. The results will provide the first complete analysis of the expression of this critical glycosyltransferase family in human ocular tissues and point to one possible cause of human dry eye disease. In the second aim we will determine which ppGaNTase isoforms are responsible for glycosylation of specific mucin types using RNA interference and inhibitors of ppGaNTase action. First, we will determine which ppGaNTase isoforms are expressed at the mRNA level in human corneal and conjuncrival cell lines by real-time PCR. Second, we will use gene silencing by small interfering RNAs (siRNA) to specifically silence each ppGaNTase isoform and then determine by Western blotting whether silencing of one ppGaNTase isoform results in altered glycosylation of each mucin type. As a positive control for ablation of mucin glycosylation, we will treat cells with inhibitors of the ppGaNTase family. The results of RNA interference will enable us to obtain the first evidence in whole ocular cells as to which ppGaNTase isoforms are required for the glycosylation of particular mucin types.