Abstract
PURPOSE: O-linked carbohydrates (O-glycans) contribute to the hydrophilic character of mucins in mucosal tissues. This study was conducted to identify the repertoire of O-glycans in the tear film and the glycosyltransferases associated with their biosynthesis, in normal subjects and patients with non-Sjögren's dry eye. METHODS: Human tear fluid was collected from the inferior conjunctival fornix. O-glycans were released by hydrazinolysis, labeled with 2-aminobenzamide, and analyzed by fluorometric, high-performance liquid chromatography (HPLC) coupled with exoglycosidase digestions. O-glycan structures identified in tears were related to potential biosynthetic pathways in human conjunctival epithelium by using a glycogene microarray database. Lectin-binding analyses were performed with agglutinins from Arachis hypogaea, Maackia amurensis, and Sambucus nigra. RESULTS: The O-glycan profile of human tears consisted primarily of core 1 (Gal beta 1-3GalNAc alpha 1-Ser/Thr)-based structures. Mono-sialyl O-glycans represented approximately 66% of the glycan pool, with alpha2-6-sialyl core 1 being the predominant O-glycan structure in human tears (48%). Four families of glycosyltransferases potentially related to the biosynthesis of these structures were identified in human conjunctiva. These included 13 polypeptide-GalNAc-transferases (GALNT), the core 1 beta-3-galactosyltransferase (T-synthase), three alpha2-6-sialyltransferases (ST6GalNAc), and two alpha2-3-sialyltransferases (ST3Gal). No significant differences in total amount of O-glycans were detected between tears of normal subjects and patients with dry eye, by HPLC and lectin blot. Likewise, no differences in glycosyltransferase expression were found by glycogene microarray. CONCLUSIONS: This study identified the most common mucin-type O-glycans in human tears and their expected biosynthetic pathways in ocular surface epithelia. Patients with non-Sjögren's dry eye showed no alterations in composition and amount of O-glycans in the tear fluid.