Abstract
Purpose: :
O-linked carbohydrates are known to confer a hydrophilic character to mucins. Alteration in the distribution of cell surface O-glycans correlates with epithelial damage in dry eye patients. By contrast, the character of secreted O-glycans in the tear film remains poorly characterized. The purpose of this study was to identify the repertoire of mucin-type O-glycans present in human tears, and the glycosyltransferases associated with their biosynthesis, in normal subjects and patients with non-Sjögren’s dry eye.
Methods: :
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 sequential exoglycosidase digestions. O-glycan structures identified in tears were related to potential biosynthetic pathways in human conjunctival epithelium using a glycogene microarray database. Lectin binding analyses of tear fluid samples were performed using agglutinins from Arachis hypogaea, Maackia amurensis and Sambucus nigra.
Results: :
The O-glycosylation profile of the human tear fluid consisted mostly of core 1-based (Galß1-3GalNAc1-Ser/Thr) structures. Mono-sialyl core 1 glycans represented approximately 74% of the glycan pool, being 2-6-sialyl core 1 the predominant O-glycan structure in human tears (54%). Four families of glycosyltranferases potentially related to the biosynthesis of these structures were identified in human conjunctiva. These included thirteen polypeptide GalNAc transferases (GalNAcT), the core 1 ß-3-galactosyltransferase (T-synthase), three 2-6-sialyltransferases (ST6GalNAc) and two 2-3-sialyltransferases (ST3Gal). No significant differences in the total amount of O-glycans were detected in tears of normal subjects and dry eye patients by HPLC and lectin blot. Likewise, no differences in glycosyltransferase expression were found by glycogene microarray analysis.
Conclusions: :
The most common mucin-type O-glycans in human tears and their biosynthetic pathways in the ocular surface epithelia have been identified. As compared to cell surface O-glycans, no changes in secreted O-glycans were detected in patients with dry eye.
Keywords: cornea: surface mucins • cornea: tears/tear film/dry eye • glycoconjugates/glycoproteins