Polydispersity of normal human conjunctival mucins.

Abstract

PURPOSE: To isolate all constituent mucins from human conjunctival mucus. METHODS: Mucins were extracted from human conjunctiva in guanidine hydrochloride and protease inhibitors. The mucins were isolated by density gradient centrifugation, gel filtration, and ion exchange chromatography. Throughout purification, the mucin profile was monitored by agarose electrophoresis and vacuum blotting. Blots were probed for peptide and carbohydrate epitopes. The latter included IE3 and TKH2 specific for Tn and sialyl-Tn, respectively, considered tumor-related antigens. In vivo impression cytology specimens of normal conjunctival goblet cells also were probed with the same reagents. Oligosaccharides were released from isolated mucins by alkaline beta-elimination and then size fractionated. RESULTS: Human conjunctival mucins consist of at least three size populations; the largest is excluded on Sepharose CL2B. The two largest populations are polydisperse. Their overall electrophoretic pattern is conserved between individuals. Similar charge distributions were detected in different buoyant density ranges from the density gradient centrifugation: a less charged population containing three components and a highly charged population with two components on agarose electrophoresis. Cross-reaction with IE3 and TKH2 was detected throughout purification in the largest mucins, which were presumably mature, and in impression cytology. Oligosaccharides from mucins in each buoyant density were largely in the monosaccharide and disaccharide range, consistent with Tn and sialyl-Tn standards. CONCLUSIONS: Secreted human conjunctival mucins are polydisperse, with discrete components appearing consistently in pooled and individual samples. They have a unique oligosaccharide pattern containing Tn and sialyl-Tn. This indicates normal roles in normal human ocular mucins for these antigens, which are disease markers in other tissues.