Analysis of secreted canine ocular mucin subunits by ion exchange chromatography

Purpose. Ocular mucus abnormalities in both human and canine patients with dry eye (keratoconjunctivitis sicca, KCS) may be caused by changes in ocular mucin glycosylation, core protein expression or post-synthetic modifications. We use the canine model to examine differences seen in ocular mucins in normal and disease conditions. In our previous investigations, we have purified secreted ocular mucins from the normal and the KCS canine ocular surface [1,2]. Reduction and alkylation of normal mucins leads to the separation of components exhibiting a range of electrophoretic mobilities, suggesting the presence of different subunit structures. Here we examine these structures further, by anion exchange separation of radiolabelled mucin subunits, as a prelude to a similar study on mucins from dogs with KCS. Methods. Ocular mucus was collected by suction from the ocular surface of 28 beagle dogs (26 female, 2 male); dispersed in a cocktail of proteinase inhibitors; and three secreted mucin fractions were derived by CsCl density gradient centrifugation and and further purified by gel filtration [1], Samples were reduced in the presence of I4C iodoacetamide and alkylated. Labelled subunits were separated by anion exchange chromatography on Q Sepharose HP. Eluted peaks were examined by agarose gel electrophoresis. Results and Conclusions. Discrete patterns of mucin subunits were identified on agarose gel electrophoresis after anion exchange chromatography. Fractionation was dependent on charge and molecular size. Supported by The Wellcome Trust Project Grant 037530/Z/93/Z/1.5/WRE/JL. [1] Hicks SJ, Carrington SD, Kaswan RL, Adam S, Bara J, Corfield AP. (1996) Exp. Eye Res. (in press). [2] Hicks SJ, Carrington SD, Kaswan RL, Stem ME, Corfield AP. (1995) IOVS 36(4); 4613. None.