Biochemical analysis of ocular surface mucin abnormalities in dry eye: the canine model

This study examines the canine model of keratoconjunctivitis sicca (KCS, 'dry eye') in order to establish the biochemical basis of altered ocular mucin secretion in this condition. It follows a previous examination of ocular mucins in the normal dog. Mucus was collected by suction from the ocular surface of dogs with KCS, and dispersed in guanidine hydrochloride containing a cocktail of protease inhibitors. Caesium chloride density gradient centrifugation was used to separate floating 'rafts' of cell membranes from gradients containing secreted mucins. Gradient fractions were collected into pools on the basis of differential staining by Periodic Acid Schiff, Wheat Germ Agglutinin, and antibodies to MUC5AC peptide. High molecular weight glycoproteins were purified from the pooled material by gel filtration chromatography. Membrane-associated glycoproteins were also derived from the membrane rafts using octyl glucoside extraction and/or reduction and alkylation. Secreted mucins and membrane extracts from KCS samples were compared to equivalent material obtained from normal eyes. Density gradient staining profiles for normal and KCS mucus were similar over the buoyant density range typical for secreted mucins, enabling the collection of identical pools of gradient fractions for direct comparison. The following differences were observed in KCS secreted mucins compared to normal samples: an increase in the proportion of mucin with low buoyant density; a decrease in mannose content detected with Concanavalin A lectin; an increase in N-acetylglucosamine structures detected with Lycopersicon esculentum lectin; increased migration and lack of evidence for distinct subunit structure on agarose gels. In membrane extracts, the main difference was the presence of T antigen (Gal beta 1-3GalNAc) in KCS. These results demonstrate alterations in the subunit linkage of mucins in KCS, and suggest that glycosylation, core protein expression and/or post-synthetic modification of ocular surface mucins may also be changed.