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

This study examines the canine model of keratoconjunctivitis sicca (KCS,'dr y eye') in order to establish the biochemical basis of altered ocular mucin secretion in this condition. It follows a previous examination of ocular m ucins in the normal dog. Mucus was collected by suction from the ocular sur face of dogs with KCS, and dispersed in guanidine hydrochloride containing a cocktail of protease inhibitors. Caesium chloride density gradient centri fugation was used to separate floating 'rafts' of cell membranes from gradi ents containing secreted mucins. Gradient fractions were collected into poo ls on the basis of differential staining by Periodic Acid Schiff, Wheat Ger m Agglutinin, and antibodies to MUC5AC peptide. High molecular weight glyco proteins were purified from the pooled material by gel filtration chromatog raphy. Membrane-associated glycoproteins were also derived from the membran e rafts using octyl glucoside extraction and/or reduction and alkylation. S ecreted mucins and membrane extracts from KCS samples were compared to equi valent material obtained from normal eyes, Density gradient staining profil es for normal and KCS mucus were similar over the buoyant density range typ ical for secreted mucins, enabling the collection of identical pools of gra dient fractions for direct comparison. The following differences were obser ved in KCS secreted mucins compared to normal samples: an increase in the p roportion of mucin with low buoyant density; a decrease in mannose content detected with Concanavalin A lectin; an incease in N-acetylglucosamine stru ctures detected with Lycopersicon esculentum lectin: increased migration an d lack of evidence for distinct subunit structure on agarose gels. In membr ane extracts. the main difference was the presence of T antigen (Gal beta 1 -3GalNAc) in KCS. These results demonstrate alterations in the subunit link age of mucins in KCS, and suggest that glycosylation, cope protein expressi on and/or post-synthetic modification of ocular surface mucins may also be changed. (C) 1998 Academic Press.