DEMONSTRATION OF DISCRETE SECRETED AND MEMBRANE-BOUND OCULAR MUCINS IN THE DOG

Our aims were to separate and characterize secreted canine ocular muci ns, and to provide definitive evidence of membrane-bound mucins at the canine ocular surface. Mucus was collected by suction fi om the ocula r surface of normal dogs and dispersed in guanidine hydrochloride and a cocktail of protease inhibitors. Caesium chloride density gradient c entrifugation separated secreted mucins fi-om membranes, which were co llected from the top of the gradients. Membranes were extracted with o ctyl glucoside and screened using lectins and anti-mucin antibodies. G radient fractions containing secreted mucins were constituted into poo ls on the basis of differential lectin and antibody staining. High mol ecular weight material from each pool was purified by gel filtration. This material, and the membrane extract, were reduced and alkylated. V acuum blotting of separated materials after agarose gel electrophoresi s was used to compare subunit structure. Density gradient profiles ind icated three principal secreted glycoprotein peaks: one staining stron gly with anti-mucin antibodies. Gel filtration demonstrated that each contained high molecular weight material. Vacuum blots demonstrated th e presence of two secreted glycoproteins with differently sized subuni ts. On the basis of buoyant density, one of these may be lipid complex ed. Membrane extracted material stained with anti-mucin antibodies, an d vacuum blotting of this material provided evidence for two membrane- bound components. Ln conclusion, we have shown that normal canine ocul ar mucus contains two secreted mucins, each exhibiting different subun it structure; one of these mucins may undergo lipid complexation. Norm al canine ocular mucus also contains two membrane-bound mucins: one of which is unique among membrane mucins in showing subunit structure. ( C) 1997 Academic Press Limited.