OCULAR SURFACE PROTECTION BY SIALOMUCIN COMPLEX

University Of Miami School Of Medicine, Coral Gables FL

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Abstract

The overall goal of this research is to understand the roles of a novel membrane/soluble glycoprotein complex (sialomucin complex, SMC) at the ocular surface and in the ocular tear film. SMC was originally identified and isolated from membranes of ascites sublines of the highly metastatic 13762 rat mammary adenocarcinoma and is composed of a mucin subunit ASGP-1 (ascites sialoglycoprotein-1) linked to the plasma membrane via an N glycosylated transmembrane subunit ASGP-2. The complex is encoded by a single gene, expressed as a 9.2 kb transcript and synthesized as a precursor of 300 kDa (pSMC-1). Molecular cloning and sequencing have revealed the complete sequence of the transcript and proteins. The transmembrane subunit has two EGF-like domains and can act selectively as a ligand for the receptor tyrosine kinase ErbB2. These results, plus the anti- adhesive activity of the mucin subunit ASGP-1, suggest that the complex is not only heterodimeric, but also bifunctional. In the rat SMC 2is found at epithelial apical cell surfaces in the airway, uterus, brain and oral cavity, in secretory granules in goblet cells in the colon, and in lactating mammary gland. Furthermore, SMC is found in some of these tissues in both soluble and membrane forms, suggesting dual protective roles as a mucin. SMC is a constituent of both milk and saliva. Western blotting, immunofluorescence and Northern analyses have demonstrated the presence of SMC at the ocular surface of both rats and humans. It is particularly abundant in the corneal epithelium compared to the conjunctival epithelium and other rat epithelia in which it is expressed. SMC also appears to be an important component of human tear fluid. Interestingly, immunolocalization using a carbohydrate-dependent antibody suggests that its synthesis is differentiation dependent in the stratified epithelia. We have proposed that SMC serves a protective function at the ocular surface. These studies are intended to test that hypothesis by investigating the protection mechanisms in which SMC may be involved. For these investigations we propose first to complete the characterization of SMC expression, forms and localization at the ocular surface in the rat and human to determine the localization of SMC in the tear film and whether SMC functions as a membrane barrier molecule and/or as an ErbB2 ligand in addition to its role as a soluble mucin. Second, we will analyze the properties of SMC in human tears in comparison to other tear film mucins to evaluate its role as a tear film mucin. Third, we will use corneal explants and epithelial cell cultures to examine the mechanisms involved in the synthesis and secretion of SMC and to determine factors which regulate SMC expression at the ocular surface. Finally, we will examine the ability of topical antisense oligonucleotide and antibody treatments to alter the role(s) of SMC at the rat ocular surface as tests of its function(s). We expect that the results of these investigations will bring unique insights into the role of this novel molecule in ocular surface protection and may lead to new opportunities for understanding and treating diseases of the ocular surface.