A relatively thick apical glycocalyx or fuzzy coat covers the lumenal surface of enterocytes. Both intestinal mucin-type sialoglycoproteins (IMTGP) and galectins are found associated with the apical glycocalyx indicating that they are strategically located to perform a protective function for the enterocyte. The long-term goal of our research is to determine the biological role that the IMTGPs and galectins play in the protection of enterocytes from harmful interactions with digestive enzymes, microorganisms and toxins. In relation to this goal, we propose the following hypotheses: 1)IMTGPs are major structural components of the protective apical glycocalyx (fuzzy coat) found on the lumenal surface of enterocytes; and 2)Galectins interact with these IMTGPs in order to form a network of crosslinked macromolecules that limits the access of microorganisms, microbial toxins, and digestive enzymes to the proteins and lipids on the lumenal surface of enterocytes. To address these hypotheses, we propose studies with the following specific aims: 1)To determine if the IMTGPs are a major structural component of the apical glycocalyx of porcine enterocytes by separating the apical glycocalyx from the enterocytes and quantitating the amount of IMTGPs in each fraction; 2)To determine if intestinal galectins are found in the apical glycocalyx of porcine enterocytes by producing galectin- specific monoclonal antibodies for use in immunofluorescence and enzyme-linked immunosorbent assays; 3)To determine which glycoconjugate ligands (IMTGPs, secreted mucins, pancreatic and brush border digestive enzymes) within the apical glycocalyx that the galectins bind to. Characterize the binding affinity of galectins for the IMTGPs using kinetic binding studies; and 4)To determine the contribution of the IMTGPs and galectins to the protection of an integral membrane protein from pancreatic proteases in a liposome-based system.
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