Studies of the role(s) of galectin in quail intestinal mucin organization and secretion
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AbstractA program of studies was undertaken to explore the role(s) played by galectins at the mucosal surface. The first phase of this project involved purification and characterization of intestinal galectins from rabbit, rat, and quail. A family of galectins were found in mammalian intestinal tissues, however, only a single galectin was identified from the quail. In the second stage of this project, quail intestinal mucin was purified and the biochemical and immunological properties were characterized. Quail mucin was purified by two sequential isopycnic density-gradient centrifugations in CsCl followed by gel filtration chromatography. Purified quail mucin was then characterized by SDS-PAGE, amino acid and carbohydrate composition analyses, immuno-cross reactivity with mammalian mucin antibodies and sensitivity to disulphide-reduction, H2O2 , and trypsin. Quail mucin was revealed to be high molecular weight polymer composed of large heterogeneous glycoprotein monomers and a 110 kD 'link' protein, held together by disulphide bonds. Quail mucin was further identified as a putative ligand for quail galectin in binding studies and through immunohistochemical co-localization in goblet cells. The quail galectin binds quail mucin through the B-galactose residues on the carbohydrate side chains of quail mucin as shown by hapten inhibition studies. A putative membrane receptor of quail galectin on the brush border membrane of the quail small intestine was also detected by radioactive ligand binding assays. The regulation of mucin secretion from the quail small intestine was investigated using in vitro organ culture. Mucin secretion was enhanced by cholera toxin and regulated in a similar fashion to mammalian mucin; being stimulated by cholinergic but not adrenergic stimuli and being sensitive to the activation of protein kinase C and changes in Ca2 + levels. Quail galectin seems to serve as an adhesion molecule for extracellular mucus gel adherence to the epithelial surfaces and maybe also serve as a control for mucin release, which is evidenced by the following discoveries: (1) lactose could release both quail galectin and quail mucin from intestinal tissue; (2) blocking of galectin binding to the mucosal surfaces with antiserum against quail galectin could induce quail mucin release; and (3) binding the mucosal receptors for quail galectin with plant lectins could specifically reduce the rate of baseline secretion of quail mucin. However, lactose-induced mucin release was independent of the muscarinic receptor, protein kinase C and Ca2 +, suggesting that quail galectin may control mucin release through a unique pathway.
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