Continuous glycoprotein (GP) secretion, primarily mucin, helps to protect the adult colonic epithelium from bacterial adherence and colonization. This role depends on the physicochemical properties of mucin and thus its chemical composition. In the neonate, (GP) synthesis, composition, and secretion may be particularly important during the establishment of the bacterial flora. The goals of our studies are to characterize and localize the secretory products of different mucous cell types in the developing colon and to define the mechanism(s) controlling their release. To define the cell types involved in O-linked (mucin type) biosynthesis, explants of neonatal rat colon will be incubated in the presence of 3H-N-acetylmannosamine (3HManNac). N-linked synthesis will be inhibited by Tunicamycin. Labelled sialo- glycoproteins (SG) will be localized by light and electron microscopic autoradiography. The presence and specific position of O-acetyl substitutions on the sialic acid residues will then be determined histochemically. Secretory cells will also be defined by 3H-fucose incorporation and binding of ferritin conjugated UEA. The latter is a fucose specific lectin that binds specifically to a population of cells in the neonatal colon corresponding to adult deep crypt secretory cells. To characterize secreted GP, media from explants cultured in the presence of HManNAc will be applied to Sepharose 4B columns and the radioactive peaks determined. The peaks will be pooled and the oligosaccharide side chains cleaved by sodium borohydride elimination for O-linked sugars and by N-glycanase for N-linked chains. The released side chains will be evaluated on P-10 or P-30 columns. Relative carbohydrate content of intact GP will be assessed by buoyant densities on cesium chloride gradients. The molecular weights and number of GP in tissue and media samples cultured in the presence of 3H-Man-NAc will be evaluated by SDS-PAGE gels. Fluorography will show the location of labelled SG, while selected transfer gels probed with lectins with and without prior enzymatic digestion will provide additional data on carbohydrate composition. Finally, the intracellular distribution of cytoskeleton elements will be studied by immunofluorescence and the role of microtubules in GP secretion assessed in explants cultured in the presence of the inhibitor colchicine. These studies will define selected aspects of GP structure, composition, and secretion, important prerequisites to understanding the function of these GP in the developing colon.
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