The primary objective is to examine the role of protein glycosylation in directing glycoproteins to specific intra- and extra-cellular sites. Recognition of individual oligosaccharides requires both unique oligosaccharide structures and specific receptors. Alterations in either oligosaccharide structure or receptor specificity may lead to pathologic manifestations such as developmental defects, altered regulation of growth, or altered metabolism. Two lectins with markedly different properties form a focus for our studies and are used as prototypes for basic processes involved in recognition and transport. The Gal/GalNAc-specific receptor is a membrane protein which mediates the uptake of Gal bearing glycoproteins from plasma by hepatocytes. Endogenous ligands will be identified and characterized using a blotting technique relying on recognition of ligand by the purified receptor. The structure of the oligosaccharides, their origin and functional significance will be established. The relationship of subunit assembly to ligand binding and regulation of receptor movement during endocytosis will be examined with solubilized receptor and viable cells. Components of the endocytic vesicles mediating uptake will be identified by vectorial labeling with lactoperoxidase conjugates delivered to these vesicles through the receptor. Endocytic vesicles and plasma membrane vesicles will be isolated by high magnetic gradient separation following delivery of 20-40 nm magnetic iron-dextran particles and will be used to characterize the process of uptake by the Gal/GalNAc specific receptor. The Core-specific lectin, synthesized and secreted by hepatocytes, is a soluble lectin found in plasma. Its synthesis, post-translational modification, and assembly will be examined. The basis for the slow kinetics of movement from the Golgi to the medium will be established to determine if this lectin has an intracellular as well as an extracellular function. Endogenous ligands for this lectin will be identified in either Golgi or plasma and characterized. Structural characterization of a number of glycoproteins will be carried out including the endogenous ligands for the Gal/GalNAc-specific receptor and the core specific lectin, a platelet Alpha-granule specific membrane protein expressed at the platelet surface only after stimulation of the release reaction, and endosome specific membrane proteins. The presence of unique structures may reflect their highly restricted location and may have functional significance.
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