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Evidence is presented suggesting that hepatocytes contain a receptor that binds glycoproteins specifically through fucose in alpha1-->3 linkage to N-acetylglucosamine. Human lactoferrin, which contains this type of linkage, is rapidly cleared from the circulation of mice after intravenous injection, and greater than 90% of the injected material is found in hepatocytes. Binding of lactoferrin is mediated through its carbohydrate groups, since its clearance is prolonged after periodate oxidation or after its oligosaccharide groups are extensively degraded with glycosidases. In addition, glycopeptides from lactoferrin inhibit lactoferrin clearance. That lactoferrin clearance is mediated through binding to its fucosyl groups is suggested for several reasons. First, transferrin and asialotransferrin, whose oligosaccharide groups are essentially structurally identical to those of lactoferrin but devoid of fucose, are not cleared on intravenous injection. Second, when fucose is incorporated into asialotransferrin by alpha1-->3 N-acetylglucosamine fucosyl transferase, the resulting fucosylated derivative is cleared rapidly. Neither mannan nor derivatives of orosomucoid that are cleared by binding to receptors for galactose, N-acetylglucosamine, or mannose, inhibit clearance of lactoferrin although clearance is inhibited by fucoidin. Finally, glycoproteins containing fucose in alpha1 --> 2 linkage to galactose or alpha1 --> 6 linkage to N-acetylglucosamine do not inhibit lactoferrin clerance by the liver. Since clearance of other glycoproteins, such as human lactoperoxidase, also appears to be mediated through binding to the same hepatocyte receptor as lactoferrin, it is concluded that the fucose-specific receptor studied here may fulfill other functions than binding lactoferrin. Preliminary studies with liver homogenates and detergent extracts of liver show binding in vitro.

Using galactosylated bovine serum albumin coupled to colloidal gold (galBSA-CG) as a probe, the receptor-mediated endocytosis pathway has been analyzed qualitatively and quantitatively at the ultrastructural level in cultured rat hepatocytes. The results showed that galBSA-CG was specifically recognized by the asialoglycoprotein receptor of the hepatocytes, thus confirming biochemical findings. The probe was preferentially bound in coated pits of the cell surface. When bound elsewhere on the plasma membrane, it apparently moved towards coated regions. In both cases, it was then internalized via coated vesicles. The galBSA-CG passed through pleiomorphic tubular structures and in endosomes, a pool of smooth-surfaced vesicles of various size (50-350 nm), before transiently accumulating in multivesicular bodies. The latter then fused with lysosomes where the glycoproteinic moiety of the probe was degraded, as judged by the flocculated aspect of the accumulated gold particles. About 10% of the internalized ligand was recycled back to the cell surface via secreting vesicles containing lipoprotein-like particles without having apparently passed through lysosomes, which suggests the existence of a pre-lysosomal sorting mechanism of the endocytosed material. Functional recovery of the morphologically restored biliary polarity of hepatocytes in culture was indicated by the fact that galBSA-CG finally appeared in the reconstituted bile canaliculi.

The hepatic uptake and degradation of human diferric 125I-lactoferrin by the liver of the intact rat were studied. After intravenous injection, the tracer was rapidly cleared by the liver, probably by adsorptive pinocytosis, as inferred from observations with a 3,470-fold dose range. Endocytosed lactoferrin was transferred, with a delay, from a light-density subcellular particle to an organelle that had a density similar to lysosomes. The loss of protein bound 125I from the liver was very slow (half-life 2.7 h), and its rate matched closely that of human asialotransferrin type 3. Lactoferrin was found to be a poor substrate for lysosomal hydrolases in vitro. Fucoidin effected the release of a portion of lactoferrin from the liver back into the plasma. By using this agent, indirect evidence was obtained suggesting that a fraction of lactoferrin is being repeatedly endo- and exocytosed (diacytosed) by the liver over prolonged periods of time. Fucosylation failed to impart lactoferrinlike properties on human asialotransferrin type 1, although the derivatized protein exhibited a less than or equal to 10-fold increase in affinity for the liver relative to the parent molecule.