Uptake, intracellular transport, and degradation of polyethylene glycol-modified asialofetuin in hepatocytes

Polyethylene glycol (PEG) is attached to proteins in order to increase their half-life in the circulation and reduce their immunogenicity in vivo. For many applications involving "targeting" molecules, it is important to know how PEG modification of the molecule affects its interaction with a receptor and the subsequent internalization, intracellular transport, and lysosomal degradation. As a model system, we used asialofetuin, which binds to the galactose receptor of hepatocytes, because removal of sialic acid exposes galactose residues. We modified asialofetuin by attaching various amounts of PEG of molecular weight 1900 or 5000. The preparations were labeled with 125I so that endocytosis and degradation could be followed in suspended hepatocytes. Depending on the number of PEG molecules attached, receptor-mediated uptake was affected to varying degrees. If two-thirds of the exposed amino groups of the asialofetuin molecule were modified, the rate of uptake decreased to less than one-fourth of controls; degradation of endocytosed molecules was 12% of controls. The reduction in endocytic uptake was due to a reduced rate of formation of the receptor-ligand complex. Subcellular frationation in density gradients showed that PEG-modified asialofetuin is transported intracellularly and degraded in the same manner as the native protein, but the rate of proteolysis is reduced. This observation explains the paradoxical result of experiments with injection of modified asialofetuin into rats in vivo: even though the clearance of one preparation of PEG-asialofetuin was much slower than that of the native protein, accumulation of radioactivity in the liver from the modified protein was twice as high. The hepatocytes accounted for 85% of the hepatic accumulation of either PEG-modified or native asialofetuin in vivo.