Binding and Endocytosis of Glycoproteins by Isolated Chicken Hepatocytes

The binding and endocytosis of glycoproteins containing different terminal sugars by isolated chicken hepatocytes were studied. At 2 °C, where there is no endocytosis, the hepatocyte surface bound 30800 GlcNAc₄₄-AI-BSA molecules [a bovine serum albumin (BSA) derivative which contains 44 residues of N-octylglucosamine (GlcNAc)] [Lee, Y. C, Stowell, C. P., & Krantz, M. J. (1976) Biochemistry 15, 3956-3963] and 32 900 asialoagalactoorosomucoid (AGOR) molecules per cell with estimated dissociation constants of 5 × 10^-10 and 4 × 10^-9 M, respectively. In the presence of digitonin or Triton X-100, each hepatocyte bound 7-18 times more ligand than in the absence of these detergents. Bound ¹²⁵I-AGOR could be dissociated from the cell surface by 5.5 × 10_-5 M GlcNAc₄₄-AI-BSA with a t_1/2 of 30 min, while GlcNAc (10 mM) or ethylene glycol bis(β-aminoethyl ether)-N,N,N’,N′-tetraacetic acid (4 mM) could dissociate over 98% of the surface-bound radioactivity within 10 min. Several neoglycoproteins inhibited the binding of ¹²⁵I-AGOR, requiring for 50% inhibition 2.1 × 10^-9, 4.0 × 10^-7, 1.6 × 10^-6, and 2 × 10^-6 M for GlcNAc₄₄-, Glc₃₇-, Man₄₃-, and L-Fuc₂₈-AI-BSA, respectively. The bound AGOR and neoglycoproteins were internalized and degraded at 37 °C. [125I]Iodide was the only labeled degradation product found. When the hepatocytes were exposed to 250 nM AGOR at 37 °C, ca. 100000 molecules of AGOR were associated with the cell surface at the steady state of endocytosis. This is about a 3-fold increase over the corresponding value at 2 °C. Kinetic simulation of synchronous processing of surface-bound AGOR or G1cNAc₄₄-AI-BSA suggests that significant recycling of internalized ligand to the surface occurs during endocytosis at 37 °C.