Binding and endocytosis of glycoproteins by isolated chicken hepatocytes

Abstract

The binding and endocytosis of glycoproteins containing different terminal sugars by isolated chicken hepatocytes were studied. At 2.degree. C, where there is no endocytosis, the hepatocyte surface bound 30,800 GlcNAc44-AI-BSA molecules [a bovine serum albumin (BSA) derivative which contains 44 residues of N-acetylglucosamine (GlcNAc)] and 32,900 asialoagalactoorosomucoid (AGOR) molecules per cell with estimated dissociation constants of 5 .times. 10-10 and 4 .times. 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 .times. 10-5 M GlcNAc44-AI-BSA with a t1/2 [half-time] of 30 min, while GlcNAc (10 mM) or ethylene glycol bis(.beta.-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 .times. 10-9, 4.0 .times. 10-7, 1.6 .times. 10-6 and 2 .times. 10-6 M for GlcNAc44-, Glc37-, Man43- and L-Fuc28-AI-BSA, respectively. The bound AGOR and neoglycoproteins were internalized and degraded at 37.degree. C. [125I]Iodide was the only labeled degradation product found. When the hepatocytes were exposed to 250 nM AGOR at 37.degree. C, .apprx. 100,000 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.degree. C. Kinetic stimulation of synchronous processing of surface-bound AGOR or GlcNAc44-AI-BSA suggests that significant recycling of internalized ligand to the surface occurs during endocytosis at 37.degree. C.