Differentiation of intrahepatic membrane-bound and secretory apolipoprotein B by monoclonal antibodies: membrane-bound apolipoprotein B is more glycosylated.

Most apolipoprotein B (apoB) in rat hepatocytes membrane is membrane-bound. The purpose of this study was to determine whether differences existed between membrane-bound and plasma apolipoprotein B, which could be detected using monoclonal antibodies. Detergent-solubilized microsomal membrane-bound apoB was probed with two previously characterized monoclonal antibodies (LRB 200, LRB 220) and compared to a monospecific polyclonal antibody. LRB 200 (capable of binding 71% of plasma apoB) was able to recognize less than 20% of microsomal apoB compared to LRB 220 (a pan-apoB monoclonal antibody capable of binding 100% plasma apoB). To test the hypothesis that the immunologic difference detected by the monoclonal antibodies was due to increased glycosylation of the membrane-bound apolipoprotein B, plasma lipoproteins were incubated with neuraminidase. A progressive increase was found in antibody binding by LRB 200 but not by LRB 220 or the polyclonal antibodies. Inhibition of N-glycosylation by tunicamycin also increased the binding of monoclonal antibody LRB 200 to hepatocyte apoB. Inhibition of trimming of N-linked sugar by incubating hepatocytes with the inhibitors of glucosidase I and mannosidase I eliminated antibody binding by LRB 200 but not by LRB 220 or the polyclonal antibody. When N-linked sugars were removed by peptide: N-glycosidase F, antibody binding by monoclonal antibody LRB 200 was increased. Double-labeling experiments using 3H-mannose and 35S-methionine showed that cellular apoB contained twice the amount of mannose as medium apoB. These data suggest that membrane-bound apoB is more glycosylated than plasma lipoprotein apoB.