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.
Wong
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L
Department of Physiology, Louisiana State University Medical Center, New Orleans 70112-2822.
Torbati
A
A
D000581:Amidohydrolases / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D002236:Carbohydrate Conformation; D010450:Endopeptidases / Q000494:pharmacology; D006031:Glycosylation; D006596:Hexosaminidases / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D051381:Rats; D014415:Tunicamycin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B; D009844:Oligosaccharides; D014415:Tunicamycin; D008715:Methionine; D006596:Hexosaminidases; C060638:glycopeptide alpha-N-acetylgalactosaminidase; D010450:Endopeptidases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D008358:Mannose