Secretion of a major phosphorylated glycoprotein by hepatocytes. Characterization of specific antibodies and investigations of the processing, excretion kinetics, and phosphorylation.
Isolated rat hepatocytes secreted a major phosphorylated glycoprotein (PP63) with apparent Mr = 63,000 and isoelectric point ranging from 4.8 to 5.3. Specific antibodies were raised in a rabbit using material obtained from plasma as an antigen. The biosynthesis of PP63 was studied in vitro in a cell-free system and in intact hepatocytes incubated with or without tunicamycin. The mRNA translation product had a Mr = 43,000 and was of the same size as the major unglycosylated precursor found in intact cells. This precursor was rapidly processed into two major intracellular forms of Mr = 53,000 and 56,000. These species were insensitive to neuraminidase but susceptible to endoglycosidase H, indicating that they contained oligosaccharide side chains of the high mannose-type. Terminal glycosylation gave rise to the mature Mr = 63,000 protein that contained sialic acid and fucose. This species represented the exportable form of the protein and was the only one to be phosphorylated. The charge heterogeneity observed for the mature protein already existed in all the precursors, indicating that it could not be ascribed to sialylation or to phosphorylation. However, these covalent modifications were mainly responsible for the acidic character of PP63. PP63 secretion was altered by tunicamycin. Pulse-chase experiments showed that the phosphorylated glycoprotein was secreted according to kinetics similar to that described for other liver glycoprotein, with slower kinetics than albumin. Permanent phosphorylation did not appear mandatory for excretion since the dephosphorylated PP63 was excreted with an efficacy comparable to that of the phosphorylated protein. Phosphorylation of PP63 was shown to occur on a single tryptic peptide, at a serine residue.
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D005643:Fucose / Q000378:metabolism; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism*; D006596:Hexosaminidases / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D009439:Neuraminidase / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D014357:Trypsin / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D000906:Antibodies; D006023:Glycoproteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D014415:Tunicamycin; D005643:Fucose; D006596:Hexosaminidases; D009439:Neuraminidase; D014357:Trypsin