Characterization of nascent and secreted thyroxine-binding globulin in cultured human hepatoma (Hep G2) cells.

Thyroxine-binding globulin (TBG) synthesis by human hepatoma (Hep G2) cells was demonstrated by pulse labeling with [35S]methionine or [3H]mannose and subsequent immunoprecipitation in the medium or cell lysate. Secreted TBG was glycosylated and had the same apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as TBG purified from human serum. Pulse-chase experiments failed to show any precursor form intracellularly. Treatment of cells with the amino acid analogs, canavanine and thialysine, did not cause secretion of large-molecular-weight moieties, in contrast to what was observed in the case of albumin. Thyroxine-binding activity, as assessed by [125I]thyroxine immunoprecipitation with anti-TBG serum, was detectable in the media of oocytes injected with RNA from Hep G2 cells. Translation of this RNA in rabbit reticulocyte lysate, followed by immunoprecipitation with anti-TBG serum, revealed a protein having the same electrophoretic mobility as deglycosylated TBG purified from human serum (Mr approximately 45,000). Since deglycosylated TBG still contains 3% of its weight as carbohydrate, it appears that the translation product contains an additional fragment (signal peptide) of about 1,500 daltons. It is unlikely, however, that TBG is synthesized via a larger-molecular-weight precursor.