Biosynthesis of a novel thyroxine-binding protein (27K protein) in human hepatoma (Hep G2) cells

Human hepatoma (Hep G2) cells were shown to synthesize and secrete a novel T4-binding protein, called 27K protein for its apparent mol wt on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The mRNA coding for this protein was characterized by immunoprecipitation of [125I]T4 bound to 27K protein secreted into the medium of oocytes injected with total Hep G2 RNA. Sucrose gradient fractionation of RNA from Hep G2 cells showed that TBG mRNA and 27K mRNA had different sizes, indicating that TBG and 27K protein are two distinct proteins. In vitro translation of RNA in a rabbit reticulocyte lysate demonstrated that the translation product immunoprecipitated by anti-27K serum had the same mol wt as the immunoprecipitated protein from whole cells labeled with [35S]methionine, thus suggesting that 27K protein is neither derived from TBG nor synthesized through a larger mol wt precursor, and also that it does not contain carbohydrates. The absence of carbohydrates was further supported by the observation that [3H]mannose was not covalently bound to the 27K protein when Hep G2 cells were labeled with [3H]mannose, nor was there a shift in apparent mol wt when the cells were treated with the glycosylation inhibitor tunicamycin. The kinetics of secretion of 27K protein were similar to those of albumin and faster than those of TBG, which is also in keeping with the nonglycoprotein nature of 27K protein.