The role of glycosylation in the molecular conformation and secretion of thyroxine-binding globulin.
T4-binding globulin (TBG), the principal carrier of thyroid hormone in serum, is a glycoprotein containing 20% carbohydrate. The importance of the carbohydrate moiety has been previously studied by enzymatic deglycosylation, which showed that deglycosylated TBG retains its original immunological an T4-binding properties. However, the structure and properties of TBG before glycosylation and the steps involved in carbohydrate addition have not been explored. In the present report, we used a human hepatoma cell line (Hep G2) which synthesizes and secretes TBG into the medium. This TBG binds T4 and possesses immunoreactivity and microheterogeneity identical to those of native TBG (nTBG) from serum. Cells were pulsed with [35S]methionine, [3H]mannose, and [3H]glucosamine in the absence or presence of 5 micrograms tunicamycin/ml medium. Materials from cells and media were immunoprecipitated with antibodies specific for nTBG and denatured TBG molecule. They were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cells incubated with [35S]methionine contained two forms of labeled TBG, with apparent mol wt of 60K (TBG1) and 54K (TBG2). Medium contained only the TBG1 form, which is identical to nTBG in serum. In contrast, in the presence of tunicamycin, the predominant intracellular form of TBG had an apparent mol wt of 44K (TBG3). At no time was this material detected in the medium. [3H]Mannose and [3H]glucosamine labeled both TBG1 and TBG2, but not TBG3. TBG1 and TBG2 reacted with anti-nTBG serum, whereas TBG3 reacted only with anti-dentured TBG serum, specific for the unfolded TBG molecule. Intracellular TBG was rich (80-90%) in high mannose (seven to nine mannose residues) oligosaccharides and was relatively poor (10-20%) in complex-type species, resistant to endoglycosidase H. These results indicate that 1) the precursor nonglycosylated 44K TBG3 is glycosylated to produce TBG2 (54K) and TBG1 (60K); 2) TBG2 contains oligosaccharides rich in mannose and appears to be a major intracellular intermediate in the synthesis of TBG1; 3) only the endoglycosidase H-resistant TBG1 is secreted; and 4) prior glycosylation of TBG appears to be required for the molecule to assume its tertiary structure and, ultimately, for its secretion. However, once the peptide chain is folded, removal of the carbohydrate moieties does not alter the tertiary structure.
Murata
Y
Y
Magner
J A
JA
Refetoff
S
S
D002236:Carbohydrate Conformation; D050260:Carbohydrate Metabolism; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005470:Fluorometry; D005944:Glucosamine / Q000378:metabolism; D006801:Humans; D008358:Mannose / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis; D011487:Protein Conformation; D013975:Thyroxine-Binding Proteins / Q000378:metabolism*; D013997:Time Factors
D009844:Oligosaccharides; D013975:Thyroxine-Binding Proteins; D008715:Methionine; D005944:Glucosamine; D008358:Mannose