Further studies on the role of glycosylation in thyroxine-binding globulin secretion by human hepatoma (HEP G2) cells.
To investigate the role of glycosylation of T4-binding globulin (TBG) in the secretion of the protein, human hepatoma (Hep G2) cells were continuously labeled with [35S] methionine or [3H]mannose or pulse-chase labeled with [35S] methionine in the absence or presence of 1 microgram/ml swainsonine, an inhibitor of Golgi alpha-mannosidase II and lysosomal alpha-mannosidase. In the presence of this alkaloid, TBG was released into the medium at a faster rate than in control cells (50% being secreted after 35 min and 47 min, respectively), owing to accelerated intracellular transport of the newly synthesized protein. TBG secreted from swainsonine-treated cultures moved faster in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, probably because of the reduced sialylation of TBG consequent to the perturbed processing of the oligosaccharide units. Furthermore, secreted TBG was sensitive to endo-beta-N-acetylglucosaminidase H digestion as shown by the shift in the apparent molecular size in sodium dodecyl sulfate-polyacrylamide gel electro-phoresis from 50,000 to 45,000 daltons. Sensitivity to endo H indicated the presence of hybrid-type oligosaccharide chains with high mannose structures. This was also suggested by the higher incorporation of [3H]mannose in swainsonine-treated cultures. In conclusion, the results of the present study demonstrate that swainsonine accelerates the release of TBG from Hep G2 cells and that complete processing of oligosaccharide moieties is not required for TBG secretion.
Bartalena
L
L
Cattedra di Endocrinologia e Medicina Costituzionale, University of Pisa, Italy.
Robbins
J
J
Pacchiarotti
A
A
Martino
E
E
Pinchera
A
A
D000470:Alkaloids / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002460:Cell Line; D055598:Chemical Phenomena; D002621:Chemistry; D006031:Glycosylation; D006801:Humans; D007158:Immunologic Techniques; D007700:Kinetics; D008113:Liver Neoplasms / Q000378:metabolism*; D017026:Swainsonine; D013975:Thyroxine-Binding Proteins / Q000378:metabolism*
D000470:Alkaloids; D013975:Thyroxine-Binding Proteins; D017026:Swainsonine