Glycosylation of Thyroid-Stimulating Hormone in Pituitary Tumor Cells: Influence of High Mannose Oligosaccharide Units on Subunit Aggregation, Combination, and Intracellular Degradation

Abstract

We have studied the glycosylation of TSH in cell culture and have examined the influence of carbohydrate on subunit aggregation, intracellular degradation, and combination. Dispersed mouse thyrotropic tumor cells were labeled by pulsechase methods with [³⁵S]methionine and various ³H-labeled carbohydrates; cell lysates and media were precipitated with antisera to TSHα and TSHβ, and the products were analyzed by sodium dodecyl sulfate gradient gel electrophoresis without or with preexposure to Endoglycosidase (Endo) H. At early pulses, both intracellular α and β were mainly composed of one Endo H-sensitive (high mannose) carbohydrate unit and a small amount of nonglycosylated forms; α only had the posttranslational addition of a second high mannose unit. With increasing chase times up to 18 h, intracellular subunits showed a slow but progressive increase in Endo H-resistant (complex) forms, and media subunits were completely resistant. Preincubation of cells with tunicamycin caused production of nonglycosylated subunits that showed a high degree of aggregation, especially after heating at 37 C under nonreducing conditions. Unlike glycosylated subunits, which were not degraded, nonglycosylated subunits were 50–65% degraded intracellularly before secretion; the degradation caused by tunicamycin was specific for TSH subunits and not noted for other ³⁵S-labeled proteins. Incubation of various ³⁵S-labeled α forms with excess unlabeled TSHβ showed high combining activity for intracellular α with two high mannose units, intermediate activity for media a with two complex units, and low activity for intracellular α with one high mannose unit or nonglycosylated media α. These data suggest that the initial glycosylation with high mannose carbohydrate units prevents intracellular aggregation and degradation of TSH subunits and enhances attainment of the conformation necessary for α- and β-subunit combination.