Glycosylation of thyroid-stimulating hormone in pituitary tumor cells: influence of high mannose oligosaccharide units on subunit aggregation, combination, and intracellular degradation.

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 pulse-chase methods with [35S]methionine and various 3H-labeled carbohydrates; cell lysates and media were precipitated with antisera to TSH alpha and TSH beta, 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 alpha and beta were mainly composed of one Endo H-sensitive (high mannose) carbohydrate unit and a small amount of nonglycosylated forms; alpha 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 alpha forms with excess unlabeled TSH beta showed high combining activity for intracellular alpha with two high mannose units, intermediate activity for media alpha with two complex units, and low activity for intracellular alpha with one high mannose unit or nonglycosylated media alpha. 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 alpha- and beta-subunit combination.