The degradation and turnover of fucosylated glycoproteins in the plasma membrane of a neuroblastoma-cell line

Abstract

When monolayer cultures of neuroblastoma N2a cells [mouse] were prelabeled with [3H]fucose to steady state, and then reincubated in complete medium in the presence of unlabeled 40 mM L-fucose, there was a rapid metabolism of fucosylated cellular macromolecules and the specific radioactivity of the acid-insoluble material decreased by 22% within 2 h. After this the remaining radioactive glycoproteins appeared to be more stable and the rate of loss of specific radioactivity markedly decreased. Since fucose is associated predominantly with plasma-membrane components, the analysis of fucosylated glycoproteins was characterized in plasma-membrane fractions by polyacrylamide-gel electrophoresis. Two experimental approaches were used to measure glycoprotein degradation and turnover in the cell-surface membranes. In 1 set of experiments, with a similar incubation procedure to that used with intact cells, 3 membrane components were rapidly degraded (150,000, 130,000 and 48,000 daltons), but another surface glycoprotein (68,000 daltons) appeared to be more slowly metabolized than the mean rate of glycoprotein degradation. The relationship of the degradation of membrane glycoproteins to their turnover was analyzed by dual-label experiments that used both [14C]fucose and [3H]fucose. Glycoproteins of the surface membrane of neuroblastoma cells turned over at heterogeneous rates. The components mentioned above that exhibited significantly rapid rates of degradation, turned over more rapidly than the average surface component. In addition to the membrane components detected by the use of only [3H]fucose, dual-label experiments illustrated that numerous surface glycoproteins were metabolized more rapidly or slowly than most of the cell-surface constituents.