GLYCOSYLATION EVENTS IN THE PROCESSING AND SECRETION OF PRO-ACTH-ENDORPHIN IN MOUSE PITUITARY-TUMOR CELLS

The glycosylation of pro-ACTH-endorphin and of its cleavage products was studied in a mouse pituitary tumor cell line (AtT-20/D16v) by pulse labeling with 3H-labeled sugars and amino acids followed by immunoprecipitation and sodium dodecyl sulfate slab gel electrophoresis. The results of this analysis indicate that there are 2 major intracellular species of pro-ACTH-endorphin (29K and 32K) and 2 minor species (30K and 34K). All of these species label with [3H]mannose, [3H]glucosamine and [3H]galactose. In addition, the 30K and 34K species label with [3H]fucose and are the major secreted forms of pro-ACTH-endorphin. Only the 29K and 32K forms are labeled after a short pulse with [35S]methionine. The 30K and 34K forms become labeled during a subsequent chase. Glycosidase digestion of Pronase glycopeptides derived from pro-ACTH-endorphin shows that the 29K and 32K forms contain high mannose type oligosaccharides. The 30K and 34K forms contain complex oligosaccharides with both core and branch sugars. The 29K and 32K forms apparently are converted to 30K and 34K forms by processing of mannose-rich oligosaccharides. Pulse labeling with 3H-labeled sugars shows that pro-ACTH-endorphin is the 1st species labeled with both core and branch sugars. The label appears later in 21-26K and 12-15K ACTH, suggesting that glycosylation is completed before proteolytic cleavages begin. In support of this model, it is shown that intracellular and secreted forms of ACTH and N-terminal fragments have the same apparent MW on sodium dodecyl sulfate slab gels. Glycosidase digestion shows that the intracellular and secreted forms of the latter protein contain complex oligosaccharides. The N-terminal portion of pro-ACTH-endorphin gives rise during processing to 2 major fragments (14.5K and 16-17.5K fragments) with very similar tryptic peptide contents. The 14.5K fragment contains a single oligosaccharide while the 16-17.5K fragment contains two.