Inhibition of asparagine-linked glycosylation of pro-opiomelanocortin in mouse pituitary cells by DL-threo-beta-fluoroasparagine.

The actions of DL-threo-beta-fluoroasparagine (DL-beta-F-Asn) on the glycosylation of proteins were examined in AtT-20/D16v cells which synthesize several forms of the glycoprotein prohormone, pro-opiomelanocortin (POMC). Treatment with threo-beta-F-Asn(5-10 mM) resulted in: 1) a reduction in the amount of the more highly glycosylated form of POMC (Mr = 32,000) relative to the less glycosylated form (Mr = 29,000) and 2) the appearance of a new species of POMC (Mr = 27,000). 35S]Methionine-labeled tryptic peptides prepared from 27,000 POMC were identical to those from 29,000 and 32,000 POMC; however, 27,000 POMC was found to contain 10% as much [3H]glucosamine relative to [35S]methionine as the 32,000 molecule. Furthermore, 27,000 POMC comigrated with a previously characterized unglycosylated form of this prohormone produced by treatment of cells with tunicamycin. These findings indicate that treatment of cells with threo-beta-F-Asn results in the production of a species of POMC which contains little or no carbohydrate. The effects of beta-F-Asn were specific for the threo diastereomer, were reversible by equimolar concentrations of Asn, but not Asp, and were dose-dependent. Evidence that threo-beta-F-Asn can replace Asn in proteins was obtained by showing that an identified Asn-containing tryptic peptide from threo-beta-F-Asn-treated cells displayed an altered mobility during electrophoresis consistent with threo-beta-F-Asn substitution within this peptide. We conclude that threo-beta-F-Asn can inhibit the glycosylation of proteins in intact cells and that this effect is due to its ability to replace Asn at glycosylation sites.