Abstract
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.
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|