title: Incorporation of beta-fluoroasparagine into peptides prevents N-linked glycosylation. In vitro studies with synthetic fluoropeptides.
abstract: Previously, we reported that incorporation of threo-beta-fluoroasparagine into cellular protein inhibits N-linked glycosylation. We now show that short synthetic peptides which contain N-acetyl-threo-beta-fluoroasparagine fail to undergo glycosylation in a cell-free system except at extremely high substrate concentrations. An N-benzoyl-threo-beta-fluoroasparagine-containing peptide has a 100-fold lower Vmax/Km than the analogous N-benzoyl-asparagine-containing peptide. Substitution of a fluorine for a hydrogen on the beta-carbon of asparagine weakens the ability of the peptide to bind the oligosaccharyltransferase. A 100-fold excess of acetyl-threo-beta-fluoroasparaginyl-leucyl-threonine methylamide over acetyl-asparaginyl-leucyl-threonine methylamide inhibited glycosylation of the latter peptide by less than 10%. Both threo-beta-fluoroasparagine and erythro-beta-fluoroasparagine-containing peptides are glycosylated at the same rate. Glycofluoropeptides generated from beta-fluoroasparagine-containing peptides were N-glycosylated. These cell-free studies with synthetic fluoropeptides suggest that incorporation of beta-fluoroasparagine into cellular protein inhibits N-linked glycosylation by rendering protein substrates ineffective for glycosylation. In the course of this work, we also demonstrate that the N-linked glycosylating enzyme acts only on L-asparagine-containing peptides and not on D-asparagine peptides.
mesh_terms: D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000031:analogs & derivatives* / Q000378:metabolism; D050260:Carbohydrate Metabolism; D006602:Hexosyltransferases; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008565:Membrane Proteins; D011402:Pronase / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013552:Swine; D014166:Transferases / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D001216:Asparagine; C023464:beta-fluoroasparagine; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D011402:Pronase