title: Site-directed mutagenesis of the N-linked glycosylation site in platelet-derived growth factor B-chain results in diminished intracellular retention.
abstract: Pulse-chase analysis of human platelet-derived growth factor (PDGF) B-chain was conducted in stably transfected Chinese hamster ovary cells to determine precisely the kinetics of processing, intracellular trafficking and secretion. Newly synthesized 31 kDa monomers of the B-chain (p31) dimerized rapidly via disulfide bonds to a p54 species (t1/2 < 30 min). The p54 dimer was processed to a group of intracellular, cell surface (suramin-releasable) and secreted forms whose rates of appearance and disappearance from the cell were measured over a 48 h period. The newly synthesized p31 species was quantitatively converted to p27 by treatment with endoglycosidase H, consistent with efficient N-glycosylation at a site in the N-terminal propeptide region (Asn63-Met64-Thr65). Interruption of B-chain glycosylation by oligodeoxynucleotide-directed mutagenesis resulted in a significant increase in suramin-releasable forms at the cell surface (p34-38) and a concomitant decrease in accumulation of an intracellular p24 species. The glycosylation-defective mutant exhibited slight increases in receptor binding and mitogenic activity. Our results suggest that N-linked glycosylation of the B-chain is not important for formation of mitogenically active protein, but that it plays a role in early intracellular sorting and proteolytic processing events.
authors.affiliation: Department of Pharmacology, College of Medicine, Chandler Medical Center, University of Kentucky, Lexington 40536, USA. dmkaetzel@pop.uky.edu
authors.forename: D M
authors.initials: DM
authors.lastname: Kaetzel
authors.forename: D
authors.initials: D
authors.lastname: Morgan
authors.forename: J D
authors.initials: JD
authors.lastname: Reid
authors.forename: R A
authors.initials: RA
authors.lastname: Fenstermaker
mesh_terms: D000818:Animals; D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D007231:Infant, Newborn; D016297:Mutagenesis, Site-Directed; D010982:Platelet-Derived Growth Factor / Q000235:genetics* / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D010982:Platelet-Derived Growth Factor; D011498:Protein Precursors; D011994:Recombinant Proteins; D001216:Asparagine; D017479:Receptors, Platelet-Derived Growth Factor