Regulation of N-glycosylation. Long term effect of cyclic AMP mediates enhanced synthesis of the dolichol pyrophosphate core oligosaccharide.
The influence of 8-bromo-cAMP on N-glycosylation in JEG-3 choriocarcinoma cells was investigated using the octanoyl-tripeptide (OTP; N-octanoyl-asparagyl-125I-tyrosyl-threonine amide) as glycosyl acceptor. In cells pretreated with 8-bromo-cAMP (2.5 nM to 1 mM), the amount of glycosylated OTP released into the culture medium was increased up to 35-fold. Under the same conditions, a 23-fold higher quantity of the glycoprotein hormone human chorionic gonadotropin was secreted. Preincubation of 10-90 min with 250 microM 8-bromo-cAMP caused only a 2-fold increase of the total amount of glycosylated OTP, whereas it was approximately 20-fold higher when the pretreatment was extended to 40 h. This strongly suggests involvement of gene activation rather than cAMP-mediated phosphorylation. The specific activity of the oligosaccharyltransferase, as well as the mRNA levels of ribophorin I and II (presumptive subunits of the enzyme), remained unchanged. In pulse-chase experiments, [3H]mannose incorporation into dolichol-linked Glc3Man9(GlcNAc)2 was up to 20-fold higher in cells pretreated with 8-bromo-cAMP (250 microM, 40 h). The radioactivity was chased from the lipid-linked oligosaccharide pool and shifted to the glycoprotein fraction 10 times more rapidly in the pretreated cells. The flux of [3H]mannose through the dolichol phosphate mannose pool was only slightly affected by the 8-bromo-cAMP pretreatment. Our investigations show that the oligosaccharyltransferase activity in JEG-3 cells is not rate-limiting. N-Glycosylation seems to be controlled by the amount of lipid-linked core oligosaccharide. The size of the lipid-linked core oligosaccharide pool, as well as the flux through, is markedly increased by pretreatment with 8-bromo-cAMP.
Konrad
M
M
Department of Biochemistry II, University of Heidelberg, Germany.
Merz
W E
WE
D000595:Amino Acid Sequence; D000242:Cyclic AMP / Q000502:physiology*; D004288:Dolichol Phosphates / Q000378:metabolism*; D006001:Glycoconjugates / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis*; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D014407:Tumor Cells, Cultured
D004288:Dolichol Phosphates; D006001:Glycoconjugates; D006023:Glycoproteins; D009842:Oligopeptides; C026406:dolichol pyrophosphate; D000242:Cyclic AMP