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 choriocarcin oma cells was investigated using the octanoyl-tripeptide (OTP; N-octan oyl-asparagyl-I-125- tyrosyl-threonine amide) as glycosyl acceptor. In cells pretreated with 8-bromo-cAMP (2.5 nM to 1 mM), the amount of gl ycosylated OTP released into the culture medium was increased up to 35 -foId. Under the same conditions, a 23-fold higher quantity of the gly coprotein hormone human chorionic gonadotropin was secreted. Preincuba tion of 10-90 min with 250 mu M 8-bromo-cAMP caused only a 2-fold incr ease of the total amount of glycosylated OTP, whereas it was approxima tely 20-fold higher when the pretreatment was extended to 40 h. This s trongly suggests involvement of gene activation rather than cAMP-media ted phosphorylation. The specific activity of the oligosaccharyltransf erase, as well as the mRNA levels of ribophorin I and II (presumptive subunits of the enzyme), remained unchanged. In pulse-chase experiment s, [H-3]mannose incorporation into dolichol-linked Glc(3)Man(9)(GlcNAc )(2) was up to 20-fold higher in cells pretreated with 8-bromo-cAMP (2 50 mu M, 40 h). The radioactivity was chased from the lipid-linked oli gosaccharide pool and shifted to the glycoprotein fraction 10 times mo re rapidly in the pretreated cells. The flux of [H-3]mannose through t he dolichol phosphate mannose pool was only slightly affected by the 8 -bromo-cAMP pretreatment. Our investigations show that the oligosaccha ryltransferase activity in JEG-3 cells is not rate-limiting. N-Glycosy lation seems to be controlled by the amount of lipid-linked core oligo saccharide. The size of the lipid-linked core oligosaccharide pool, as well as the flux through, is markedly increased by pretreat ment with 8-bromo cAMP.