Long-chain cis-isoprenyltransferase activity is induced early in the developmental program for protein N-glycosylation in embryonic rat brain cells.

A large developmental increase in Glc3Man9-GlcNAc2-P-P-dolichol (Oligo-P-P-Dol) synthesis and protein N-glycosylation in primary cultures of embryonic rat brain cells has been reported previously. In vitro enzyme studies and metabolic labeling experiments now show that there is a coordinate induction of long-chain cis-isoprenyltransferase (IPTase) activity, an activity required for the chain-elongation stage of dolichyl monophosphate (Dol-P) biosynthesis de novo, and Oligo-P-P-Dol biosynthesis in embryonic rat brain. Different developmental patterns were observed for IPTase and beta-hydroxy-beta-methyl-glutaryl-CoA (HMG-CoA) reductase activity as well as Dol-P and cholesterol biosynthesis, indicating that these pathways are regulated independently in rat brain. Three separate experimental approaches provide evidence that the amount of Dol-P available in the rough endoplasmic reticulum (RER) is a rate-limiting factor in the expression of the lipid intermediate pathway. First, metabolic labeling experiments show that the biosynthesis of Dol-P is induced at the same time or just prior to the induction of Oligo-P-P-Dol biosynthesis. Second, the time of induction and rate of Oligo-P-P-Dol synthesis are accelerated when Dol-P is supplemented in the culture medium. Third, in vitro assays of mannosylphosphoryldolichol synthase and N-acetylglucosaminylpyrophosphoryldolichol synthase indicate that there are only minor increases in the levels of these enzymes during development, but the amount of endogenous Dol-P in the RER that is accessible to the glycosyltransferases increases when IPTase activity is induced. In summary, the current studies with embryonic rat brain cells document the coordinate induction of IPTase activity and Oligo-P-P-Dol synthesis, support the hypothesis that the availability of Dol-P in the RER is one rate-limiting factor in Oligo-P-P-Dol synthesis, and strongly suggest that increases in IPTase activity and the rate of de novo Dol-P biosynthesis enhance the capacity of embryonic rat brain cells for lipid intermediate synthesis early in the developmental program for N-linked glycoprotein biosynthesis.