Long‐Chain os‐Isoprenyltransferase Activity Is Induced Early in the Developmental Program for Protein N‐Glycosylation in Embryonic Rat Brain Cells

Abstract

A large developmental increase in Gl c,Man,-GlcNAc,-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-Do1 biosynthesis in embryonic rat brain. Different developmental patterns were observed for IPTase and P-hydroxy-P-methylglutaryl-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-Do1 biosynthesis. Second, the time of induction and rate of Oligo-P-P-Do1 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-Do1 synthesis, support the hypothesis that the availability of Dol-P in the RER is one rate-limiting factor in Oligo-P-P-Do1 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.

Recombinant antibody 2G12 produced in maize endosperm efficiently neutralizes HIV‐1 and contains predominantly single‐GlcNAc N‐glycans

“…Approximately 10% of HC was non‐glycosylated, which is far lower than that reported recently for a KDEL‐tagged single‐chain Fv‐Fc antibody produced in Arabidopsis seeds (Van Droogenbroeck et al ., 2007). However, it falls within the normal range of glycosylation site occupancy observed with recombinant glycoproteins derived from mammalian cells, depending on the culture environment, in particular the availability of dolichol (Rosenwald et al ., 1990; Crick and Waechter, 1994) and the ambient glucose concentration (Hayter et al ., 1992, 1993; Tachibana et al ., 1994). Most surprisingly, more than half of the N ‐glycans comprised only a single GlcNAc residue.…”

Genetic defects in dolichol metabolism

“…The availability of Dol-P in the ER is considered as a rate-limiting factor in Dol-PP-glycan biosynthesis since an increase in CPT activity and enhanced rate of de novo Dol-P biosynthesis increases the capacity of LLO synthesis in embryonic rat brain cells (Crick and Waechter 1994 ).…”