Nogo-B receptor is necessary for cellular dolichol biosynthesis and protein N-glycosylation.

Dolichol monophosphate (Dol-P) functions as an obligate glycosyl carrier lipid in protein glycosylation reactions. Dol-P is synthesized by the successive condensation of isopentenyl diphosphate (IPP), with farnesyl diphosphate catalysed by a cis-isoprenyltransferase (cis-IPTase) activity. Despite the recognition of cis-IPTase activity 40 years ago and the molecular cloning of the human cDNA encoding the mammalian enzyme, the molecular machinery responsible for regulating this activity remains incompletely understood. Here, we identify Nogo-B receptor (NgBR) as an essential component of the Dol-P biosynthetic machinery. Loss of NgBR results in a robust deficit in cis-IPTase activity and Dol-P production, leading to diminished levels of dolichol-linked oligosaccharides and a broad reduction in protein N-glycosylation. NgBR interacts with the previously identified cis-IPTase hCIT, enhances hCIT protein stability, and promotes Dol-P production. Identification of NgBR as a component of the cis-IPTase machinery yields insights into the regulation of dolichol biosynthesis.

Phosphorylation of lysosomal enzymes in fibroblasts. Marked deficiency of N-acetylglucosamine-1-phosphotransferase in fibroblasts of patients with mucolipidosis III.

N-Acetylglucosamine-1-phosphotransferase activity was assayed in human skin fibroblasts using [beta-32P]UDP-N-acetylglucosamine as donor and dephosphorylated beta-N-acetyl-D-hexosaminidase as acceptor. An optimal transfer rate of N-acetylglucosamine 1-phosphate required CDP-choline and ADP in order to inhibit the breakdown of [beta-32P]UDP-N-acetylglucosamine and a combination of leupeptin and iodoacetamide to protect the transferase. The transferase required Mg2 or Mn2. Using doubly labelled UDP-N-acetylglucosamine, simultaneous transfer of N-acetyl-[6-3H]glucosamine and [32P]phosphate to endogenous acceptors was demonstrated. Membranes prepared from fibroblasts from patients with mucolipidosis III were defective in transfer of N-acetylglucosamine 1-phosphate. A residual transferase activity of less than 10% of controls was detectable in fibroblast membranes of eight patients with mucolipidosis III. In membranes from fibroblasts from patients with mucolipidosis II,N-acetylglucosamine-1-phosphotransferase activity was not detectable. Our results indicate that the primary defect in mucolipidoses II and III is a deficiency in N-acetylglucosamine-1-phosphotransferase, the residual activity being higher in mucolipidosis III than in mucolipidosis II.