The ins(ide) and outs(ide) of dolichyl phosphate biosynthesis and recycling in the endoplasmic reticulum

Abstract

The precursor oligosaccharide donor for protein N-glycosylation in eukaryotes, Glc3Man9GlcNAc(2)-P-P-dolichol, is synthesized in two stages on both leaflets of the rough endoplasmic reticulum (ER). There is good evidence that the level of dolichyl monophosphate (Dol-P) is one rate-controlling factor in the first stage of the assembly process. In the current topological model it is proposed that ER proteins (flippases) then mediate the transbilayer movement of Man-P-Dol, Glc-P-Dol, and Man5GlcNAc(2)-P-P-Dol from the cytoplasmic leaflet to the lumenal leaflet. The rate of flipping of the three intermediates could plausibly influence the conversion of Man5GlcNAc(2)-P-P-Dol to Glc3Man(9)GlcNAc(2)-P-P-Dol in the second stage on the lumenal side of the rough ER. This article reviews the current understanding of the enzymes involved in the de novo biosynthesis of Dol-P and other polyisoprenoid glycosyl carrier lipids and speculates about the role of membrane proteins and enzymes that could be involved in the transbilayer movement of the lipid intermediates and the recycling of Dol-P and Dol-P-P discharged during glycosylphosphatidylinositol anchor biosynthesis, N-glycosylation, and O- and C-mannosylation reactions on the lumenal surface of the rough ER.

N-Linked Protein Glycosylation in the Endoplasmic Reticulum

“…The mechanism by which dolichol chain length is regulated is not completely understood, although regions and amino acid residues of bacterial cis-prenyltransferases were identified that seem to regulate the final length of the polyprenyl-pyrophosphate product (Chen et al 2005;Kharel et al 2006). Models for the final steps of the dolichol biosynthesis suggest that the polyprenyl-pyrophosphate product is first dephosphorylated (Schenk et al 2001) before the a-isoprene unit is reduced by a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent a-reductase (Sagami et al 1993;Szkopinska et al 1996;Cantagrel et al 2010). The a-saturated isoprene is then phosphorylated to form dolichyl phosphate (Dol-P).…”

“…The availability of Dol-P at the cytosolic side of the ER membrane is crucial for the process of N-glycosylation and it might become a ratelimiting factor in the biosynthesis of LLO Schenk et al 2001). Dol-P can be generated de novo by the biosynthetic pathway described above, but is also a product of glycosyltransferases using Dol-PMan and Dol-P-Glc as substrate in the ER lumen.…”

Apposite Insulin-like Growth Factor (IGF) Receptor Glycosylation Is Critical to the Maintenance of Vascular Smooth Muscle Phenotype in the Presence of Factors Promoting Osteogenic Differentiation and Mineralization

“…of cellular mevalonate by HMG-CoA reductase inhibitors leads to depletion of dolichyl phosphate, the initial donor of carbohydrate in protein N-linked glycosylation (26). It has also been reported that aberrant glycosylation of the IGFR results in a buildup of the IGF proreceptor due to the inability of the cell to cleave the proprotein to the mature ␣-and ␤-subunits (27).…”

HPLC and mass spectrometry analysis of dolichol-phosphates at the cell culture scale

“…Prior to the NADPH-dependent reduction of the α-isoprene unit, the entirely unsaturated polyprenol-pyrophosphate intermediates are dephosphorylated by mono-or pyrophosphate phosphatases. In a terminatory step, the resulting Dol are phosphorylated by the CTP-dependent Dol-kinase [3]. Dolichol-phosphates (Dol-P) serve as lipid carriers of mono-and oligosaccharides involved in several protein glycosylation pathways [4; 5] and in the formation of the glycosylphosphatidylinositol anchor [6].…”