Dolichol pyrophosphate

Synthesis of dolichol pyrophosphate, a required cofactor in N-hnked glycosylation of proteins in the endoplasmic reticulum... 

The synthesis of N-linked glycosides also occurs in the lumen of the ER and in the Golgi. However, these structures undergo additional processing steps, and require the participation of a lipid (dolichol) and its phosphorylated derivative, dolichol pyrophosphate (Figure 14.16). 

Structures of dolichol phosphate, GlcNAc-P-P-Dol, and Man-P-Dol. GlcNAc-P-P-Dol is the first compound formed in the biosynthesis of the lipid-linked oligosaccharide shown in Figure 16-5. Replacement of GlcNAc by -OH in GIcNAc-P-P-Dol yields dolichol diphosphate (dolichol pyrophosphate). Replacement of Man by Glc in Man-P-Dol yields Glc-P-Dol. 

The pathway for de novo synthesis of dolichol phosphate is the same as for cholesterol until production of famesyl pyrophosphate (Chapter 19). Two salvage pathways provide for the reutilization of dolichol and dolichol pyrophosphate once transfer of oligosaccharide from the dolichol to glycoprotein has occurred. A significant portion of the dolichol in liver is present as the free alcohol or as a fatty acid ester, and dolichol absorbed from the diet may enter this pool. Phosphorylation of dolichol is catalyzed by a dolichol kinase via cytidine triphosphate (CTP). Removal of one phosphate... 

The lipid-soluble intermediate that accumulates is dolichol pyrophosphate, whose terminal phosphate comes from glucose 1-phosphate. (See Figure 11.23 in Section 11.3.3 in the text.) Bacitracin is an antibiotic that forms a 1 1 complex with dolichol pyrophosphate, preventing its hydrolysis to dolichol phosphate and inorganic phosphate. Thus, in the presence of bacitracin, the label will remain in dolichol pyrophosphate. In the absence of bacitracin, the terminal phosphate will be released as inorganic phosphate. 

The existence of mannosylated saccharides linked to dolichol via a pyrophosphate was already well established. Studies by Behrens etal. (1973) had shown that rat liver microsomes could catalyse the synthesis of various lipid-linked oUgosaccharides from GDP mannose and dolichol phosphate, among which were some that were soluble in chloroform-methanol-water (1 1 0.3, v/v). These were more polar than dolichol monophosphate derivatives and could be bound to DEAE-cellulose in the presence of the organic solvent. In an ammonium formate gradient (also in the organic solvent) these polyprenyl saccharides eluted at the position of dolichol pyrophosphate derivatives. Waechter et al (1973) independently showed that a similar class of compounds was produced from GDP-[ C]maimose in the presence of microsomes from hen oviduct and that the labelled products... 

Following the transfer of the glucosylated mannan cores from dolichol pyrophosphate to the peptide acceptor and the subsequent removal of terminal glucosyl and certain mannosyl residues (see above), there follows a series of reactions in which N-acetylglucosamine is added to the exposed mannosyl terminals and some further removal of mannosyl residues occurs. The general pattern and sequence of these processes has now been substantially proven by the studies of the Kornfelds, Schachter and others. The addition of phosphate to mannose cores occurs in some lysosomal acid hydrolases and is part of a targeting mechanism, considered in detail in Chapter 6. 

Coumarin is the only inhibitor yet known to act at the level of protein glycosylation from a dolichol pyrophosphate derivative. 

The biosynthesis of the mannan inner core involves dolichol pyrophosphate derivatives containing JV-acetyl glucosamine and several mannose residues [38]. The participation of the polyprenol (dolichol) in the biosynthesis of the mannan inner core in yeast is analagous to its role in animal tissues as a mechanism for the glycosylation of proteins. 

Incubation of Dol-P-( C)-Glc with liver microsomes was found to lead to the transfer of the label to an endogenous acceptor which on the basis of somewhat indirect evidence was assumed to be a dolichol pyrophosphate oligosaccharide (Behrens et al., 1971). 

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