Dolichol diphosphate

In the first Section, the dolichol pathway of protein glycosylation is introduced, and the reader is made familiar with the various reactions in the formation of the lipid and carbohydrate moieties of lipid-linked saccharides. Three different classes of compound are known so far (a) isoprenoid alcohol esters of monosaccharide monophosphates, such as D-mannosyl and D-glucosyl (dolichol phosphate), (b) such isoprenoid alcohol esters of saccharide diphosphates as dolichol diphosphate linked to 2-acetamido-2-deoxy-D-glucose and to oligosaccharides, and (c) retinol (D-mannosyl phosphate). The dolichol-linked sugars occur in all eukaryotes. 

As shown in Scheme 1, two other pathways lead to the formation of dolichol phosphate. These are (a) phosphorylation of the free poly-prenol, and (b) dephosphorylation of dolichol diphosphate. A dolichol phosphate-cleaving phosphatase has been described as occurring in Tetrahymena pyriformis,15 human lymphocytes,18 calf brain,17 and rat-nerve tissue.18 The protozoal enzyme was a soluble acid phosphatase, although it was clearly different from the bulk phosphatase activity, whereas the mammalian enzymes had optima at neutral pH and were membrane-bound. 

A particulate, enzyme preparation from human lymphocytes contains a phosphatase that converts dolichol diphosphate into dolichol phosphate and phosphate.15 This alkaline phosphatase may play a role in the recycling of dolichol phosphate, if this compound is to act as a coenzyme. However, the specificity of the phosphatase for dolichol diphosphate, 2,3-dehydrodolichol diphosphate, or dolichol diphosphate-linked oligosaccharides27 has not yet been determined. 

Although it could be established that saturation of the a-isoprene unit of polyprenol phosphate is important for these compounds to act as effective acceptors of glycosyl moieties in eukaryotic cells, little chain-length specificity was observed in such studies.39-41 This observation found support in the result that the same set of prenylogs occurs in total-yeast dolichol and in dolichol diphosphate-linked 2-acetamido-2-deoxy-D-glucose synthesized in vitro with yeast-cell homogenates.42 As already noted by Chojnacki and coworkers,40 dur-... 

How, exactly, retinol affects protein glycosylation is not yet known, but the formation of retinol phosphate, the D-mannosylation of retinol phosphate by GDP-Man, and the transfer of Man from D-mannosyl (retinol phosphate) to pronase-sensitive glycoconjugates are well established.5857,59 On the other hand, the formation and role of other gly-cosyl esters of retinol phosphate are still controversial.2,80 It is very probable that the D-mannosylation of retinol phosphate, and also the transfer of Man from this lipid derivative to protein, are catalyzed by enzymes different from those involved in the dolichol pathway,81-83 and this finding points to the different physiological roles of Man-P-Dol and D-mannosyl (retinol phosphate). Thus, whereas Man-P-Dol can serve to D-mannosylate dolichol diphosphate-linked oligosaccharides (see Section III), such a role has not been found for D-mannosyl (retinol phosphate).81,83... 

The biological function of this dolichol-linked oligosaccharide was revealed in pulse-chase experiments which showed that the intact oligosaccharide was transferred, as such, to protein. Subsequent studies, to be discussed, confirmed that the formation of the oligosaccharide linked to the Asn residues of glycoproteins is probably initiated by transfer of the oligosaccharide from the dolichol diphosphate intermediate to the asparagine residue of a nascent polypeptide (see Refs. 35, 49, 50, and 67 for reviews). 

Figure 20-6 Biosynthesis of the dolichol diphosphate-linked oligosaccharide precursor to glycoproteins. The site of inhibition by tunicamycin is indicated.

UDP-D-xylose, GDP-D-mannose, GDP-L-Fucose, or CMP-D-A-acetylneuraminic acid. Mammalian GTs also use dolichol-diphosphate-GlcNAc2Man9GlC3, dolichol-phosphate-mannose and dolichol-phosphate-glucose (Fig. 2). 

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. 

CDG-type I syndrome [264,266,267] is due to a deficiency in the oligosaccharidyltrans-ferase which transfers en bloc onto the nascent protein the oligosaccharide linked to dolichol diphosphate. Later, van Schaftingen and Jaeken [1995, FEES Lett. 377, 318-320] demonstrated that the syndrome was due in fact to a phosphomannomutase deficiency, an enzyme which provides the mannose-1-phosphate required for the initial steps of protein glycosylation. This leads to four transferrin isoforms non-glycosylated, glycosylated in Asn-413 or in Asn-611 and in both Asn-413 and 611. 

Lehle, L. Tanner, W. (1976) The Specific Site of Tunicamycin Inhibition in the Formation of Dolichol-bound -Acetylglucosamine Derivatives , FEBS Letters, 71,167-70 Lehle, L. Tanner, W. (1978) Biosynthesis and Characterization of Large Dolichol Diphosphate-linked Oligosaccharides in Saccharomyces cerevisiad, Biochimica et Biophysica Acta, 539, 218-29... 

Transfer to protein. The first dolichol diphosphate oligosaccharide which was found to act as donor of oligosaccharide to protein was the glucose labelled compound (Parodi et al., 1972). The enzyme as well as the acceptor protein was found in the microsomal fraction of liver. The reaction requires manganese ions. 

Transfer from dolichol diphosphate-mannose labelled oligosaccharide to protein has been studied with enzymes from liver (Behrens et al., 1973), hen oviduct (Lucas et al., 1975) and myeloma cells (Hsu et al., 1974). The results were similar to those described before in that not one but many proteins seem to become labelled. In the case of hen oviduct which produces mainly ovalbumin only less than 10% of the label was recovered in ovalbumin (Lucas et al., 1975). For reviews on the subject see Behrens (1974), Lennarz (1975), Parodi Leloir (1975) and Waechter Lennarz (1976). 

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