Mannosyl dolichyl phosphate

From these results it would appear that some mannosyl transfers take place from dolichyl mannosyl phosphate, with some from GDPMan, to generate 1,6 branches. The enzyme partially characterised by Forsee et al. (1977) is involved in transfers from dolichyl mannosyl phosphate. Jensen and Schutzbach (1982) have recently shown that the second mannosyl tranferase has a requirement for phospholipid. 

In a companion paper to that of Chen et al (1975), Pless and Lennarz (1975) examined the glycoproteins, labelled from GDP[ " C]Man and the " C-labelled oligosaccharide attached to dolichyl pyrophosphate, by means of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. They found that dolichyl mannosyl phosphate was a precursor of label in a glycoprotein with an apparent molecular weight of 25,000 daltons, while about two-thirds of the label from GDPMan entered this glycoprotein plus two other large ones (of 55,000 and 75,000 daltons). All the acceptor proteins appeared to be membrane-bound. 

The transfer of mannose to phenyl phosphate was probably mediated by the same enzyme that synthesised dolichyl mannosyl phosphate. It showed similar ionic requirements, subcellular distribution and thermosensitivity. Phenyl phosphate and dolichyl phosphate showed apparent competition, as if for one enzyme. However, the reverse reaction, to synthesise GDPMan from phenyl mannosyl phosphate, could not be demonstrated, unlike the case with dolichyl mannosyl phosphate. Phenyl mannosyl phosphate was water soluble and, unlike dolichyl phosphate, was not retained in the microsomal membranes. Thus phenyl phosphate might trap mannose both by directing it, irreversibly, into the synthesis of a non-metabolite and by draining it away from microsomal membranes into the incubation medium. In the microsomal system it was shown to inhibit the transfer of mannose from dolichyl mannosyl phosphate to p-nitrophenyl a-D-mannoside, in a manner which depended on the concentration of phenyl phosphate. A study of mannosyl transfer to larger oligosaccharides on dolichyl pyrophosphate is needed and detailed kinetic analysis will be required to determine whether the inhibitor exerts its main... 

Martin and Thorne (1974) showed that microsomes from rat liver could synthesise both retinyl mannosyl phosphate and dolichyl mannosyl phosphate in vitro and with comparable efficiency, and Rosso et al (1975) found that retinol phosphate would stimulate retinyl mannosyl phosphate synthesis from GDP [ Man in a similar system, if ATP,Mn and lipid extract were added. 

In summary, retinyl mannosyl phosphate has the P-anomeric configuration, like the dolichyl mannosyl phosphate, since it yielded mannose-2-phosphate upon mild alkaline cleavage (Rosso et al, 1976). Its properties... 

A possible transfer of mannose from retinyl mannosyl phosphate to polypeptide was first indicated by the studies of Maestri and De Luca (1973), though these did not prove that retinyl mannosyl phosphate was the mannosyl donor. In a more detailed analysis Rosso etaL (1977) used a radioactive label largely free of dolichyl mannosyl phosphate in a cell-free system and obtained a clear-cut incorporation of mannose from retinyl mannosyl phosphate into a substance with the solubility properties of protein and which was labile to proteolysis. Some free mannose, its 1-phosphate and retinyl mannosyl phosphate were also present at the end of the incubation, but no large mannolipid was detected. Dolichy CJmannosyl phosphate did not give any transfer of [ C] mannose to protein and further support for a distinctive retinol pathway came from the effects of EDTA. This totally failed to block transfer from retinyl [ mannosyl phosphate, under conditions where block... 

This enzyme [EC 2.4.1.83] which catalyzes the following reaction GDP-mannose + dolichyl phosphate to yield GDP -h dolichyl D-mannosyl phosphate. 

Amphomycin specifically inhibits the formation of dolichyl D-mannosyl phosphate in pig aorta microsomal preparations, but does not inhibit the further transfer of b-mannose from dolichyl D-mannosyl phosphate or from lipid-linked oligosaccharides. However, some of the D-mannosyl residues in the lipid-linked oligosaccharides come directly from GDP-D-mannose without the involvement of dolichyl D-mannosyl phosphate. 

The biosynthesis and functions of the membrane glycoproteins of eukaryotic cells have been reviewed. The general features of the biosynthesis of these glycoproteins closely resemble those involved in the biosynthesis of complex bacterial glycans, especially the O-antigen of lipopolysaccharides. Syntheses of polyisoprenyl jS-o-mannopyranosyl phosphate and P -di-A-acetylchitobiosyl P -dolichyl pyrophosphate have been reported. A preparation from human-lymphocyte membranes was able to transfer D-mannose from GPD-o-mannose to the chitobiosyl derivative, whereas dolichyl D-mannosyl phosphate did not act as a D-mannosyl donor. It appears that D-mannosyl residues at various positions of the glycoprotein have different anomeric configurations, so that different D-mannosyl donors are required in their synthesis. 

Membrane preparations from the alga Prototheca zopfii incorporate D-mannose and 2-acetamido-2-deoxy-D-glucose from their respective sugar nucleotides into acid labile glycolipids whose properties are consistent with those of dolichyl D-mannopyranosyl phosphate. The intermediate appears to be involved in the formation of glycoproteins containing high proportions of D-mannosyl residues of the type present in more-evolved eukaryotes. 

The elucidation of the mechanism by which the trisaccharide derivative Dol.PP.aGlcNAc.pGlcNAc.pMan is synthesised was, in some respects, rather more difficult, since it was not immediately obvious what the mannosyl donor might be. The existence of Dol.P.Man was already known from the studies of Behrens etal (1971) in liver, while GDPMan had long been widely implicated in mannosyl transfers of various kinds. In 1974, Wedgewood etal incubated GDPMan, dolichyl phosphate and a membrane preparation... 

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