Lipid-linked oligosaccharide

The partially methylated monosaccharides obtained on depolymerization of the permethylated sample are preferably analyzed as acetates by g.l.c.-m.s., as shown by Bjomdal and coworkers.41,42 The neutral sugars and the amino sugars obtained in acetolysis-acid hydrolysis are reduced, and acetylated for the analysis, and the amino-hexitol and the neuraminic acid residues are acetylated after methanolysis. Identification with the aid of g.l.c.-m.s. has been described for all of the common components of protein- and lipid-linked glycans and oligosaccharides from animal cells, namely, the neutral sugars,41-43 hexitols,44 hexosamines,29,43,45,46 aminohexitols,31,32 and neuraminic acids.33,34,47... 

Glycolipid 3-ce-mannosyltransferase [EC 2.4.1.132], also known as mannosyltransferase II, catalyzes the transfer of an ce-D-mannosyl residue from GDP-mannose into a lipid-linked oligosaccharide, forming an o -l,3-D-manno-syl-D-mannose linkage. The 1,3-linked mannosyl residue in the mammalian lipid-linked oligosaccharide of the structure Glc3Man9GlcNAc2 is produced by this enzyme. 

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. 

Assembly of the Lipid-linked Oligosaccharide, and its Transfer to Protein... 

Still another approach to solving the problem of detecting intenne-diates in the assembly of the lipid-linked oligosaccharide comes from the use of cell mutants having defects in the synthesis of an essential intermediate. This approach has proved productive (see later). 

That some cell-free systems were incomplete became evident when the role of UDP-Glc was appreciated (see Refs. 127-135). Only in the presence of UDP-Glc (in addition to UDP-GlcNAc and GDP-Man, when no endogenous acceptors are present) was a lipid-linked oligosaccharide obtained that had the same chemical properties as the D-... 

As already indicated, control points in the regulation of the rate of glyeosylation of proteins may be (a) the availability of Dol-P (Section 11,1,a), (b) the D-glucosylation of the lipid-linked oligosaccharide, or its transfer to protein, or both (Section 1,2,a), and, as already discussed, (c) the metabolic fate of GDP-Man. Furthermore, GDP-Man inhibits fonnation of Glc-P-Dol in cell-free preparations from liver,157 and activates formation of GlcNAe-PP-Dol in cell-free preparations from tissues of chick embryo.156... 

Other mutants of Chinese-hamster ovary-cells having decreased amounts of lipid-linked oligosaccharides are known, although they are less well characterized. G-Protein of vesicular stomatitis virus grown in one of these mutants appeared to contain fewer, but full-sized rather than truncated, oligosaccharide side-chains. There may be insufficient amounts of oligosaccharide precursor available for transfer to nascent glycoproteins.172... 

It is clear that mutants of the lipid pathway are useful in elucidating the individual steps in the assembly (see Section II,2,a) and transfer of the lipid-linked oligosaccharides, and possibly will become important in future work on the synthesis of lipid-linked oligosaccharides. 

D-Mannosylation of lipid-linked oligosaccharides and of proteins has been found to occur in rough, endoplasmic reticulum.188 192 In hen-... 

Thus, the effects of glycosylation inhibitors on intact cells may also be studied best with virus-infected cells. Before release of virus, the glycoproteins are detected in the water-insoluble, membranous fraction. Furthermore, the lipid-linked oligosaccharides may be rather specifically extracted from whole cells, and monosaccharide-lipids may also be determined.3-116 It is thus seen that the various tools of virology and of lipid and carbohydrate biochemistry have proved productive in establishing the mode of action of inhibitors of lipid-depen-dent glycosylation of proteins. 

In contrast to these results, 25-hydroxycholesterol (and also, 20-hy-droxycholesterol, 7-ketocholesterol, and diosgenin) in aortic, smooth-muscle cells effectively blocks the incorporation of acetate into lipid-linked oligosaccharides (and, also, into cholesterol7). Thus, less of the lipid-linked oligosaccharides were available for glycosylation of proteins. In harmony with the presumed, inhibitory mechanism was the observation that incorporation of mevalonate into lipid-linked oligosaccharides was not inhibited, and that mevalonate itself (the product formed by HMG-CoA reductase from HMG-CoA and NADPH) could reverse the inhibition of glycosylation of protein (see Scheme 1). 

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