Lipid-linked

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... 

Acid-labile linkages of carbohydrates are commonly those of furano-sidic sugar residues or of deoxy monosaccharides.50,63 Consequently, neuraminic acid and fucose residues, which occur as terminal monosaccharides of protein- and lipid-linked glycans, are removed by hydrolysis with a dilute acid. The linkages formed by these two sugar residues can conveniently be differentiated by subjecting a portion of... 

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. 

The dolichol-linked D-mannosyl, D-glucosyl, and 2-acetamido-2-deoxy-D-glucosyl residues are used in the glycosylation of proteins and in the biosynthesis of D-m an nan.2,35 GlcNAc-PP-Dol may also play a role in the synthesis of glycosaminoglycans. Reaction 4 is the first step in the formation of lipid-linked precursors for cellulose.46... 

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... 

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