Polyprenyl glycosyl phosphates

II. Glycosyl Esters of Nucleotides and Polyprenyl Glycosyl Phosphates in Polysaccharide Biosynthesis... 

In abbreviations for polyprenyl glycosyl phosphates, p stands for phosphate, and pp" for diphosphate groups. The abbreviation Bpr means a bacterial undecaprenyl group. For the moraprenyl group (see Ref. 57), the abbreviation Mpr is used, and the symbol Pre stands for any unidentified, lipid acceptor having the properties of polyprenol. 

For the study of polyprenyl glycosyl phosphates as intermediates in the synthesis of complex glycans, several techniques have been developed, and these have been described elsewhere in detail.18 20 Two important features should be emphasized. First, the very small amounts of polyprenyl phosphates that are present in most tissues for this reason, the use of radioactive techniques for the detection of products is obligatory. Second, on account of the hydrophobic nature of these compounds, and as the enzymes involved in the reactions are membrane-bound, the use of detergents and organic solvents becomes necessary. 

It is difficult to identify the lipid moiety as a polyprenol, because of the small amounts present in tissues. Nevertheless, some information can be obtained by using glycosylated lipids labelled in the sugar moiety. The first indication of the presence of polyprenyl glycosyl phosphates comes from study of the properties just mentioned, namely, lability to mild acid, stability to mild alkali, and acidic properties. Information on the polyprenyl nature of the lipid may be obtained by labelling the lipid with radioactive 2,4-dideoxyO-C-methyl-D-g/ycero-pentono-l.S-lactone (mevalonic acid ). 

The ability of nucleoside glycosyl diphosphates to serve as glycosyl phosphate donors in the biosynthesis of polyprenyl glycosyl diphosphates (see Section 11,3) is well documented. It is not yet known whether they may function as immediate precursors of glycosyl phosphate residues not infrequently present as components of bacterial polymers, or whether some other intermediates are involved. 

It seemed of interest to check whether the type of biosynthetic mechanism involved may be related to the structure of the polymeric chain formed. In the block mechanism of chain assembly, formation of polyprenyl glycosyl diphosphates through transfer of glycosyl phosphate is a necessary step of the process. Thus far, only UDP-activated monosaccharides have been found to participate in this reaction (see Section 11,3). Consequently, the presence of a monosaccharide residue of this group in the main chain of a polymer may be regarded as a necessary condition for assembly of the chain through the block mechanism. 

The first stage of the synthesis is the preparation of the substrate for enzymatic polymerization, the polyprenyl pyrophosphate-galactosy1-rhamnosy1-mannose XXI. Since the most convenient way to control the polycondensation reaction is the use of isotopic methods, a procedure for incorporation of tritium into trisaccharide I was developed (2 5). Labelled trisaccharide was then converted into the glycosyl phosphate XIX through interaction of its peracetate III with anhydrous phosphoric acid (26). Conditions were found under which the reaction is accompanied by minimal destruction and yields the Ot-phosphate of the trisaccharide. 

The first evidence showing that plant polyprenols involved in glycosyl-transfer reactions are not allylic, but a-saturated, like animal dolichol (2), came from Pont Lezica and coworkers.23 The authors postulated that the presence of sugar acceptors having the properties of a-saturated polyprenyl phosphates may be a general feature of eukaryotic cells, in contrast to the a-unsaturated polyprenyl phosphates characteristic of prokaryotic cells. 

Further work in plant systems confirmed, by different methods (biosynthesis from mevalonate,27 28 mass spectrometry,33 and the techniques already mentioned30), that the polyprenyl phosphate involved in glycosyl-transfer reactions is dolichyl phosphate. Table II shows different sources from which dolichyl phosphate has been identified in plants. 

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