Incubation of SV40-transformed human lung fibroblasts with [3H]glucosamine for 1 h. followed by chloroform:methanol extraction and thin layer chromatographic analysis, revealed the presence of a major radioactive lipid that was isolated and characterized as GIcUA-(1 leads to 4)-GlcNAc-P-P-dolichol. An identical lipid was formed in smaller quantities under similar incubation conditions in several fibroblastic lines, HeLa cells, and in mouse L cells. Rat lung microsomal preparations catalyze the synthesis of the disaccharide lipid in the following sequence of reactions: UDP-[3H]GlcNAc + dolichol-P leads to [3H]GlcNAc-P-P-dolichol (1) [3H]GlcNAc-P-P-dolichol + UDP-[14C]GlcUA leads to [14C]GlcUA-[3H]GlcNAc-P-P-dolichol (2) The double-labeled lipid was identical to the lipid isolated from SV40-transformed fibroblasts with regard to its behavior on thin layer and silicic acid chromatography. Further, the double-labeled disaccharide released from the lipid by mild acid hydrolysis was identical to GlcUA-(1 leads to 4)-GlcNAc in its chromatographic and electrophoretic behavior and in its composition. The occurrence of a polyprenol derivative of GlcUA-(1 leads to 4)-GlcNAc suggests a possible role for this lipid in the biosynthesis of the repeating disaccharide units of proteoglycans, such as heparin.

Animals↗

PubMed1977

Proposal for a common oligosaccharide intermediate in the synthesis of membrane glycoproteins.

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Acetylglucosaminidase↗

PubMed1977

Comparative rates of transfer of lipid-linked oligosaccharides to endogenous glycoprotein acceptors in vitro.

We have shown previously that particulate preparations of Nil 8 fibroblasts catalyze the synthesis of two oligosaccharide-lipids: one composed of N-acetylglucosamine and mannose residues and the other containing, in addition to mannose and N-acetylglucosamine, one or two glucose residues. These two oligosaccharide-lipids were purified and added to fresh microsomal preparations. In comparative studies, we find that the glucose-containing lipid-linked oligosaccharide is transferred much more rapidly to endogenous protein acceptors than the glucose-free compound. With materials of comparable specific activities, as much as 41% of the glucose-containing oligosaccharide was transferred to protein as compared to 5% for the glucose-free compound. These results suggest that the attachment of glucose to mannosyl lipid-linked oligosaccharide serves an important role in the transfer of these compounds from lipid carrier to protein acceptor.

Cells, Cultured↗

PubMed1977