Biosynthesis of lipid-linked oligosaccharides in cotton fibers. Stimulation by lipids from pig liver.

An acidic lipid fraction isolated from pig liver (Forsee, W. T. & Elbein, A.D. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2574-2578) stimulated the incorporation of mannose from GDP-[14C]mannose into lipid-linked oligosaccharides using a particulate enzyme fraction from maturing cotton bolls. This lipid fraction did not stimulate mannose incorporation into the mannosylphosphorylpolyprenol. The 14C-oligosaccharides, formed in the presence or absence of the pig liver "acceptor" lipid, were released from the lipid-linked oligosaccharides by mild acid hydrolysis and were isolated by paper chromatography. Both sets of 14C-oligosaccharides had similar mobilities suggesting that they were qualitatively similar, except that those formed in the presence of "acceptor" lipid had much more radioactivity. The individual oligosaccharides were purified on a calibrated column of Sephadex G-25 and were then subjected to various treatments to obtain information about their structures. The molecular weights of the larger oligosaccharides ranged from about L1210 to 1720 indicating they probably contained from 6 to 10 sugar residues. Strong acid hydrolysis of the oligosaccharide with a molecular weight of 1350, followed by reduction with NaB3H4, gave a ratio of [3H]hexitol to [3H]hexosaminitol that was compatible with the molecular weight (about 5:2). However, the hexitol fraction contained small amounts of [3H]glucitol in addition to [3H]mannitol. Thus, this oligosaccharide may contain small amounts of glucose in addition to mannose and GlcNAc. When the smaller oligosaccharides (i.e. those having 5 or 6 sugars) were treated with alpha-mannosidase, essentially of all the 14C was released as mannose, but only 30 to 50% of the radioactivity could be released from the larger oligosaccharides by this enzyme. Acetolysis of the oligosaccharide with a molecular weight of 1720 released the radioactivity as mannobiose and mannotriose suggesting that these oligosaccharides contain di- and trisaccharide branches linked to the main chain in 1 leads to 6 linkages.