Membrane-derived Oligosaccharides Research Articles

Diglyceride Kinase Mutants of Escherichia coli : Inner Membrane Association of 1,2-Diglyceride and Its Relation to Synthesis of Membrane-Derived Oligosaccharides

Mutants of Escherichia coli defective in diglyceride kinase contain 10 to 20 times more sn-1,2-diglyceride than normal cells. This material constitutes about 8% of the total lipid in such strains. We now report that this excess diglyceride is recovered in the particulate fraction, primarily in association with the inner, cytoplasmic membrane. The diglyceride kinase of wild-type cells was recovered in the same inner membrane fractions. The conditions employed for the preparation of the membranes did not appear to cause significant redistribution of lipids and proteins. The biochemical reactions leading to the formation of diglyceride in E. coli are not known. To determine whether diglyceride formation requires concurrent synthesis of the membrane-derived oligosaccharides (H. Schulman and E. P. Kennedy, J. Biol. Chem. 252:4250-4255, 1977), we have constructed a double mutant defective in both the kinase (dgk) and phosphoglucose isomerase (pgi). When oligosaccharide synthesis was inhibited in this organism by growing the cells on amino acids as the sole carbon source, the diglyceride was no longer present in large amounts. When glucose was also added to the medium, the pgi mutation was bypassed, oligosaccharide synthesis resumed, and diglyceride again accumulated. These findings suggest that diglyceride may arise during the transfer of the sn-glycero-1-P moiety from phosphatidylglycerol (and possibly cardiolipin) to the oligosaccharides. In wild-type cells the kinase permits the cyclical reutilization of diglyceride molecules for phospholipid biosynthesis.

Localization of membrane-derived oligosaccharides in the outer envelope of Escherichia coli and their occurrence in other Gram-negative bacteria.

The glucose-containing, membrane-derived oligosaccharides of Escherichia coli are localized in the external envelope of that organism, most probably in the periplasmic space. The membrane-derived oligosaccharides appear to be generally occurring cell constituents of gram-negative (but not gram-positive) bacteria.

A novel phosphodiesterase from Aspergillus niger and its application to the study of membrane-derived oligosaccharides and other glycerol-containing biopolymers

A novel phosphodiesterase has been found in commercially available extracts of Aspergillus niger and has been partially purified by fractionation with acetone and chromatography on carboxymethylcellulose. The enzyme attacks glycerophosphodiester bonds with the liberation of free glycerol only. The synthetic substrate glucose 6-phospho-sn-1'(3')-glycerol is hydrolyzed with production of equivalent amounts of free glycerol and glucose 6-phosphate. Similarly, the enzymic hydrolysis of sn-glycero-3-phosphocholine liberates glycerol and phosphocholine. The hydrophilic head groups of membrane phospholipids of Escherichia coli are continuously transferred to a closely related family of oligosaccharides ("membrane-derived oligosaccharides") containing glucose as the sole sugar (van Golde, L. M. G., Schulman, H., and Kennedy, E. P. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1368--1372). Oligosaccharide A-2 contains sn-1-glycerophosphate residues (derived from phosphatidylglycerol) in phosphodiester linkage. Treatment of this oligosaccharide with the phosphodiesterase led to the liberation of nearly all of the glycerol as free glycerol. Subsequent partial acid hydrolysis of the enzyme-treated oligosaccharide led to the recovery of glucose 6-phosphate in almost quantitative yield. The sn-1-glycerophosphate residues are therefore linked to position 6 of glucose units of the oligosaccharide. The activity of the enzyme is not restricted to glycerophosphodiesterases since it will hydrolyze phosphodiesters containing other polyols such as the synthetically prepared glucose 6-phospho-DL-1'(2'-hydroxy-3'-ethoxy)propane.

Identification of UDP-glucose as an intermediate in the biosynthesis of the membrane-derived oligosaccharides of Escherichia coli.

The membrane-derived oligosaccharides of Escherichia coli constitute a closely related family of oligosaccharides containing approximately 9 glucose units variously substituted with sn-glycero-1-phosphate and phosphoethanolamine residues derived from the head groups of membrane phospholipids, and also with succinate in O-ester linkage (Kennedy, E.P., Rumley, M.K., Schulman, H., and van Golder, L.M.G. (1976) J. Biol. Chem. 251, 4208-4213). Studies with mutant strains defective in the synthesis of various nucleoside diphosphate sugars have now revealed that UDP-glucose is an essential intermediate in the biosynthesis of these oligosaccharides. Mutants unable to synthesize UDP-glucose do not contain significant amounts of the membrane-derived oligosaccharides. In contrast, a strain unable to synthesize ADP-glucose, the glucosyl donor for glycogen synthesis in E. coli, contained normal amounts of the membrane-derived oligosaccharides, although with a somewhat different pattern of distribution of the various subspecies. In confirmation of these genetic studies, pulse-label isotope tracer studies have been carried out with glucose of high specific activity, under conditions in which UDP-glucose comprises a large fraction of the total radioactivity in the low molecular weight pool. Subsequent "chase" experiments clearly revealed the conversion of UDP-glucose to the higher molecular weight membrane-derived oligosaccharides.

Relation of turnover of membrane phospholipids to synthesis of membrane-derived oligosaccharides of Escherichia coli.

Relation of turnover of membrane phospholipids to synthesis of membrane-derived oligosaccharides of Escherichia coli.

Identification of sn-glycero-1-phosphate and phosphoethanolamine residues linked to the membrane-derived Oligosaccharides of Escherichia coli.

A previous report from this laboratory (van Golde, L.M.G., Schulman, H., and Kennedy, E.P. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1368-1372) described the discovery in Escherichia coli of a novel class of oligosaccharides, containing glucose as the sole sugar, substituted with glycerophosphate units derived from membrane phospholipids, and with succinic acid in O-ester linkage. These membrane-derived oligosaccharides, comprising about 0.5 to 1.0% of the dry weight of E. coli, represent a family of closely related oligosaccharides that may be subfractionated on anion exchange resins. The present paper describes studies of the oligosaccharide A-1 described by van Golde et al. in the previous report. The glycerophosphate linked to the oligosaccharide in phosphodiester bond is the sn-glycero-1-P enantiomer. This finding strongly supports the previous conclusion that the oligosaccharides are the acceptors of the polar headgroups of membrane phospholipids, since the unesterified glycerophosphate of phosphatidyl glycerol is an sn-glycero-1-P residue, otherwise rare in nature. The glycerophosphate residues in the membrane-derived oligosaccharide are not substituted in the sn-2 or sn-3 positions, since they are readily oxidized by periodate under mild conditions. Alkaline hydrolysis liberates glycerophosphate, and only negligible amounts of free glycerol, consistent with the view that the glycerophosphate residues are linked to glucose units through position 6, unfavorable for the formation of glucose cyclic phosphate intermediates that would eliminate free glycerol. Oligosaccharide A-1 (but not Fraction A-2) contains phosphoethanolamine residues equivalent to 30 to 40% of the total phosphorus. The phosphoethanolamine residues are linked to position 6 of glucose units, as proved by the isolation of glucose 6-phosphate as a product of partial acid hydrolysis.