Accumulation of a polyisoprene-linked amino sugar in polymyxin-resistant Salmonella typhimurium and Escherichia coli: structural characterization and transfer to lipid A in the periplasm.

Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A. We now report the purification of this lipid from a pss(-) pmrA(C) mutant of E. coli and assign its structure as undecaprenyl phosphate-alpha-l-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M - H](-) at m/z 977.5, consistent with undecaprenyl phosphate-alpha-l-Ara4N (M(r) = 978.41). (31)P NMR spectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit. One- and two-dimensional (1)H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IV(A) to lipid II(A), which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-alpha-l-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.

An Escherichia coli mutant defective in lipid export.

Escherichia coli phospholipids and lipopolysaccharide, made on the inner surface of the inner membrane, are rapidly transported to the outer membrane by mechanisms that are not well characterized. We now report a temperature-sensitive mutant (WD2) with an A270T substitution in a trans-membrane region of the ABC transporter MsbA. As shown by (32)P(i) and (14)C-acetate labeling, export of all major lipids to the outer membrane is inhibited by approximately 90% in WD2 after 30 min at 44 degrees C. Transport of newly synthesized proteins is not impaired. Electron microscopy shows reduplicated inner membranes in WD2 at 44 degrees C, consistent with a key role for MsbA in lipid trafficking.

A water-soluble analogue of glucosaminylphosphatidylinositol distinguishes two activities that palmitoylate inositol on GPI anchors.

2-Palmitoylation of the inositol residue occurs during biosynthesis of glycosylphosphatidylinositol (GPI) anchors, but the enzymology of this step has been enigmatic. With endogenously synthesized glucosamine-PI (GlcN-PI; a GPI intermediate), a CoA-dependent palmitoyl-CoA-independent acyltransfer activity (AT-1) has been reported in rodent preparations. In contrast, a palmitoyl-CoA-dependent GlcN-PI acyltransferase activity (AT-2) was reported in both rodent and yeast preparations with a novel water-soluble dioctanoyl GlcN-PI analogue, GlcN-PI(C8). We report that AT-1, as well as AT-2, can be detected in rodent microsomes with GlcN-PI(C8), thus demonstrating the coexistence of these activities in a single membrane preparation and the general utility of GlcN-PI(C8) for studying the GPI pathway. Unexpectedly, AT-2 was peripherally associated with microsomes, a property atypical for GPI biosynthetic enzymes.

Regulation of the dolichol pathway in human fibroblasts by the endoplasmic reticulum unfolded protein response.

Accumulation of unfolded proteins within the endoplasmic reticulum (ER) of eukaryotic cells triggers the unfolded protein response (UPR), which activates transcription of several genes encoding ER chaperones and folding enzymes. This study reports that conversion of dolichol-linked Man(2-5)GlcNAc(2) intermediates into mature Glc(3)Man(9)GlcNAc(2) oligosaccharides in primary human adult dermal fibroblasts is also stimulated by the UPR. This stimulation was not evident in several immortal cell lines and did not require a cytoplasmic stress response. Inhibition of dolichol-linked Glc(3)Man(9)GlcNAc(2) synthesis by glucose deprivation could be counteracted by the UPR, improving the transfer of Glc(3)Man(9)GlcNAc(2) to asparagine residues on nascent polypeptides. Glycosidic processing of asparagine-linked Glc(3)Man(9)GlcNAc(2) in the ER leads to the production of monoglucosylated oligosaccharides that promote interaction with the lectin chaperones calreticulin and calnexin. Thus, control of the dolichol-linked Glc(3)Man(9)GlcNAc(2) supply gives the UPR the potential to maintain efficient protein folding in the ER without new synthesis of chaperones or folding enzymes.

Acylation of glucosaminyl phosphatidylinositol revisited. Palmitoyl-CoA dependent palmitoylation of the inositol residue of a synthetic dioctanoyl glucosaminyl phosphatidylinositol by hamster membranes permits efficient mannosylation of the glucosamine residue.

Two critical steps in the assembly of yeast and mammalian glycosylphosphatidylinositol (GPI) anchor precursors are palmitoylation of the inositol residue and mannosylation of the glucosamine residue of the glucosaminyl phosphatidylinositol (GlcNalpha-PI) intermediate. Palmitoylation has been reported to be acyl-CoA dependent in yeast membranes (Costello, L. C., and Orlean, P. (1992) J. Biol. Chem. 267, 8599-8603) but strictly acyl-CoA independent in rodent membranes (Stevens, V. L., and Zhang, H. (1994) J. Biol. Chem. 269, 31397-31403), and thus poorly conserved. In addition, it was suggested that acylation must precede mannosylation in both yeast (Costello, L. C., and Orlean, P. (1992) J. Biol. Chem. 276, 8599-8603) and rodent (Urakaze, M., Kamitani, T., DeGasperi, R., Sugiyama, E., Chang, H.-M., Warren, C. D., and Yeh, E. T. H. (1992) J. Biol. Chem. 267, 6459-6462) cells because GlcNalpha-acyl-PI accumulates in vivo when mannosylation is blocked. However, GlcNalpha-acyl-PI accumulation would also be expected if mannosylation and acylation were independent of each other. These issues were addressed by the use of a synthetic dioctanoyl GlcNalpha-PI analogue (GlcNalpha-PI(C8)) as an in vitro substrate for GPI-synthesizing enzymes in Chinese hamster ovary cell membranes. GlcNalpha-PI(C8) was acylated in an manner requiring acyl-CoA. Thus, the process involving acyl-CoA reported for yeast has been conserved in mammals. Furthermore, both GlcNalpha-PI(C8) and GlcNalpha-acyl-PI(C8) could be mannosylated in vitro, but mannosylation of the latter was significantly more efficient. This provides direct support for the earlier suggestion that acylation precedes mannosylation in rodents cells. A similar result was also observed with the Saccharomyces cerevisiae mannosyltransferase. In contrast, it has been reported that mannosylation of endogenous GlcNalpha-PI by Trypansoma brucei membranes occurs without prior acylation. The same result was obtained with GlcNalpha-PI(C8), confirming that the mannosyltransferase of trypanosomes is divergent from those in yeasts and rodents.

Endotoxin and cytokines increase hepatic messenger RNA levels and serum concentrations of apolipoprotein J (clusterin) in Syrian hamsters.

Infection and inflammation induce alterations in hepatic synthesis and plasma concentrations of the acute phase proteins. Our results show that apolipoprotein (apo) J is a positive acute phase protein. Endotoxin (LPS), tumor necrosis factor (TNF), and interleukin (IL)-1 increased hepatic mRNA and serum protein levels of apo J in Syrian hamsters. Hepatic apo J mRNA levels increased 10- to 15-fold with doses of LPS from 0.1 to 100 micrograms/100 g body weight within 4 h and were elevated for > or = 24 h. Serum apo J concentrations were significantly increased by 16 h and further elevated to 3.3 times that of control, 24 h after LPS administration. Serum apo J was associated with high density lipoprotein and increased fivefold in this fraction, after LPS administration. Hepatic apo J mRNA levels increased 3.5- and 4.6-fold, with TNF and IL-1, respectively, and 8.2-fold with a combination of TNF and IL-1. Serum apo J concentrations were increased 2.3-fold by TNF, 79% by IL-1, and 2.9-fold with a combination of TNF and IL-1. These results demonstrate that apo J is a positive acute phase protein.

Tumor necrosis factor acutely increases plasma levels of very low density lipoproteins of normal size and composition.

Tumor necrosis factor (TNF) has been proposed to mediate the hypertriglyceridemic response to infection by either increasing hepatic lipid synthesis or decreasing clearance of triglyceride-rich particles through inhibition of lipoprotein lipase. We demonstrate that within 90 min of administration of recombinant human TNF alpha to rats there is a rapid increase in plasma levels of very low density lipoproteins (VLDL) of normal ultracentrifugal flotation rate, apoprotein and lipid composition, and particle size, as assessed by nondenaturing gradient gel electrophoresis. Clearance of radioiodinated VLDL 90 min after TNF administration did not differ from that in control animals, consistent with other observations that increases in plasma triglyceride concentrations after TNF precede detectable reductions in tissue lipoprotein lipase activity. Between 90 min and 16 h after TNF, VLDL levels decline, and there are increases in intermediate (IDL) and low (LDL) density lipoproteins consistent with lipolytic processing of VLDL, although the findings are also compatible with direct secretion of IDL and LDL subspecies. By 16 h, there is a 50% increase in protein mass of LDL of normal composition and subspecies distribution, as assessed by nondenaturing gradient gel electrophoresis. These data suggest that the initial locus of TNF's metabolic effects is the liver, and the resulting increases in secretion and metabolic processing of VLDL may represent an early manifestation of the acute phase response.