Life with too much polyprenol: polyprenol reductase deficiency.

Congenital disorders of glycosylation (CDG) are caused by a dysfunction of glycosylation, an essential step in the manufacturing process of glycoproteins. This paper focuses on a 6-year-old patient with a new type of CDG-I caused by a defect of the steroid 5α reductase type 3 gene (SRD5A3). The clinical features were psychomotor retardation, pathological nystagmus, slight muscular hypotonia and microcephaly. SRD5A3 was recently identified encoding the polyprenol reductase, an enzyme catalyzing the final step of the biosynthesis of dolichol, which is required for the assembly of the glycans needed for N-glycosylation. Although an early homozygous stop-codon (c.57G>A [W19X]) with no functional protein was found in the patient, about 70% of transferrin (Tf) was correctly glycosylated. Quantification of dolichol and unreduced polyprenol in the patient's fibroblasts demonstrated a high polyprenol/dolichol ratio with normal amounts of dolichol, indicating that high polyprenol levels might compete with dolichol for the initiation of N-glycan assembly but without supporting normal glycosylation and that there must be an alternative pathway for dolichol biosynthesis.

Lipid IVA inhibits synthesis and release of tumor necrosis factor induced by lipopolysaccharide in human whole blood ex vivo.

Tumor necrosis factor (TNF) released by lipopolysaccharide (LPS)-stimulated mononuclear phagocytes is a critical mediator of sepsis. We examined the capacities of rough mutant Salmonella typhimurium LPS (Rc) and LPS partial structures lipid A, monophosphoryl lipid A (MPLA), lipid IVA, and lipid X to induce production of TNF in whole blood. Rc LPS (0.0001-10 ng/ml) produced a dose-dependent release of TNF as determined by cytotoxicity of actinomycin D-sensitized L929 murine fibroblasts. Lipid A, MPLA, lipid IVA, and lipid X exhibited decreasing capacities to stimulate production of TNF in whole blood, respectively. Fractional deacylation of LPS by incubation with acyloxyacyl hydrolase isolated from human leukocytes produced a reduction in the capacity of LPS to induce TNF release in whole blood. Maximal enzymatic deacylation reduced activity of LPS by greater than 100-fold. Coincubation with lipid IVA inhibited TNF release induced by Rc LPS or lipid A, but not by phorbol ester. In contrast, MPLA, lipid X, and deacylated LPS failed to inhibit LPS-stimulated release of TNF. Corresponding to the inhibition of the release of TNF protein, lipid IVA also inhibited the accumulation of TNF mRNA in LPS-stimulated mononuclear cells. These results suggest that lipid IVA may act as a competitive antagonist of LPS, perhaps at the receptor level.

A left-handed parallel beta helix in the structure of UDP-N-acetylglucosamine acyltransferase.

UDP-N-acetylglucosamine 3-O-acyltransferase (LpxA) catalyzes the transfer of (R)-3-hydroxymyristic acid from its acyl carrier protein thioester to UDP-N-acetylglucosamine. LpxA is the first enzyme in the lipid A biosynthetic pathway and is a target for the design of antibiotics. The x-ray crystal structure of LpxA has been determined to 2.6 angstrom resolution and reveals a domain motif composed of parallel beta strands, termed a left-handed parallel beta helix (L beta H). This unusual fold displays repeated violations of the protein folding constraint requiring right-handed crossover connections between strands of parallel beta sheets and may be present in other enzymes that share amino acid sequence homology to the repeated hexapeptide motif of LpxA.

A phospholipid derivative of cytosine arabinoside and its conversion to phosphatidylinositol by animal tissue.

We have synthesized an analog (ara-CDP-DL-dipalmitin) of cytidine diphosphate diglyceride (CDP-diglyceride) in which the antitumor drug, cytosine arabinoside, is substituted for the cytidine moiety. Enzymes in rat and human liver convert this analog to phosphatidylinositol, thereby releasing cytosine arabinoside-5'-monophosphate, an obligatory intermediate in the activation of cytosine arabinoside. Unlike cytidine diphosphate diglyceride, however, ara-CDP-DL-diapalmitin is not an efficient substrate for phosphatidylglycerophosphate synthesis in liver or phosphatidylserine in Escherichia coli. The antitumor activity of ara-CDP-DL-dipalmitin in mice bearing L5178Y leukemia is described.

Antibacterial agents that inhibit lipid A biosynthesis.

Lipid A constitutes the outer monolayer of the outer membrane of Gram-negative bacteria and is essential for bacterial growth. Synthetic antibacterials were identified that inhibit the second enzyme (a unique deacetylase) of lipid A biosynthesis. The inhibitors are chiral hydroxamic acids bearing certain hydrophobic aromatic moieties. They may bind to a metal in the active site of the deacetylase. The most potent analog (with an inhibition constant of about 50 nM) displayed a minimal inhibitory concentration of about 1 microgram per milliliter against Escherichia coli, caused three logs of bacterial killing in 4 hours, and cured mice infected with a lethal intraperitoneal dose of E. coli.

Biochemical and immunological characterization of mutant L-M cells with altered levels of dibutyryl cyclic AMP-inducible alkaline phosphatase.

Phosphotyrosine-Sepharose 4B was synthesized and used to purify L-cell alkaline phosphatase. Antibodies to this enzyme interacted with the alkaline phosphatase of strains A-1-2 and A-3-3, mutants that express the enzyme constitutively. This and thermal stability studies suggest that these mutants contain the same alkaline phosphatase isozyme as their parent strain.

Antibacterial and anti-inflammatory agents that target endotoxin.

Antibiotic-resistant bacterial infections are a major clinical problem. Lipid A, the active part of lipopolysaccharide endotoxins in Gram-negative bacteria, is an intriguing target for new antibacterial and anti-inflammatory agents. Inhibition of lipid A biosynthesis kills most Gram-negative bacteria, increases bacterial permeability to antibiotics and decreases endotoxin production.

Bacterial polysaccharide synthesis and gene nomenclature.

Gene nomenclature for bacterial surface polysaccharides is complicated by the large number of structures and genes. We propose a scheme applicable to all species that distinguishes different classes of genes, provides a single name for all genes of a given function and greatly facilitates comparative studies.

Association of lipid A disaccharide synthase with aerobic glycerol-3-phosphate dehydrogenase in extracts of Escherichia coli.

Variants of the Escherichia coli UDP-GlcNAc O-acyltransferase (LpxA) and of the lipid A disaccharide synthase (LpxB) containing affinity chromatography tags (C-terminal histidine 8 [H8] tails) were constructed in order to investigate whether or not these enzymes interact with other E. coli proteins. These variants (LpxA-H8 and LpxB-H8) had specific activities in vitro that were similar to wild-type enzymes. Crude extracts made from E. coli cells expressing LpxA-H8 or LpxB-H8 were chromatographed over Ni(2+)-NTA-Agarose, and proteins purifying with the tagged proteins were identified by SDS-PAGE, followed by blotting and N-terminal microsequencing. At high levels of LpxB-H8 expression, two heat-shock proteins (DnaK and GroEL) were associated with the disaccharide synthase, but not with the acyltransferase. Another major protein recovered with LpxB-H8 (both at low and high levels of expression) was the aerobic glycerol-3-phosphate dehydrogenase (GlpD). The latter interaction was specific, since GlpD did not bind the affinity resin when the affinity tag was present on the UDP-GlcNAc O-acyltransferase (LpxA-H8). Velocity centrifugation experiments indicated that both wild-type LpxB and GlpD sedimented together under some conditions, but these aggregates were smaller than and distinct from inner membranes. Our findings suggest a possible new mechanism by which the biosynthetic pathways for lipid A and glycerophospholipids may be coordinated.

Escherichia coli membrane vesicles with elevated phosphatidic acid levels. A detergent-free system for in vitro phospholipid synthesis.

A new method for studying phospholipid biosynthesis in Escherichia coli is described. The method makes use of the previously reported observation that E. coli cytidine auxotrophs accumulate phosphatidic acid when starved of cytidine (Ganong, B. and Raetz, C.R.H. (1982) J. Biol. Chem. 257, 389-394). We now show that phosphatidic acid that accumulates in these cells is competent for further biosynthetic use in vivo, if cytidine is re-supplied to the cells. Furthermore, phosphatidic acid-rich membranes prepared from such cells can be used for in situ assays of the later steps of phospholipid biosynthesis. Since this system does not require detergent, our in situ assays more accurately reflect the conditions of an intact membrane. We have used this system to probe the regulation of the branch-point of the biosynthetic pathway for phospholipid polar headgroups. Phosphatidic acid-rich membranes prepared from cells that overproduce either phosphatidylserine synthase or phosphatidylglycerolphosphate synthase do not have increased rates of lipid synthesis in our in situ assays. This correlates with synthetic rates measured in vivo and, thus, our in situ assays accurately reflect conditions in a growing cell's membrane.

Signal transduction triggered by lipid A-like molecules in 70Z/3 pre-B lymphocyte tumor cells.

The lipid A (endotoxin) moiety of lipopolysaccharide (LPS) elicits rapid cellular responses from many cell types, including macrophages, lymphocytes, and monocytes. In CD14 transfected 70Z/3 pre-B lymphocyte tumor cells, these responses include activation of the MAP kinase homolog, p38, activation of NF-kappaB, and transcription of kappa light chains, leading to the assembly of surface IgM. In this work, we explored the specificity of the response with regard to lipid structure, and the requirement for p38 kinase activity prior to NF-kappaB activation in control and CD14 transfected 70Z/3 (CD14-70Z/3) cells. A p38-specific inhibitor, SB203580, was used to block p38 kinase activity in cells. CD14-70Z/3 cells were incubated with 1-50 microM SB203580, and then stimulated with LPS. Nuclear extracts were prepared, and NF-kappaB activation was measured using an electrophoretic mobility shift assay. SB203580 did not inhibit LPS induced NF-kappaB activation. In addition, LPS failed to activate p38 tyrosine phosphorylation in 70Z/3 cells lacking CD14, in spite of rapid NF-kappaB activation and robust surface IgM production with appropriate higher doses of LPS. LPS stimulation of p38 phosphorylation, NF-kappaB activation, and surface IgM expression were all blocked completely by lipid A-like endotoxin antagonists whether or not CD14 was present. Acidic glycerophospholipids and ceramides did not mimic lipid A-like molecules either as agonists or antagonists in this system. Our data support the hypothesis that lipid A-mediated activation of cells requires stimulation of a putative lipid A sensor that is downstream of CD14, but upstream of p38 and NF-kappaB.

A rapid selection for animal cell mutants with defective peroxisomes.

Chinese hamster ovary (CHO) cells take up and incorporate 9-(1'-pyrene)nonanol (P9OH) into phospholipids and neutral lipids. Exposure of P9OH-labeled cells to long wavelength ultraviolet (UV) light causes cell death, because excitation of the pyrene moiety generates reactive oxygen species. CHO mutant cells deficient in plasmalogen biosynthesis and peroxisome assembly (Zoeller, R.A. and Raetz, C.R.H. (1986) Proc. Natl. Acad. Sci. USA 83, 5170-5174) are much more resistant to P9OH/UV treatment than are wild-type cells. This phenotype is explained by a 7.5-fold reduction of P9OH incorporation into the ethanolamine-linked phospholipids in the mutant cells and 2.4- to 6-fold reduction of P9OH incorporation into all other phospholipids and triglycerides, suggesting a general defect in fatty alcohol metabolism. [U-14C]Hexadecanol incorporation into the phospholipids of the mutant cells is also impaired. In contrast, the fatty acid analog, 9-(1'-pyrene)nonanoic acid, is incorporated into cells two times more rapidly by the mutants than by the wild type. Resistance to P9OH/UV treatment affords a simple, new method for the selection of animal cell mutants defective in peroxisome biogenesis.

Increased cholesterol synthesis in Chinese hamster ovary cells deficient in peroxisomes.

In a previous study we have shown that Chinese hamster ovary (CHO) cells deficient in intact peroxisomes, lack the nonspecific lipid transfer protein (nsL-TP; sterol carrier protein 2) (van Heusden, G.P.H., Bos, K., Raetz, C.R.H. and Wirtz, K.W.A. (1990) J. Biol. Chem. 265, 4105-4110). The consequences of the absence of peroxisomes and of nsL-TP on intracellular cholesterol metabolism have been investigated in two peroxisome-deficient CHO cell lines (CHO-82 and CHO-78). Compared with wild-type cells (CHO-K1), the incorporation of [3H]acetate into cholesterol was 3-fold higher in the CHO-82 cells and 2-fold higher in the CHO-78 cells. In agreement with an increased synthesis of cholesterol, a 2-3-fold higher 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was measured in both mutant cell lines. On the other hand, addition of low density lipoprotein (LDL), mevalonate (30 mM) or 25-hydroxycholesterol (2 micrograms/ml) to cells grown in lipoprotein-deficient serum, demonstrated that in both mutant cell lines the down-regulation of HMG-CoA reductase and of cholesterol synthesis were comparable to that in wild-type cells. These results strongly suggest that, in addition to down-regulation by LDL-derived cholesterol, mevalonate and 25-hydroxycholesterol, HMG-CoA reductase activity is under control of peroxisomes and/or nsL-TP.

Regulated covalent modifications of lipid A.

Regulated covalent modifications of lipid A are implicated in virulence of pathogenic Gram-negative bacteria. The Salmonella PhoP/PhoQ-activated gene pagP is required for resistance to cationic antimicrobial peptides and for biosynthesis of hepta-acylated lipid A species containing palmitate. Interestingly, pagP encodes an unusual enzyme of lipid A biosynthesis localized in the outer membrane, whereas all previously characterized lipid A enzymes are cytoplasmic or associated with the inner membrane. PagP is not unique, however, as pagL encodes another outer membrane enzyme in Salmonella that deacylates the 3 position of lipid A.S. typhimurium also synthesizes S-2-hydroxymyristate modified lipid A in a PhoP/PhoQ-dependent manner. We postulated that 2-hydroxylation might be catalyzed by a novel dioxygenase. Using well-characterized dioxygenase sequences as probes, tBLASTn searches revealed unassigned open reading frame(s) with similarity to mammalian aspartyl beta-hydroxylases in bacteria known to make 2-hydroxyacylated lipid A. The S. typhimurium aspartyl beta-hydroxylase homologue (lpxO) was cloned and expressed in Escherichia coli K-12, which does not contain lpxO. Analysis of the resulting construct revealed that lpxO expression induces O(2)-dependent formation of 2-hydroxymyristate-modified lipid A in E. coli. LpxO may be an inner membrane enzyme that catalyzes Fe(2+)/ascorbate/alpha-ketoglutarate dependent hydroxylation of lipid A. We propose that 2-hydroxymyristate released from LPS inside infected animal cells might be converted to 2-hydroxymyristoyl coenzyme A, a potent inhibitor of protein N-myristoyl transferase.

Pulmonary pressor responses in sheep to chemically defined precursors of E. coli endotoxin.

The toxicity of various monosaccharide and disaccharide endotoxin precursors has now been studied in sheep. We measured the early pulmonary arterial pressure responses after injections of the monosaccharides lipid X (2,3-diacylglucosamine 1-phosphate) and MAGP (2-monoacylglucosamine 1-phosphate), of the tetraacyl disaccharide diphosphate precursor of lipid A, IV-A (Federation Proc. 43: 1567, 1984), and of Escherichia coli bacterial endotoxin (lipopolysaccharide). We also measured the response of lipid X after prior administration of indomethacin and MAGP. Lipid X, at a total cumulative dose of 40 micrograms/kg, produced an immediate, but transient dose-dependent pulmonary arterial vasoconstrictive response. MAGP, at a total dose of 40 micrograms/kg, had no pulmonary pressure activity but did increase extravascular lung water and produce some histological changes in the lung. Disaccharide precursor IV-A, at a total dose of 40 micrograms/kg, produced an immediate dose-dependent pulmonary arterial vasoconstrictive response that was prolonged for greater than 2 h. E. coli endotoxin caused a delayed (15-min) increase in the pulmonary arterial pressure but one that also persisted for greater than 2 h. Prior administration of indomethacin blocked the pulmonary pressor activity of lipid X, whereas prior administration of MAGP increased both the magnitude and the duration of the pulmonary pressure response of lipid X. We conclude that the initial pulmonary hypertension seen after lipid X injection may involve cyclooxygenase-dependent formation of prostaglandins and that the genesis of this pulmonary pressor activity is at least in part dependent on the ester-linked hydroxymyristoyl moiety at position 3 of the lipid X molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Aggregation behavior of lipid IVA in aqueous solutions at physiological pH. 1: Simple buffer solutions.

We have investigated the aggregation behaviour of lipid IVA (a bioactive precursor of lipid A and the lipid anchor of lipopolysaccharide) in aqueous solutions in the physiological pH range using dynamic light scattering, nuclear magnetic resonance, fluorescence, surface pressure, electron microscopy and force field simulation studies. The sonication of lipid IVA in PBS, Tris and Hepes produces vesicles which are stable in the concentration range of 10(-3) - 10(-7) M, possibly even at lower concentrations. The vesicle size is not sensitive to the nature of the buffer, only to the pH and to some extent to the ionic strength. The long time stability of the small unilamellar vesicles as well as the structureless 1H-NMR spectra might be attributed to a rigid surface structure. This structure is also supported by the simulation studies. We have tentatively proposed a coexistence of micelles and/or other aggregates with the bilayered vesicles at higher lipid concentrations in order to explain some of the experimental observations.

A convenient synthesis of 4-amino-4-deoxy-L-arabinose and its reduction product, 1,4-dideoxy-1,4-imino-L-arabinitol.

Methyl beta-D-xylopyranoside in a mixture of N,N-dimethylformamide and 2-methoxypropene containing a little hydrogen chloride gave preponderantly the 2,3-O-isopropylidene derivative, which was readily converted into its 4-trifluoromethanesulfonate. The facile displacement of the triflate group gave a 4-azido-4-deoxy-alpha-L-arabinopyranoside derivative, and this, on mild acid treatment, was hydrolyzed to the 2,3-diol, or under more vigorous conditions to 4-azido-4-deoxy-L-arabinose. Methyl 2,3-di-O-acetyl-4-azido-4-deoxy-alpha-L-arabinopyranoside, from the diol, appears (1H-n.m.r. data) to exist as an equilibrating mixture of the 4C1 and 1C4 conformers in chloroform solution. The reduction of the azido sugar by hydrogen over Pd/C in .6M HCl yielded 4-amino-4-deoxy-L-arabinopyranose as its hydrochloride; in 0.1M HCl, further reactions occurred to give 1,4-dideoxy-1,4-imino-L-arabinitol as the final product. The aminodeoxypentose from lipid A precursor IIA, isolated from a Salmonella mutant by Raetz et al. in 1985, was shown to be identical with the synthetic aminoarabinose by t.l.c., 1H-n.m.r. spectroscopy, and g.l.c. of the acetylated reduction products.