Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine.
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Certain strains of Escherichia coli and Salmonella contain lipopolysaccharide (LPS) modified with a phosphoethanolamine (pEtN) group at position 7 of the outer 3-deoxy-d-manno-octulosonic acid (Kdo) residue. Using the heptose-deficient E. coli mutant WBB06 (Brabetz, W., Muller-Loennies, S., Holst, O., and Brade, H. (1997) Eur. J. Biochem. 247, 716-724), we now demonstrate that the critical parameter determining the presence or absence of pEtN is the concentration of CaCl(2) in the medium. As judged by mass spectrometry, half the LPS in WBB06, grown on nutrient broth with 5 mm CaCl(2), is derivatized with a pEtN group, whereas LPS from WBB06 grown without supplemental CaCl(2) is not. Membranes from E. coli WBB06 or wild-type W3110 grown on 5-50 mm CaCl(2) contain a novel pEtN transferase that uses the precursor Kdo(2)-[4'-(32)P]lipid IV(A) as an acceptor. Transferase is not present in membranes of E. coli grown with 5 mm MgCl(2), BaCl(2), or ZnCl(2). Hydrolysis of the in vitro reaction product, pEtN-Kdo(2)-[4'-(32)P]lipid IV(A), at pH 4.5 shows that the pEtN substituent is located on the outer Kdo moiety. Membranes from an E. coli pss knockout mutant grown on 50 mm CaCl(2), which lack phosphatidylethanolamine, do not contain measurable transferase activity unless exogenous phosphatidylethanolamine is added back to the assay system. The induction of the pEtN transferase by 5-50 mm CaCl(2) suggests possible role(s) in establishing transformation competence or resisting environmental stress, and represents the first example of a regulated covalent modification of the inner core of E. coli LPS. (+info)
Phosphatidylcholine synthesis influences the diacylglycerol homeostasis required for SEC14p-dependent Golgi function and cell growth.
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Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids in eukaryotic cells and thus have major roles in the formation and maintenance of vesicular membranes. In yeast, diacylglycerol accepts a phosphocholine moiety through a CPT1-derived cholinephosphotransferase activity to directly synthesize phosphatidylcholine. EPT1-derived activity can transfer either phosphocholine or phosphoethanolamine to diacylglcyerol in vitro, but is currently believed to primarily synthesize phosphatidylethanolamine in vivo. In this study we report that CPT1- and EPT1-derived cholinephosphotransferase activities can significantly overlap in vivo such that EPT1 can contribute to 60% of net phosphatidylcholine synthesis via the Kennedy pathway. Alterations in the level of diacylglycerol consumption through alterations in phosphatidylcholine synthesis directly correlated with the level of SEC14-dependent invertase secretion and affected cell viability. Administration of synthetic di8:0 diacylglycerol resulted in a partial rescue of cells from SEC14-mediated cell death. The addition of di8:0 diacylglycerol increased di8:0 diacylglycerol levels 20-40-fold over endogenous long-chain diacylglycerol levels. Di8:0 diacylglcyerol did not alter endogenous phospholipid metabolic pathways, nor was it converted to di8:0 phosphatidic acid. (+info)
Separate myocardial ethanolamine phosphotransferase activities responsible for plasmenylethanolamine and phosphatidylethanolamine synthesis.
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Ethanolamine phosphotransferase (EPT) is a key enzyme responsible for the synthesis of ethanolamine glycerophospholipids. Plasmenylethanolamine is a predominant molecular subclass of ethanolamine glycerophospholipids in the heart. The present study was designed to identify the selective use of 1-O-alk-1'-enyl-2-acyl-sn-glycerol as a substrate for EPT as a mechanism responsible for the predominance of plasmenylethanolamine in the rabbit heart. EPT activity in rabbit myocardial membranes using 1,2-diacyl-sn-glycerol as substrate is activated by Mn2+, inhibited by dithiobisnitrobenzoic acid (DTNB) and is unaffected by Ca2+. In contrast, ethanolamine phosphotransferase activity using 1-O-alk-1'-enyl-2-acyl-sn-glycerol as substrate is inhibited by Mn2+ and Ca2+, but is activated by DTNB. Additionally, ethanolamine phosphotransferase activity using 1-O-alk-1'-enyl-2-acyl-sn-glycerol substrate was more sensitive to thermal denaturation compared with that of 1,2-diacyl-sn-glycerol. Taken together, these results suggest that separate ethanolamine phosphotransferase activities are present in heart membranes that are responsible for the synthesis of phosphatidylethanolamine and plasmenylethanolamine. (+info)
Phosphorylation of the lipid A region of meningococcal lipopolysaccharide: identification of a family of transferases that add phosphoethanolamine to lipopolysaccharide.
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A gene, NMB1638, with homology to the recently characterized gene encoding a phosphoethanolamine transferase, lpt-3, has been identified from the Neisseria meningitidis genome sequence and was found to be present in all meningococcal strains examined. Homology comparison with other database sequences would suggest that NMB1638 and lpt-3 represent genes coding for members of a family of proteins of related function identified in a wide range of gram-negative species of bacteria. When grown and isolated under appropriate conditions, N. meningitidis elaborated lipopolysaccharide (LPS) containing a lipid A that was characteristically phosphorylated with multiple phosphate and phosphoethanolamine residues. In all meningococcal strains examined, each lipid A species contained the basal diphosphorylated species, wherein a phosphate group is attached to each glucosamine residue. Also elaborated within the population of LPS molecules are a variety of "phosphoforms" that contain either an additional phosphate residue, an additional phosphoethanolamine residue, additional phosphate and phosphoethanolamine residues, or an additional phosphate and two phosphoethanolamine residues in the lipid A. Mass spectroscopic analyses of LPS from three strains in which NMB1638 had been inactivated by a specific mutation indicated that there were no phosphoethanolamine residues included in the lipid A region of the LPS and that there was no further phosphorylation of lipid A beyond one additional phosphate species. We propose that NMB1638 encodes a phosphoethanolamine transferase specific for lipid A and propose naming the gene "lptA," for "LPS phosphoethenolamine transferase for lipid A." (+info)
Phosphatidylethanolamine is the donor of the ethanolamine residue linking a glycosylphosphatidylinositol anchor to protein.
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Numerous cell surface glycoproteins from eukaryotic organisms including African trypanosomes and budding yeast (Saccharomyces cerevisiae), are anchored to the lipid bilayer by a glycophospholipid, glycosylphosphatidylinositol, covalently linked to the carboxyl terminus of the protein via a phosphoethanolamine bridge. In this paper we describe metabolic labeling experiments aimed at identifying the biosynthetic origin of the ethanolamine residue in the phosphoethanolamine bridge. Using yeast mutants generated by disruption of the ethanolaminephosphotransferase (EPT1) and cholinephosphotransferase (CPT1) genes, we report data consistent with the proposal that the ethanolamine residue is derived from phosphatidylethanolamine. (+info)
The selective utilization of substrates in vivo by the phosphatidylethanolamine and phosphatidylcholine biosynthetic enzymes Ept1p and Cpt1p in yeast.
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In yeast, the aminoalcohol phosphotransferases Ept1p and Cpt1p catalyze the final steps in the CDP-ethanolamine and CDP-choline routes leading to phosphatidylethanolamine (PE) and phosphatidylcholine (PC), respectively. To determine how these enzymes contribute to the molecular species profiles of PE and PC in vivo, wild-type, cpt1Delta, and ept1Delta cells were pulse labeled with deuterated ethanolamine and choline. Analysis of newly synthesized PE and PC using electrospray ionization tandem mass spectrometry revealed that PE and PC produced by Ept1p and Cpt1p have different species compositions, demonstrating that the enzymes consume distinct sets of diacylglycerol species in vivo. Using the characteristic phospholipid species profiles produced by Ept1p and Cpt1p as molecular fingerprints, it was also shown that in vivo CDP-monomethylethanolamine is preferentially used as substrate by Ept1p, whereas CDP-dimethylethanolamine and CDP-propanolamine are converted by Cpt1p. (+info)
A phosphoethanolamine transferase specific for the outer 3-deoxy-D-manno-octulosonic acid residue of Escherichia coli lipopolysaccharide. Identification of the eptB gene and Ca2+ hypersensitivity of an eptB deletion mutant.
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Addition of a phosphoethanolamine (pEtN) moiety to the outer 3-deoxy-D-manno-octulosonic acid (Kdo) residue of lipopolysaccharide (LPS) in WBB06, a heptose-deficient Escherichia coli mutant, occurs when cells are grown in 5-50 mM CaCl2 (Kanipes, M. I., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 1156-1163). A Ca2+-induced, membrane-bound enzyme was responsible for the transfer of the pEtN unit to the Kdo domain. We now report the identification of the gene encoding the pEtN transferase. E. coli yhjW was cloned and overexpressed, because it is homologous to a putative pEtN transferase implicated in the modification of the beta-chain heptose residue of Neisseria meningitidis lipo-oligosaccharide (Mackinnon, F. G., Cox, A. D., Plested, J. S., Tang, C. M., Makepeace, K., Coull, P. A., Wright, J. C., Chalmers, R., Hood, D. W., Richards, J. C., and Moxon, E. R. (2002) Mol. Microbiol. 43, 931-943). In vitro assays with Kdo2-4'-[32P]lipid A as the acceptor showed that YhjW (renamed EptB) utilizes phosphatidylethanolamine in the presence of Ca2+ to transfer the pEtN group. Stoichiometric amounts of diacylglycerol were generated during the EptB-catalyzed transfer of pEtN to Kdo2-lipid A. EptB is an inner membrane protein of 574 amino acid residues with five predicted trans-membrane segments within its N-terminal region. An in-frame replacement of eptB with a kanamycin resistance cassette rendered E. coli WBB06 (but not wild-type W3110) hypersensitive to CaCl2 at 5 mM or higher. Ca2+ hypersensitivity was suppressed by excess Mg2+ in the medium or by restoring the LPS core of WBB06. The latter was achieved by reintroducing the waaC and waaF genes, which encode LPS heptosyl transferases I and II, respectively. Our data demonstrate that pEtN modification of the outer Kdo protected cells containing heptose-deficient LPS from damage by high concentrations of Ca2+. Based on its sequence similarity to EptA(PmrC), we propose that the active site of EptB faces the periplasmic surface of the inner membrane. (+info)
Stearoyl-CoA desaturase 1 deficiency increases CTP:choline cytidylyltransferase translocation into the membrane and enhances phosphatidylcholine synthesis in liver.
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Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in monounsaturated fatty acid synthesis. Previously, we showed that Scd1 deficiency reduces liver triglyceride accumulation and considerably decreases synthesis of very low density lipoprotein and its secretion in both lean and obese mice. In the present study, we found that Scd1 deficiency significantly modulates hepatic glycerophospholipid profile. The content of phosphatidylcholine (PC) was increased by 40% and the activities of CTP:choline cytidylyltransferase (CCT), the rate-limiting enzyme in de novo PC synthesis, and choline phosphotransferase were increased by 64 and 53%, respectively, in liver of Scd1-/- mice. In contrast, the protein level of phosphatidylethanolamine N-methyltransferase, an enzyme involved in PC synthesis via methylation of phosphatidylethanolamine, was decreased by 80% in the liver of Scd1-/- mice. Membrane translocation of CCT is required for its activation. Immunoblot analyses demonstrated that twice as much CCTalpha was associated with plasma membrane in livers of Scd1-/- compared with wild type mice, suggesting that Scd1 mutation leads to an increase in CCT membrane affinity. The incorporation of [(3)H]glycerol into PC was increased by 2.5-fold in Scd1-/- primary hepatocytes compared with those of wild type mice. Furthermore, mitochondrial glycerol-3-phosphate acyltransferase activity was reduced by 42% in liver of Scd1-/- mice; however, the activities of microsomal glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase, and ethanolamine phosphotransferase were not affected by Scd1 mutation. Our study revealed that SCD1 deficiency specifically increases CCT activity by promoting its translocation into membrane and enhances PC biosynthesis in liver. (+info)