Phosphatidylethanolamine (GPEtn), a major phospholipid component of trypanosome membranes, is synthesized de novo from ethanolamine through the Kennedy pathway. Here the composition of the GPEtn molecular species in the bloodstream form of Trypanosoma brucei is determined, along with new insights into phospholipid metabolism, by in vitro and in vivo characterization of a key enzyme of the Kennedy pathway, the cytosolic ethanolamine-phosphate cytidylyltransferase (TbECT). Gene knockout indicates that TbECT is essential for growth and survival, thus highlighting the importance of the Kennedy pathway for the pathogenic stage of the African trypanosome. Phosphatiylserine decarboxylation, a potential salvage pathway, does not appear to be active in cultured bloodstream form T. brucei, and it is not upregulated even when the Kennedy pathway is disrupted. In vivo metabolic labelling and phospholipid composition analysis by ESI-MS/MS of the knockout cells confirmed a significant decrease in GPEtn species, as well as changes in the relative abundance of other phospholipid species. Reduction in GPEtn levels had a profound influence on the morphology of the mutants and it compromised mitochondrial structure and function, as well as glycosylphosphatidylinositol anchor biosynthesis. TbECT is therefore genetically validated as a potential drug target against the African trypanosome.
Ethanolamine kinase catalyzes the committed step in the synthesis of phosphatidylethanolamine via the CDP-ethanolamine branch of the Kennedy pathway. Regulation of the EKI1-encoded ethanolamine kinase by the essential nutrient zinc was examined in Saccharomyces cerevisiae. The level of ethanolamine kinase activity increased when zinc was depleted from the growth medium. This regulation correlated with increases in the CDP-ethanolamine pathway intermediates phosphoethanolamine and CDP-ethanolamine, and an increase in the methylated derivative of phosphatidylethanolamine, phosphatidylcholine. The β-galactosidase activity driven by the PEKI1-lacZ reporter gene was elevated in zinc-depleted cells, indicating that the increase in ethanolamine kinase activity was attributed to a transcriptional mechanism. The expression level of PEKI1-lacZ reporter gene activity in the zrt1Δzrt2Δ mutant (defective in plasma membrane zinc transport) cells grown with zinc was similar to the activity expressed in wild-type cells grown without zinc. This indicated that EKI1 expression was sensitive to intracellular zinc. The zinc-mediated regulation of EKI1 expression was attenuated in the zap1Δ mutant defective in the zinc-regulated transcription factor Zap1p. Direct interactions between Zap1p and putative zinc-responsive elements in the EKI1 promoter were demonstrated by electrophoretic mobility shift assays. Mutations of these elements to a nonconsensus sequence abolished Zap1p-DNA interactions. Taken together, this work demonstrated that the zinc-mediated regulation of ethanolamine kinase and the synthesis of phospholipids via the CDP-ethanolamine branch of the Kennedy pathway were controlled in part by Zap1p.
It has been established that yeast membrane phospholipid content is responsive to the inositol and choline content of the growth medium. Alterations in the levels of transcription of phospholipid biosynthetic enzymes contribute significantly to this response. We now describe conditions under which ethanolamine can exert significant influence on yeast membrane phospholipid composition. We demonstrate that mutations which block a defined subset of the reactions required for the biosynthesis of phosphatidylcholine (PC) via the CDP-choline pathway cause ethanolamine-dependent effects on the steady-state levels of bulk PC in yeast membranes. Such an ethanolamine-dependent reduction in bulk membrane PC content was observed for both choline kinase (cki) and choline phosphotransferase (cpt1) mutants, but it was not observed for mutants defective in cholinephosphate cytidylyltransferase, the enzyme that catalyzes the penultimate reaction of the CDP-choline pathway for PC biosynthesis. Moreover, the ethanolamine effect observed for cki and cpt1 mutants was independent of the choline content of the growth medium. Finally, we found that haploid yeast strains defective in the activity of both the choline and ethanolamine phosphotransferases experienced an ethanolamine-insensitive reduction in steady-state PC content, an effect which was not observed in strains defective in either one of these activities alone. The collective data indicate that specific enzymes of the CDP-ethanolamine pathway for phosphatidylethanolamine biosynthesis, while able to contribute to PC synthesis when yeast cells are grown under conditions of ethanolamine deprivation, do not do so when yeast cells are presented with this phospholipid headgroup precursor.
CTP synthetase is a cytosolic-associated glutamine amidotransferase enzyme that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In the yeast Saccharomyces cerevisiae, the reaction product CTP is an essential precursor of all membrane phospholipids that are synthesized via the Kennedy (CDP-choline and CDP-ethanolamine branches) and CDP-diacylglycerol pathways. The URA7 and URA8 genes encode CTP synthetase in S. cerevisiae, and the URA7 gene is responsible for the majority of CTP synthesized in vivo. The CTP synthetase enzymes are allosterically regulated by CTP product inhibition. Mutations that alleviate this regulation result in an elevated cellular level of CTP and an increase in phospholipid synthesis via the Kennedy pathway. The URA7-encoded enzyme is phosphorylated by protein kinases A and C, and these phosphorylations stimulate CTP synthetase activity and increase cellular CTP levels and the utilization of the Kennedy pathway. The CTPS1 and CTPS2 genes that encode human CTP synthetase enzymes are functionally expressed in S. cerevisiae, and rescue the lethal phenotype of the ura7Δ ura8Δ double mutant that lacks CTP synthetase activity. The expression in yeast has revealed that the human CTPS1-encoded enzyme is also phosphorylated and regulated by protein kinases A and C.
CTP; CTP synthetase; CDP-diacylglycerol; CDP-choline; CDP-ethanolamine; phospholipid synthesis; phosphorylation
Ethanolamine is important for synthesis of choline, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in plants. The latter two phospholipids are the major phospholipids in eukaryotic membranes. In plants, ethanolamine is mainly synthesized directly from serine by serine decarboxylase. Serine decarboxylase is unique to plants and was previously shown to have highly specific activity to l-serine. While serine decarboxylase was biochemically characterized, its functions and importance in plants were not biologically elucidated due to the lack of serine decarboxylase mutants. Here we characterized an Arabidopsis mutant defective in serine decarboxylase, named atsdc-1 (Arabidopsis thaliana serine decarboxylase-1). The atsdc-1 mutants showed necrotic lesions in leaves, multiple inflorescences, sterility in flower, and early flowering in short day conditions. These defects were rescued by ethanolamine application to atsdc-1, suggesting the roles of ethanolamine as well as serine decarboxylase in plant development. In addition, molecular analysis of serine decarboxylase suggests that Arabidopsis serine decarboxylase is cytosol-localized and expressed in all tissue.
serine decarboxylase; ethanolamine; choline; phosphatidylethanolamine; phosphatidylcholine; Arabidopsis thaliana
Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and β). Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform.
In this study, an antiserum against CKα was produced by immunizing rabbits with denatured, purified recombinant CKα2 full-length protein. This antiserum was highly specific for CKα when tested with extracts from different cell lines, and there was no cross reactivity with purified CKβ and other related proteins like human ethanolamine kinases (EK) and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CKα1 and α2 isoforms in MCF-7 and HepG2 cell extracts, but not in HeLa, HCT-116, and mouse embryonic stem cell extracts. Subsequent protein dot blot assay of total CKα in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CKα was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CKα expression levels were observed in kidney (11-fold higher in tumor) and liver (15-fold lower in tumor) samples.
Apart from its high sensitivity and specificity, the antiserum produced in this work, which does not require further purification, has the advantage of co-detecting both α1 and α2 isoforms in cell extracts for direct comparison of their expression levels.
Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labeling confirmed stage specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased >3-fold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian SM synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites.
The Kennedy pathway generates phosphocoline and phosphoethanolamine through its two branches. Choline Kinase (ChoK) is the first enzyme of the Kennedy branch of synthesis of phosphocholine, the major component of the plasma membrane. ChoK family of proteins is composed by ChoKα and ChoKβ isoforms, the first one with two different variants of splicing. Recently ChoKα has been implicated in the carcinogenic process, since it is over-expressed in a variety of human cancers. However, no evidence for a role of ChoKβ in carcinogenesis has been reported.
Here we compare the in vitro and in vivo properties of ChoKα1 and ChoKβ in lipid metabolism, and their potential role in carcinogenesis. Both ChoKα1 and ChoKβ showed choline and ethanolamine kinase activities when assayed in cell extracts, though with different affinity for their substrates. However, they behave differentially when overexpressed in whole cells. Whereas ChoKβ display an ethanolamine kinase role, ChoKα1 present a dual choline/ethanolamine kinase role, suggesting the involvement of each ChoK isoform in distinct biochemical pathways under in vivo conditions. In addition, while overexpression of ChoKα1 is oncogenic when overexpressed in HEK293T or MDCK cells, ChoKβ overexpression is not sufficient to induce in vitro cell transformation nor in vivo tumor growth. Furthermore, a significant upregulation of ChoKα1 mRNA levels in a panel of breast and lung cancer cell lines was found, but no changes in ChoKβ mRNA levels were observed. Finally, MN58b, a previously described potent inhibitor of ChoK with in vivo antitumoral activity, shows more than 20-fold higher efficiency towards ChoKα1 than ChoKβ.
This study represents the first evidence of the distinct metabolic role of ChoKα and ChoKβ isoforms, suggesting different physiological roles and implications in human carcinogenesis. These findings constitute a step forward in the design of an antitumoral strategy based on ChoK inhibition.
chol mutants of Saccharomyces cerevisiae are deficient in the synthesis of the phospholipid phosphatidylserine owing to lowered activity of the membrane-associated enzyme phosphatidylserine synthase. chol mutants are auxotrophic for ethanolamine or choline and, in the absence of these supplements, cannot synthesize phosphatidylethanolamine or phosphatidylcholine (PC). We exploited these characteristics of the chol mutants to examine the regulation of phospholipid metabolism in S. cerevisiae. Macromolecular synthesis and phospholipid metabolism were examined in chol cells starved for ethanolamine. As expected, when chol mutants were starved for ethanolamine, the rates of synthesis of the phospholipids phosphatidylethanolamine and PC declined rapidly. Surprisingly, however, coupled to the decline in PC biosynthesis was a simultaneous decrease in the overall rate of phospholipid synthesis. In particular, the rate of synthesis of phosphatidylinositol decreased in parallel with the decline in PC biosynthesis. The results obtained suggest that the slowing of PC biosynthesis in ethanolamine-starved chol cells leads to a coordinated decrease in the synthesis of all phospholipids. However, under conditions of ethanolamine deprivation in chol cells, the cytoplasmic enzyme inositol-1-phosphate synthase could not be repressed by exogenous inositol, and the endogenous synthesis of the phospholipid precursor inositol appeared to be elevated. The implications of these findings with respect to the coordinated regulation of phospholipid synthesis are discussed.
Phosphatidylethanolamine (PE) is the most abundant lipid on the protoplasmatic leaflet of cellular membranes. It has a pivotal role in cellular processes such as membrane fusion, cell cycle regulation, autophagy, and apoptosis. CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) is the main regulatory enzyme in de novo biosynthesis of PE from ethanolamine and diacylglycerol by the CDP-ethanolamine Kennedy pathway. The following is a summary of the current state of knowledge on Pcyt2 and how splicing and isoform specific differences could lead to variations in functional properties in this family of enzymes. Results from the most recent studies on Pcyt2 transcriptional regulation, promoter function, autophagy, and cell growth regulation are highlighted. Recent data obtained from Pcyt2 knockout mouse models is also presented, demonstrating the essentiality of this gene in embryonic development as well as the major physiological consequences of deletion of one Pcyt2 allele. Those include development of symptoms of the metabolic syndrome such as elevated lipogenesis and lipoprotein secretion, hypertriglyceridemia, liver steatosis, obesity, and insulin resistance. The objective of this review is to elucidate the nature of Pcyt2 regulation by linking its catalytic function with the regulation of lipid and energy homeostasis.
phosphatidylethanolamine; CTP:phosphoethanolamine cytidylyltransferase; Pcyt2; lipid homeostasis; cell growth; hypertriglyceridemia; liver steatosis; obesity; insulin resistance; metabolic syndrome
Choline-containing teichoic acid seems to be essential for the adsorption of bacteriophage Dp-1 to pneumococci. This conclusion is based on the following observations: In contrast to pneumococci grown in choline-containing medium, cells grown in medium containing ethanolamine or other submethylated aminoalcohols instead of choline were found to be resistant to infection by Dp-1. Live choline-grown bacteria and heat- or UV-inactivated cells and purified cell walls prepared from these cells were capable of adsorbing phage Dp-1; ethanolamine-grown pneumococci or cell wall preparations were unable to do so. Adsorption of Dp-1 to choline-containing cell walls was competitively inhibited by phosphorylcholine and by several choline-containing soluble cell surface components, such as the Forssman antigen and the teichoic acid-glycan complexes formed by autolytic cell wall degradation. Cell walls prepared from pneumococci grown in ethanolamine or phosphorylethanolamine were inactive. Electron microscopic studies with pneumococci that had segments of choline-containing cell wall material amid ethanolamine-containing regions indicated that the Dp-1 phage particles adsorbed exclusively to the choline-containing surface areas. We suggest that the choline residues of the pneumococcal teichoic acid are essential components of the Dp-1 phage receptors in this bacterium.
The CDP-ethanolamine pathway is responsible for the de novo biosynthesis of ethanolamine phospholipids, where CDP-ethanolamine is coupled with diacylglycerols to form phosphatidylethanolamine. We have disrupted the mouse gene encoding CTP:phosphoethanolamine cytidylyltransferase, Pcyt2, the main regulatory enzyme in this pathway. Intercrossings of Pcyt2+/− animals resulted in small litter sizes and unexpected Mendelian frequencies, with no null mice genotyped. The Pcyt2−/− embryos die after implantation, prior to embryonic day 8.5. Examination of mRNA expression, protein content, and enzyme activity in Pcyt2+/− animals revealed the anticipated 50% decrease due to the gene dosage effect but rather a 20 to 35% decrease. [14C]ethanolamine radiolabeling of hepatocytes, liver, heart, and brain corroborated Pcyt2 gene expression and activity data and showed a decreased rate of phosphatidylethanolamine biosynthesis in heterozygotes. Total phospholipid content was maintained in Pcyt2+/− tissues; however, this was not due to compensatory increases in the decarboxylation of phosphatidylserine. These results establish the necessity of Pcyt2 for murine development and demonstrate that a single Pcyt2 allele in heterozygotes can maintain phospholipid homeostasis.
Parathyroid hormone (PTH) and phorbol-12,13-dibutyrate (PDBu) stimulate phospholipase D (PLD) activity and phosphatidylcholine (PC) hydrolysis in UMR-106 osteoblastic cells . The current studies were designed to determine whether ethanolamine-containing phospholipids, and specifically phosphatidylethanolamine (PE), could also be substrates. In cells labeled with 14C-ethanolamine PTH and PDBu treatment decreased 14C-phosphatidylethanolamine. In cells co-labeled with 3H-choline and 14C-ethanolamine, PTH and PDBu treatment increased both 3H-choline and 14C-ethanolamine release from the cells. Choline and ethanolamine phospholipid hydrolysis was increased within 5 min, and responses were sustained for at least 60 min. Maximal effects were obtained with 10 nM PTH and 50 nM PDBu. Dominant negative PLD1 and PLD2 constructs inhibited the effects of PTH on the phospholipid hydrolysis. The results suggest that both PC and PE are substrates for phospholipase D in UMR-106 osteoblastic cells and could therefore be sources of phospholipid hydrolysis products for downstream signaling in osteoblasts.
Phosphatidylethanolamine; phosphatidylcholine; phospholipase D; parathyroid hormone; osteoblast; UMR-106
Ethanolamine can be used as a source of carbon and nitrogen by phylogenetically diverse bacteria. Ethanolamine-ammonia lyase, the enzyme that breaks ethanolamine into acetaldehyde and ammonia, is encoded by the gene tandem eutBC. Despite extensive studies of ethanolamine utilization in Salmonella enterica serovar Typhimurium, much remains to be learned about EutBC structure and catalytic mechanism, about the evolutionary origin of ethanolamine utilization, and about regulatory links between the metabolism of ethanolamine itself and the ethanolamine-ammonia lyase cofactor adenosylcobalamin. We used computational analysis of sequences, structures, genome contexts, and phylogenies of ethanolamine-ammonia lyases to address these questions and to evaluate recent data-mining studies that have suggested an association between bacterial food poisoning and the diol utilization pathways. We found that EutBC evolution included recruitment of a TIM barrel and a Rossmann fold domain and their fusion to N-terminal α-helical domains to give EutB and EutC, respectively. This fusion was followed by recruitment and occasional loss of auxiliary ethanolamine utilization genes in Firmicutes and by several horizontal transfers, most notably from the firmicute stem to the Enterobacteriaceae and from Alphaproteobacteria to Actinobacteria. We identified a conserved DNA motif that likely represents the EutR-binding site and is shared by the ethanolamine and cobalamin operons in several enterobacterial species, suggesting a mechanism for coupling the biosyntheses of apoenzyme and cofactor in these species. Finally, we found that the food poisoning phenotype is associated with the structural components of metabolosome more strongly than with ethanolamine utilization genes or with paralogous propanediol utilization genes per se.
The CDP-choline pathway of phosphatidylcholine (PtdCho) biosynthesis was first described more than 50 years ago. Investigation of the CDP-choline pathway in yeast provides a basis for understanding the CDP-choline pathway in mammals. PtdCho is considered as an intermediate in a cycle of synthesis and degradation, and the activity of a CDP-choline cycle is linked to subcellular membrane lipid movement. The components of the mammalian CDP-choline pathway include choline transport, choline kinase, phosphocholine cytidylyltransferase, and choline phosphotransferase activities. The protein isoforms and biochemical mechanisms of regulation of the pathway enzymes are related to their cell and tissue-specific functions. Regulated PtdCho turnover mediated by phospholipases or neuropathy target esterase participates in the mammalian CDP-choline cycle. Knockout mouse models define the biological functions of the CDP-choline cycle in mammalian cells and tissues. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.
The biological membranes of Trypanosoma brucei contain a complex array of phospholipids that are synthesized de novo from precursors obtained either directly from the host, or as catabolised endocytosed lipids. This paper describes the use of nanoflow electrospray tandem mass spectrometry and high resolution mass spectrometry in both positive and negative ion modes, allowing the identification of ~500 individual molecular phospholipids species from total lipid extracts of cultured bloodstream and procyclic form T. brucei. Various molecular species of all of the major subclasses of glycerophospholipids were identified including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol as well as phosphatidic acid, phosphatidylglycerol and cardolipin, and the sphingolipids sphingomyelin, inositol phosphoceramide and ethanolamine phosphoceramide. The lipidomic data obtained in this study will aid future biochemical phenotyping of either genetically or chemically manipulated commonly used bloodstream and procyclic strains of Trypanosoma brucei. Hopefully this will allow a greater understanding of the bizarre world of lipids in this important human pathogen.
Phospholipid; Trypanosoma brucei; mass spectrometry; lipidomics
The microviscosity and fluidity of erythrocyte ghost membranes from lead workers and control subjects was measured by fluorescence polarisation using the fluorophore, 1,6-diphenyl-1,3,5-hexatriene (DPH). Increased lead was associated with a significant decrease in the average microviscosity of resealed and unsealed erythrocyte membranes. Since DPH fluorescence reflects the organisation of lipids in the central core of the membrane, two aspects of phospholipid metabolism were investigated. Phospholipids were extracted from red blood cell ghost membranes and identified by high performance liquid chromatography. The ratio of phosphatidyl choline to phosphatidyl ethanolamine, an established correlate of membrane fluidity, was significantly increased in lead workers. This is attributed to the known increases in red blood cell cholesterol in lead workers and the structural incompatibility of phosphatidyl ethanolamine and cholesterol, which result in a compensatory increase of phosphatidyl choline. Erythrocyte ghost membranes from control subjects were resealed with the intermediates in phospholipid synthesis that increase with a lead inhibited decrease in red blood cell pyrimidine 5'-nucleotidase. Membrane fluidity was not modified by incubation with cytidine triphosphate, uridine triphosphate, cytidine diphosphate choline, or cytidine diphosphate ethanolamine. Alterations in the microviscosity of the lipid regions of the hydrophobic core of the erythrocyte membrane bilayer and in the phospholipid composition of the membrane may be defects which contribute to the clinical and biochemical instability of the red blood cell on exposure to lead.
The substrate selectivity of four Trypanosoma brucei sphingolipid synthases was examined. TbSLS1, an inositol phosphorylceramide (IPC) synthase and TbSLS4, a bi-functional sphingomyelin (SM)/ethanolamine phosphorylceramide (EPC) synthase, were inactivated by Ala substitutions of a conserved triad of residues His210, His253 and Asp257 thought to form part of the active site. TbSLS4 also catalyzed the reverse reaction, production of ceramide from sphingomyelin, but none of the Ala substitutions of the catalytic triad in TbSLS4 were able to do so. Site-directed mutagenesis identified residues proximal to the conserved triad that were responsible for the discrimination between charge and size of the different head groups. For discrimination between anionic (phosphoinositol) and zwitterionic (phosphocholine, phosphoethanolamine) head groups, doubly mutated V172D/S252F TbSLS1 and D172V/F252S TbSLS3 showed reciprocal conversion between IPC and bi-functional SM/EPC synthases. For differentiation of zwitterionic head group size, N170A TbSLS1 and A170N/N187D TbSLS4 showed reciprocal conversion between EPC and bi-functional SM/EPC synthases. These studies provide a mapping of the SLS active site and demonstrate that differences in catalytic specificity of the T. brucei enzyme family are controlled by natural variations in as few as three residue positions.
Phosphatidylcholine and phosphatidylethanolamine are the two main phospholipids in eukaryotic cells comprising ∼50 and 25% of phospholipid mass, respectively. Phosphatidylcholine is synthesized almost exclusively through the CDP-choline pathway in essentially all mammalian cells. Phosphatidylethanolamine is synthesized through either the CDP-ethanolamine pathway or by the decarboxylation of phosphatidylserine, with the contribution of each pathway being cell type dependent. Two human genes, CEPT1 and CPT1, code for the total compliment of activities that directly synthesize phosphatidylcholine and phosphatidylethanolamine through the CDP-alcohol pathways. CEPT1 transfers a phosphobase from either CDP-choline or CDP-ethanolamine to diacylglycerol to synthesize both phosphatidylcholine and phosphatidylethanolamine, whereas CPT1 synthesizes phosphatidylcholine exclusively. We show through immunofluorescence that brefeldin A treatment relocalizes CPT1, but not CEPT1, implying CPT1 is found in the Golgi. A combination of coimmunofluorescence and subcellular fractionation experiments with various endoplasmic reticulum, Golgi, and nuclear markers confirmed that CPT1 was found in the Golgi and CEPT1 was found in both the endoplasmic reticulum and nuclear membranes. The rate-limiting step for phosphatidylcholine synthesis is catalyzed by the amphitropic CTP:phosphocholine cytidylyltransferase α, which is found in the nucleus in most cell types. CTP:phosphocholine cytidylyltransferase α is found immediately upstream cholinephosphotransferase, and it translocates from a soluble nuclear location to the nuclear membrane in response to activators of the CDP-choline pathway. Thus, substrate channeling of the CDP-choline produced by CTP:phosphocholine cytidylyltransferase α to nuclear located CEPT1 is the mechanism by which upregulation of the CDP-choline pathway increases de novo phosphatidylcholine biosynthesis. In addition, a series of CEPT1 site-directed mutants was generated that allowed for the assignment of specific amino acid residues as structural requirements that directly alter either phospholipid head group or fatty acyl composition. This pinpointed glycine 156 within the catalytic motif as being responsible for the dual CDP-alcohol specificity of CEPT1, whereas mutations within helix 214–228 allowed for the orientation of transmembrane helices surrounding the catalytic site to be definitively positioned.
The lipid compositions of 17 spirochetes belonging to the genera Spirochaeta and Treponema were investigated and compared with data previously derived from 11 strains of Leptospira. The lipid compositions and lipid metabolism of any of these genera is sufficiently different to be characteristic of that genus and to differentiate it from the other two genera. Members of the genus Leptospira are characterized by their ability to beta-oxidize long chain fatty acids as their major carbon and energy source. With few exceptions, they are incapable of synthesizing fatty acids de novo. The major phospholipid found was phosphatidyl ethanolamine. No glycolipid or phosphatidyl choline was found in these organisms. Members of the genus Treponema studied were incapable of beta-oxidation as well as de novo synthesis of fatty acids. Phosphatidyl choline is the major phospholipid of this genus. The glycolipid, monogalactosyl diglyceride, is a major component of the Treponema. Members of the Spirochaeta did synthesize fatty acids de novo. Although these spirochetes contain a monoglycosyl diglyceride, the hexose content of the glycolipid varied from species to species. Neither phosphatidyl ethanolamine nor phosphatidyl choline was found in the Spirochaeta.
Slein, Milton W. (Fort Detrick, Frederick, Md.), and Gerald F. Logan, Jr. Characterization of the phospholipases of Bacillus cereus and their effects on erythrocytes, bone, and kidney cells. J. Bacteriol. 90:69–81. 1965.—Culture filtrates of Bacillus cereus contain phospholipases that split phosphoryl choline, phosphoryl ethanolamine, and phosphoryl inositol from the phospholipids phosphatidyl choline (PTC), sphingomyelin, phosphatidyl ethanolamine (PTE), and phosphatidyl inositol (PTI). It is possible that one enzyme catalyzes the degradation of PTE and PTC, but the other phospholipases appear to be separate entities. Some activity on phosphatidyl serine has also been noted. Quantitative paper chromatography has been used for characterizing the phospholipases that are separated on N,N′-diethylaminoethyl cellulose columns. A procedure for the analysis of inositol is included. A sensitive kidney cortex homogenate test is described that depends on the release of alkaline phosphatase for the measurement of phosphatasemia factor (PF) activity associated with the phospholipases. The effects of the phospholipases on erythrocytes, kidney, and bone cells are discussed. Hemolysin activity is inhibited by crude soybean “lecithin,” but hemolysis does not seem to be identical with PTE- or PTC-phospholipase activity. PF activity is also inhibited by the “lecithin.” Highest PF activity is associated with PTI-phospholipase. The phospholipase fractions differ in their sensitivities to trypsin. Phospholipases with similar properties have been obtained from culture filtrates of B. anthracis.
Human African Trypanosomiasis is a devastating disease caused by the parasite Trypanosoma brucei. Trypanosomes live extracellularly in both the tsetse fly and the mammal. Trypanosome surface proteins can directly interact with the host environment, allowing parasites to effectively establish and maintain infections. Glycosylphosphatidylinositol (GPI) anchoring is a common posttranslational modification associated with eukaryotic surface proteins. In T. brucei, three GPI-anchored major surface proteins have been identified: variant surface glycoproteins (VSGs), procyclic acidic repetitive protein (PARP or procyclins), and brucei alanine rich proteins (BARP). The objective of this study was to select genes encoding predicted GPI-anchored proteins with unknown function(s) from the T. brucei genome and characterize the expression profile of a subset during cyclical development in the tsetse and mammalian hosts. An initial in silico screen of putative T. brucei proteins by Big PI algorithm identified 163 predicted GPI-anchored proteins, 106 of which had no known functions. Application of a second GPI-anchor prediction algorithm (FragAnchor), signal peptide and trans-membrane domain prediction software resulted in the identification of 25 putative hypothetical proteins. Eighty-one gene products with hypothetical functions were analyzed for stage-regulated expression using semi-quantitative RT-PCR. The expression of most of these genes were found to be upregulated in trypanosomes infecting tsetse salivary gland and proventriculus tissues, and 38% were specifically expressed only by parasites infecting salivary gland tissues. Transcripts for all of the genes specifically expressed in salivary glands were also detected in mammalian infective metacyclic trypomastigotes, suggesting a possible role for these putative proteins in invasion and/or establishment processes in the mammalian host. These results represent the first large-scale report of the differential expression of unknown genes encoding predicted T. brucei surface proteins during the complete developmental cycle. This knowledge may form the foundation for the development of future novel transmission blocking strategies against metacyclic parasites.
Human African Trypanosomiasis (HAT) is a fatal disease caused by African trypanosomes and transmitted by an infected tsetse fly. Presently, there are no vaccines to prevent mammalian infections. Proteins expressed on the trypanosome surface can influence the host environment and allow for their transmission. Potentially accessible to the adaptive immune systems of vertebrate hosts, these proteins could serve as future vaccine targets. Identification and characterization of these currently unknown proteins can help us develop strategies to alter the host environment, making it inhospitable for the parasite, thereby reducing disease transmission. While there is extensive knowledge about trypanosome development in the mammalian host, less is known about the molecular events in the tsetse fly, particularly the salivary gland stages. We used an in silico approach to identify putative surface proteins from the known genome sequence of Trypanosoma brucei, and we describe the stage specific expression of these genes during development in the tsetse fly and mammalian host. Our findings show that a majority of unknown transcripts encoding predicted surface proteins are expressed by the parasites infecting tsetse salivary glands. These data will help focus future investigations into transmission-blocking approaches targeting the expressed antigens of trypanosomes infecting tsetse salivary glands.
Incubation of slices of the salt gland of the albatross with acetylcholine, which is the physiological secretogogue for this tissue, led to a 13-fold increase in the rate of incorporation of P32 into phosphatidic acid and a 3-fold increase in the incorporation of P32 and inositol-2-H3 into phosphoinositide. The incorporation of P32 into phosphatidyl choline and phosphatidyl ethanolamine was increased relatively slightly or not at all. Respiration was doubled. The "phospholipid effect" occurred in the microsome fraction, which is known to contain fragments of the endoplasmic reticulum. The enzymes, diglyceride kinase and phosphatidic acid phosphatase, which catalyze the stimulated turnover of phosphatidic acid in brain cortex, were also found in highest concentration in the microsome fraction. The phosphatides which respond to acetylcholine are bound to protein in the membrane. On the basis of these findings it appears that phosphatidic acid and possibly phosphoinositide participate in sodium transport. A scheme, termed the phosphatidic acid cycle, is presented as a working hypothesis, in which the turnover of phosphatidic acid in the membrane, catalyzed by diglyceride kinase and phosphatidic acid phosphatase, functions as a sodium pump.
Hemozoin (Hz) is a heme crystal produced by some blood-feeding organisms, as an efficient way to detoxify heme derived from hemoglobin digestion. In the triatomine insect Rhodnius prolixus, Hz is essentially produced by midgut extracellular phospholipid membranes known as perimicrovillar membranes (PMVM). Here, we investigated the role of commercial glycerophospholipids containing serine, choline and ethanolamine as headgroups and R. prolixus midgut lipids (RML) in heme crystallization. All commercial unsaturated forms of phospholipids, as well as RML, mediated fast and efficient β-hematin formation by means of two kinetically distinct mechanisms: an early and fast component, followed by a late and slow one. The fastest reactions observed were induced by unsaturated forms of phosphatidylethanolamine (uPE) and phosphatidylcholine (uPC), with half-lives of 0.04 and 0.7 minutes, respectively. β-hematin crystal morphologies were strikingly distinct among groups, with uPE producing homogeneous regular brick-shaped crystals. Interestingly, uPC-mediated reactions resulted in two morphologically distinct crystal populations: one less representative group of regular crystals, resembling those induced by uPE, and the other largely represented by crystals with numerous sharp edges and tapered ends. Heme crystallization reactions induced by RML were efficient, with a heme to β-hematin conversion rate higher than 70%, but clearly slower (t1/2 of 9.9–17.7 minutes) than those induced by uPC and uPE. Interestingly, crystals produced by RML were homogeneous in shape and quite similar to those mediated by uPE. Thus, β-hematin formation can be rapidly and efficiently induced by unsaturated glycerophospholipids, particularly uPE and uPC, and may play a role on biological heme crystallization in R. prolixus midgut.
The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein- GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI- PLC, cell-associated gp63 could not be detected in immunoblots. Pulse- chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.