A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase

Sphingolipids are key components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is largely conserved. However, in contrast to mammals, which produce sphingomyelin, organisms such as the pathogenic fungi and protozoa synthesize inositol phosphorylceramide (IPC) as the primary phosphosphingolipid. The key step involves the reaction of ceramide and phosphatidylinositol catalysed by IPC synthase, an essential enzyme with no mammalian equivalent encoded by the AUR1 gene in yeast and recently identified functional orthologues in the pathogenic kinetoplastid protozoa. As such this enzyme represents a promising target for novel anti-fungal and anti-protozoal drugs. Given the paucity of effective treatments for kinetoplastid diseases such as leishmaniasis, there is a need to characterize the protozoan enzyme. To this end a fluorescent-based cell-free assay protocol in a 96-well plate format has been established for the Leishmania major IPC synthase. Using this system the kinetic parameters of the enzyme have been determined as obeying the double displacement model with apparent V_max = 2.31 pmol min^?1 U^?1. Furthermore, inhibitory substrate analogues have been identified. Importantly this assay is amenable to development for use in high-throughput screening applications for lead inhibitors and as such may prove to be a pivotal tool in drug discovery.     

Functional analyses of differentially expressed isoforms of the Arabidopsis inositol phosphorylceramide synthase

Sphingolipids are key components of eukaryotic plasma membranes that are involved in many functions, including the formation signal transduction complexes. In addition, these lipid species and their catabolites function as secondary signalling molecules in, amongst other processes, apoptosis. The biosynthetic pathway for the formation of sphingolipid is largely conserved. However, unlike mammalian cells, fungi, protozoa and plants synthesize inositol phosphorylceramide (IPC) as their primary phosphosphingolipid. This key step involves the transfer of the phosphorylinositol group from phosphatidylinositol (PI) to phytoceramide, a process catalysed by IPC synthase in plants and fungi. This enzyme activity is at least partly encoded by the AUR1 gene in the fungi, and recently the distantly related functional orthologue of this gene has been identified in the model plant Arabidopsis. Here we functionally analysed all three predicted Arabidopsis IPC synthases, confirming them as aureobasidin A resistant AUR1p orthologues. Expression profiling revealed that the genes encoding these orthologues are differentially expressed in various tissue types isolated from Arabidopsis.     

Inositol phosphoryl transferases from human pathogenic fungi

The IPC1 gene from Saccharomyces cerevisiae, which encodes inositolphosphorylceramide (IPC) synthase, was first identified as a novel and essential gene encoding resistance to the natural product antifungal aureobasidin A (AUR1). The formation of IPC in fungi is essential for viability, suggesting inhibitors of IPC1p function would make ideal antifungal drug candidates. Homologs of the AUR1/IPC1 gene were identified from a number of human pathogenic fungi, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Cryptococcus neoformans. Comparison of these genes with other homologous genes from Candida albicans, Aspergillus fumigatus, Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe reveals a conserved structural motif for inositolphosphoryl transferases which is similar to a motif recently described for lipid phosphatases, but with unique characteristics.     

灰葡萄孢菌及其抗短梗霉素突变体AUR1基因序列及其酶活性的分析

【目的】探讨灰葡萄孢菌及其抗Ab A突变体AUR1基因序列与IPC合成酶活性的关系。【方法】通过分子生物学方法测定野生型及突变体的AUR1的基因序列,高效液相荧光色谱法测定IPC合成酶活力,苯甲酰化法测定神经酰胺含量。【结果】AUR1基因序列和IPC合成酶活性测定表明4株不同的突变体均产生了对IPC合成酶抑制剂Ab A的抗性,它们的突变类型为:(1)AUR1序列中缺失内含子;(2)AUR1序列中缺失内含子和P155S氨基酸突变;(3)AUR1序列中缺失内含子和V33A的氨基酸突变;(4)AUR1序列中缺失内含子和P155S、S177P、F237L的氨基酸突变。AUR1缺失内含子和既缺失内含子又伴随P155S氨基酸突变的突变体的Ab A抗性较强。神经酰胺含量测定表明野生型IPC合成酶被抑制,导致神经酰胺积累,而突变体则能抵抗Ab A对IPC合成酶的抑制作用。【结论】AUR1基因中的内含子对IPC合成酶的调控起重要的作用。Ab A通过抑制IPC合成酶引起神经酰胺积累,IPC合成酶是鞘脂代谢的关键酶。     

Developmentally regulated sphingolipid synthesis in African trypanosomes

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 labelling 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 more than threefold 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 sphingomyelin synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites.     

Kei1: A Novel Subunit of Inositolphosphorylceramide Synthase,Essential for Its Enzyme Activity and Golgi Localization

Fungal sphingolipids have inositol-phosphate head groups, which are essential for the viability of cells. These head groups are added by inositol phosphorylceramide (IPC) synthase, and AUR1 has been thought to encode this enzyme. Here, we show that an essential protein encoded by KEI1 is a novel subunit of IPC synthase of Saccharomyces cerevisiae. We find that Kei1 is localized in the medial-Golgi and that Kei1 is cleaved by Kex2, a late Golgi processing endopeptidase; therefore, it recycles between the medial- and late Golgi compartments. The growth defect of kei1-1, a temperature-sensitive mutant, is effectively suppressed by the overexpression of AUR1, and Aur1 and Kei1 proteins form a complex in vivo. The kei1-1 mutant is hypersensitive to aureobasidin A, a specific inhibitor of IPC synthesis, and the IPC synthase activity in the mutant membranes is thermolabile. A part of Aur1 is missorted to the vacuole in kei1-1 cells. We show that the amino acid substitution in kei1-1 causes release of Kei1 during immunoprecipitation of Aur1 and that Aur1 without Kei1 has hardly detectable IPC synthase activity. From these results, we conclude that Kei1 is essential for both the activity and the Golgi localization of IPC synthase.     

Inositolphosphoceramide metabolism in Trypanosoma cruzi as compared with other trypanosomatids

Chagas disease is caused by Trypanosoma cruzi and is endemic to North, Central and South American countries. Current therapy against this disease is only partially effective and produces adverse side effects. Studies on the metabolic pathways of T. cruzi, in particular those with no equivalent in mammalian cells, might identify targets for the development of new drugs. Ceramide is metabolized to inositolphosphoceramide (IPC) in T. cruzi and other kinetoplastid protists whereas in mammals it is mainly incorporated into sphingomyelin. In T. cruzi, in contrast to Trypanosoma brucei and Leishmania spp., IPC functions as lipid anchor constituent of glycoproteins and free glycosylinositolphospholipids (GIPLs). Inhibition of IPC and GIPLs biosynthesis impairs differentiation of trypomastigotes into the intracellular amastigote forms. The gene encoding IPC synthase in T. cruzi has been identified and the enzyme has been expressed in a cell-free system. The enzyme involved in IPC degradation and the remodelases responsible for the incorporation of ceramide into free GIPLs or into the glycosylphosphatidylinositols anchoring glycoproteins, and in fatty acid modifications of these molecules of T. cruzi have been understudied. Inositolphosphoceramide metabolism and remodeling could be exploited as targets for Chagas disease chemotherapy.     

Purification,characterization, and identification of a sphingomyelin synthase from Pseudomonas aeruginosa. PlcH is a multifunctional enzyme

Sphingomyelin synthase is the enzyme that synthesizes sphingomyelin (SM) in mammalian cells by transferring a phosphorylcholine moiety from phosphatidylcholine to ceramide. Despite its importance, the gene and/or the protein responsible for this activity has not yet been identified. Here we report the purification, identification, and biochemical characterization of an enzymatic activity that synthesizes SM in Pseudomonas aeruginosa. SM synthase-like activity was found secreted in the culture medium of P. aeruginosa, strains PA01 and PAK, whereas it could not be detected in cultures of Escherichia coli. From the medium of PAK cultures, SM synthase was purified through sequential chromatographic columns. After separation on polyacrylamide-SDS gels and visualization by silver staining, the purified enzyme showed two bands, one of approximately 75 kDa and one of 30-35 kDa. Interestingly, the highly purified SM synthase preparation also showed neutral sphingomyelinase activity. We therefore investigated whether the protein we purified as SM synthase could actually be the previously identified PlcH, a 78-kDa phospholipase C known to hydrolyze phosphatidylcholine and SM in P. aeruginosa. First, the purified SM synthase preparation contained a 78-kDa protein that reacted with monoclonal antibodies raised against purified PlcH. Second, purified PlcH showed SM synthase activity. Third, using different knockout mutant strains for the PlcH operon, PlcH was found to be necessary for SM synthase activity in P. aeruginosa. Interestingly, SM synthase activity was specific to the Pseudomonas PlcH as other bacterial phospholipases did not display SM synthase activity. Biochemical studies on the Pseudomonas SM synthase confirmed that it is a transferase, similar to the mammalian enzyme, that specifically recognizes the choline head-group and the primary hydroxyl on ceramide. This SM synthase did not have reverse transferase activity. In conclusion, the Pseudomonas PlcH also exerts SM synthase activity; therefore, for the first time, we have identified a structural gene for a SM synthase.     

Sphingomyelin synthase SMS2 displays dual activity as ceramide phosphoethanolamine synthase

Sphingolipids are vital components of eukaryotic membranes involved in the regulation of cell growth, death, intracellular trafficking, and the barrier function of the plasma membrane (PM). While sphingomyelin (SM) is the major sphingolipid in mammals, previous studies indicate that mammalian cells also produce the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or the enzyme(s) responsible for CPE biosynthesis. SM production is mediated by the SM synthases SMS1 in the Golgi and SMS2 at the PM, while a closely related enzyme, SMSr, has an unknown biochemical function. We now demonstrate that SMS family members display striking differences in substrate specificity, with SMS1 and SMSr being monofunctional enzymes with SM and CPE synthase activity, respectively, and SMS2 acting as a bifunctional enzyme with both SM and CPE synthase activity. In agreement with the PM residency of SMS2, we show that both SM and CPE synthase activities are enhanced at the surface of SMS2-overexpressing HeLa cells. Our findings reveal an unexpected diversity in substrate specificity among SMS family members that should enable the design of specific inhibitors to target the biological role of each enzyme individually.     

Conservation of NADPH utilization by chorismate synthase and its implications for the evolution of the shikimate pathway

The shikimate pathway is essential for the biosynthesis of aromatic compounds. The seventh and last step is catalysed by chorismate synthase, which has an absolute requirement for reduced FMN in its active site. There are two classes of this enzyme, which are distinguished according to the origin of the reduced cofactor. Monofunctional chorismate synthases sequester it from the cellular environment whereas bifunctional enzymes can generate reduced FMN at the expense of NADPH. These bifunctional enzymes are found in fungi and the ciliated protozoan Euglena gracilis while all bacterial and plant enzymes are monofunctional. In this study, we introduce an in vivo screen, which is based on a chorismate synthase-deficient Saccharomyces cerevisiae strain, allowing the classification of hitherto uncharacterized chorismate synthases. This analysis revealed that bifunctionality is present in the enzymes of protozoan species. In contrast, all bacterial and plant enzymes tested are monofunctional. In addition, we demonstrate that a monofunctional chorismate synthase confers prototrophy in conjunction with a NADPH : FMN oxidoreductase indicating that bifunctionality is required due to the lack of free reduced FMN in fungal and possibly protozoan species. Interestingly, the distribution of bifunctional chorismate synthase concurs with the presence of a pentafunctional enzyme complex.     

An aureobasidin A resistance gene isolated from Aspergillus is a homolog of yeast AUR1, a gene responsible for inositol phosphorylceramide (IPC) synthase activity

The AUR1 gene of Saccharomyces cerevisiae, mutations in which confer resistance to the antibiotic aureobasidin A, is necessary for inositol phosphorylceramide (IPC) synthase activity. We report the molecular cloning and characterization of the Aspergillus nidulans aurA gene, which is homologous to AUR1. A single point mutation in the aurA gene of A. nidulans confers a high level of resistance to aureobasidin A. The A. nidulans aurA gene was used to identify its homologs in other Aspergillus species, including A. fumigatus, A. niger, and A. oryzae. The deduced amino acid sequence of an aurA homolog from the pathogenic fungus A. fumigatus showed 87% identity to that of A. nidulans. The AurA proteins of A. nidulans and A. fumigatus shared common characteristics in primary structure, including sequence, hydropathy profile, and N-glycosylation sites, with their S. cerevisiae, Schizosaccharomyces pombe, and Candida albicans counterparts. These results suggest that the aureobasidin resistance gene is conserved evolutionarily in various fungi. Received: 27 August 1998 / Accepted: 19 November 1998     

Inhibition of yeast inositol phosphorylceramide synthase by aureobasidin A measured by a fluorometric assay

Inositol phosphorylceramide synthase (IPC synthase) is an essential and unique enzyme in fungal sphingolipid biosynthesis and is the target of the cyclic nonadepsipeptide antibiotic aureobasidin A. As a first step towards understanding the mechanism of aureobasidin A inhibition, we developed a fluorometric HPLC assay for IPC synthase using the Saccharomyces cerevisiae enzyme and the fluorescent substrate analog 6-[N-(7-nitro-2,1, 3-benzoxadiazol-4-yl)amino]-hexanoyl ceramide (C(6)-NBD-cer). The kinetic parameters for C(6)-NBD-cer were comparable to those for the synthetic substrate N-acetylsphinganine used previously. Aureobasidin A acted as a tight-binding, non-competitive inhibitor with respect to C(6)-NBD-cer and had a K(i) of 0.55 nM.     

Cloning and characterization of a cDNA encoding human cardiolipin synthase (hCLS1)

Cardiolipin (CL) is a phospholipid localized to the mitochondria, and its biosynthesis is essential for mitochondrial structure and function. We report here the identification and characterization of a cDNA encoding the first mammalian cardiolipin synthase (CLS1) in humans and mice. This cDNA exhibits sequence homology with members of a CLS gene family that share similar domain structure and chemical properties. Expression of the human CLS (hCLS1) cDNA in reticulocyte lysates or insect cells led to a marked increase in CLS activity. The enzyme is specific for CL synthesis, because no significant increase in phosphatidylglycerol phosphate synthase activity was observed. In addition, CL pool size was increased in hCLS1-overexpressing cells compared with controls. Furthermore, the hCLS1 gene was highly expressed in tissues such as heart, skeletal muscle, and liver, which have been shown to have high CLS activities. These results demonstrate that hCLS1 encodes an enzyme that synthesizes CL.     

Trypanosomatid and fungal glycolipids and sphingolipids as infectivity factors and potential targets for development of new therapeutic strategies

Several (glyco)(sphingo)lipids from different human pathogens have been characterized, and frequently many of these molecules are participating in host-pathogen interaction. In Leishmania (Leishmania) amazonensis, for example, amastigotes present on their surface glycosphingolipids (GSLs) with the structure Galbeta1-3Galalpha, which is recognized by 30 kDa receptor of macrophages. Furthermore, other Leishmania species, such as Leishmania (Leishmania) major and Leishmania (Viannia) braziliensis present glycosylinositolphospholipids (GIPLs) which are involved in Leishmania-macrophage interaction. It is worth to mention that these antigens are not expressed in mammalian cells. Leishmania promastigotes also present inositol phosphorylceramide (IPC), a unique sphingolipid characteristic of fungi and plants. It was observed that IPC synthesis is essential for parasite division, since Aureobasidin A, an inhibitor of IPC synthase, inhibited significantly promastigote and amastigote growths. Recently, it was also demonstrated that GIPLs, IPC and sterols are preferentially present in the parasite membrane microdomains resistant to Triton X-100 at 4 degrees C. The disruption of these microdomains by incubating parasites with methyl-beta-cyclodextrin inhibited significantly macrophage infectivity by Leishmania. Other pathogens, such as fungi, also present unique glycolipids which may have an important role for the fungal development and/or disease establishment. Taking together these results, this review will discuss different biological roles for (glyco)(sphingo)lipids of different pathogens.     

Trypanosoma rangeli expresses a β-galactofuranosyl transferase

Glycoconjugates play essential roles in cell recognition, infectivity and survival of protozoan parasites within their insect vectors and mammalian hosts. β-galactofuranose is a component of several glycoconjugates in many organisms, including a variety of trypanosomatids, but is absent in mammalian and African trypanosomes. Herein, we describe the presence of a β(1-3) galactofuranosyl transferase (GALFT), an important enzyme of the galactofuranose biosynthetic pathway, in Trypanosoma rangeli. The T. rangeli GALFT gene (TrGALFT) has an ORF of 1.2 Kb and is organized in two copies in the T. rangeli genome. Antibodies raised against an internal fragment of the transferase demonstrated a 45 kDa protein coded by TrGALFT was localized in the whole cytoplasm, mainly in the Golgi apparatus and equally expressed in epimastigotes and trypomastigotes from T. rangeli. Despite the high sequence similarity with Trypanosoma cruzi and Leishmania spp. orthologous TrGALFT showed a substitution of the metal-binding DXD motif, conserved amongst glycosyltransferases, for a DXE functionally analogous motif. Moreover, a reduced number of GALFT genes were present in T. rangeli when compared with other pathogenic kinetoplastid species.     

Identification of a family of animal sphingomyelin synthases

Sphingomyelin (SM) is a major component of animal plasma membranes. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, yielding diacylglycerol as a side product. This reaction is catalysed by SM synthase, an enzyme whose biological potential can be judged from the roles of diacylglycerol and ceramide as anti- and proapoptotic stimuli, respectively. SM synthesis occurs in the lumen of the Golgi as well as on the cell surface. As no gene for SM synthase has been cloned so far, it is unclear whether different enzymes are present at these locations. Using a functional cloning strategy in yeast, we identified a novel family of integral membrane proteins exhibiting all enzymatic features previously attributed to animal SM synthase. Strikingly, human, mouse and Caenorhabditis elegans genomes each contain at least two different SM synthase (SMS) genes. Whereas human SMS1 is localised to the Golgi, SMS2 resides primarily at the plasma membrane. Collectively, these findings open up important new avenues for studying sphingolipid function in animals.     

LASS5 is the predominant ceramide synthase isoform involved in de novo sphingolipid synthesis in lung epithelia

Ceramide is a key bioactive mediator that inhibits surfactant phosphatidylcholine (PtdCho) synthesis in lung epithelia. Ceramide availability is governed by sphingomyelin (SM) hydrolysis, but less is known regarding its de novo synthesis. In this study, we observed that ceramide synthesis within murine lung epithelia was associated with high-level ceramide synthase (dihydroceramide synthase) activity. Longevity assurance homolog 5 (LASS5) was the predominant ceramide synthase isoform detected in lung epithelia, whereas relatively lower level expression was detected for the other five mammalian homologs. Pulmonary LASS5 was developmentally regulated, but its expression was spatially and gender nonspecific. Exogenously expressed LASS5 in lung epithelia was membrane-associated, triggering increased ceramide synthesis, whereas knockdown studies using fumonisin B1 or LASS5 small, interfering RNA reduced ceramide synthase activity by 78% or 45%, respectively. Overexpression of LASS5 also reduced PtdCho synthesis, but maximal inhibition was achieved when LASS5 was coexpressed with a plasmid encoding a neutral sphingomyelinase involved in SM hydrolysis. These results demonstrate that LASS5 is the major ceramide synthase gene product involved in sphingolipid production that may also regulate PtdCho metabolism in pulmonary epithelia.     

Cloning, expression, and functional characterization of the Dunaliella salina 5-enolpyruvylshikimate-3-phosphate synthase gene in Escherichia coli

5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EC 2.5.1.19) is the sixth enzyme in the shikimate pathway which is essential for the synthesis of aromatic amino acids and many secondary metabolites. The enzyme is widely involved in glyphosate tolerant transgenic plants because it is the primary target of the nonselective herbicide glyphosate. In this study, the Dunaliella salina EPSP synthase gene was cloned by RT-PCR approach. It contains an open reading frame encoding a protein of 514 amino acids with a calculated molecular weight of 54.6 KDa. The derived amino acid sequence showed high homology with other EPSP synthases. The Dunaliella salina EPSP synthase gene was expressed in Escherichia coli and the recombinant EPSP synthase were identified by functional complementation assay.     

Complex sphingolipid synthesis in plants: characterization of inositolphosphorylceramide synthase activity in bean microsomes

Complex glycophosphosphingolipids present in plants are composed of ceramide, inositolphosphate, and diverse polar oligosaccharide substituents. The activity of inositolphosphorylceramide (IPC) synthase (phosphatidylinositol:ceramide inositolphosphate transferase), the enzyme proposed to catalyze the initial committed step in the formation of these complex sphingolipids, was characterized in wax bean hypocotyl microsomes. Enzyme activity was assayed by monitoring the incorporation of fluorescent NBD-C(6) ceramide or [3H]inositolphosphate from radiolabeled phosphatidylinositol (PI) into product identified by TLC. IPC synthase was found to utilize nonhydroxy fatty acid-containing ceramide, hydroxy fatty acid-containing ceramide, and NBD-C(6) ceramide as substrate. Maximum product formation was observed at PI concentrations in excess of 600 microM (with half-maximum activity at approximately 200 microM). Both endogenous PI and ceramide appeared to serve as substrates. Aureobasidin A and rustmicin, two potent inhibitors of fungal IPC synthase, inhibited enzyme activity in bean microsomes with values for IC(50) of 0.4-0.8 and 16-20 nM, respectively. IPC synthase activity appeared most closely associated with the Golgi based on results using selected marker enzymes. Enzyme activity was detected in a variety of plant tissues. This report, the first to characterize IPC synthase in plant tissues, demonstrates the similarities between the plant enzyme and its yeast counterpart, and provides insight into plant glycophosphosphingolipid biology.