Lysoglycosphingolipids have the ability to induce cell death through direct PI3K inhibition.

Sphingolipidoses are inherited metabolic disorders associated with glycosphingolipids accumulation, neurodegeneration, and neuroinflammation leading to severe neurological symptoms. Lysoglycosphingolipids (lysoGSLs), also known to accumulate in the tissues of sphingolipidosis patients, exhibit cytotoxicity. LysoGSLs are the possible pathogenic cause, but the mechanisms are still unknown in detail. Here, we first show that lysoGSLs are potential inhibitors of phosphoinositide 3-kinase (PI3K) to reduce cell survival signaling. We found that phosphorylated Akt was commonly reduced in fibroblasts from patients with sphingolipidoses, including GM1/GM2 gangliosidoses and Gaucher's disease, suggesting the contribution of lysoGSLs to the pathogenesis. LysoGSLs caused cell death and decreased the level of phosphorylated Akt as in the patient fibroblasts. Extracellularly administered lysoGM1 permeated the cell membrane to diffusely distribute in the cytoplasm. LysoGM1 and lysoGM2 also inhibited the production of phosphatidylinositol-(3,4,5)-triphosphate and the translocation of Akt from the cytoplasm to the plasma membrane. We also predicted that lysoGSLs could directly bind to the catalytic domain of PI3K by in silico docking study, suggesting that lysoGSLs could inhibit PI3K by directly interacting with PI3K in the cytoplasm. Furthermore, we revealed that the increment of lysoGSLs amounts in the brain of sphingolipidosis model mice correlated with the neurodegenerative progression. Our findings suggest that the down-regulation of PI3K/Akt signaling by direct interaction of lysoGSLs with PI3K in the brains is a neurodegenerative mechanism in sphingolipidoses. Moreover, we could propose the intracellular PI3K activation or inhibition of lysoGSLs biosynthesis as novel therapeutic approaches for sphingolipidoses because lysoGSLs should be cell death mediators by directly inhibiting PI3K, especially in neurons.

Characterization of thraustochytrid-specific sterol O-acyltransferase: modification of DGAT2-like enzyme to increase the sterol production in Aurantiochytrium limacinum mh0186.

IMPORTANCE: Since the global market for sterols and vitamin D are grown with a high compound annual growth rate, a sustainable source of these compounds is required to keep up with the increasing demand. Thraustochytrid is a marine oleaginous microorganism that can synthesize several sterols, which are stored as SE in lipid droplets. DGAT2C is an unconventional SE synthase specific to thraustochytrids. Although the primary structure of DGAT2C shows high similarities with that of DGAT, DGAT2C utilizes sterol as an acceptor substrate instead of diacylglycerol. In this study, we examined more detailed enzymatic properties, intracellular localization, and structure-activity relationship of DGAT2C. Furthermore, we successfully developed a method to increase sterol and provitamin D3 productivity of thraustochytrid by more than threefold in the process of elucidating the function of the DGAT2C-specific N-terminal region. Our findings could lead to sustainable sterol and vitamin D production using thraustochytrid.

Vibrio-binding gangliosides in fish intestinal tracts.

It has been clarified that pathogens bind to glycosphingolipid (GSL) receptors in mammals, but there have been very few reports on pathogen-binding GSLs in fish. Vibrios are facultative anaerobic bacteria ubiquitous in marine and brackish environments. They are members of the normal intestinal microflora of healthy fish, but some species can cause a disease called vibriosis in fish and shellfish when the hosts are physiologically or immunologically weakened. The adherence of vibrios to host intestinal tracts is a significant event not only for survival and growth but also in terms of pathogenicity. We show in this mini-review that sialic acid-containing GSLs (gangliosides), GM4 and GM3, are receptors to which vibrios adhere to epithelial cells in the intestinal tract of fish. We also describe the enzymes responsible for synthesizing these Vibrio-binding gangliosides in fish.

Significance of mitochondrial fatty acid ß-oxidation for the survivability of Aurantiochytrium limacinum ATCC MYA-1381 during sugar starvation.

Thraustochytrids are marine protists that accumulate large amounts of palmitic acid and docosahexaenoic acid in lipid droplets. Random insertional mutagenesis was adopted for Aurantiochytrium limacinum ATCC MYA-1381 to search for genes that regulate lipid metabolism in thraustochytrids. A mutant strain, M17, was selected because of its significant decrease in myristic acid, palmitic acid, and triacylglycerol contents and cell growth defect. Genome analysis revealed that the gene encoding for mitochondrial electron-transfer flavoprotein ubiquinone oxidoreductase (ETFQO) was lacking in the M17 strain. This mutant strain exhibited a growth defect at the stationary phase, possibly due to stagnation of mitochondrial fatty acid ß-oxidation and branched-chain amino acid degradation, both of which were caused by lack of ETFQO. This study shows the usability of random insertional mutagenesis to obtain mutants of lipid metabolism in A. limacinum and clarifies that ETFQO is integral for survival under sugar starvation in A. limacinum.

Determination of globotriaosylceramide analogs in the organs of a mouse model of Fabry disease.

Fabry disease is a heritable lipid disorder caused by the low activity of α-galactosidase A and characterized by the systemic accumulation of globotriaosylceramide (Gb3). Recent studies have reported a structural heterogeneity of Gb3 in Fabry disease, including Gb3 isoforms with different fatty acids and Gb3 analogs with modifications on the sphingosine moiety. However, Gb3 assays are often performed only on the selected Gb3 isoforms. To precisely determine the total Gb3 concentration, here we established two methods for determining both Gb3 isoforms and analogs. One was the deacylation method, involving Gb3 treatment with sphingolipid ceramide N-deacylase, followed by an assay of the deacylated products, globotriaosylsphingosine (lyso-Gb3) and its analogs, by ultra-performance LC coupled to tandem MS (UPLC-MS/MS). The other method was a direct assay established in the present study for 37 Gb3 isoforms and analogs/isoforms by UPLC-MS/MS. Gb3s from the organs of symptomatic animals of a Fabry disease mouse model were mainly Gb3 isoforms and two Gb3 analogs, such as Gb3(+18) containing the lyso-Gb3(+18) moiety and Gb3(-2) containing the lyso-Gb3(-2) moiety. The total concentrations and Gb3 analog distributions determined by the two methods were comparable. Gb3(+18) levels were high in the kidneys (24% of total Gb3) and the liver (13%), and we observed Gb3(-2) in the heart (10%) and the kidneys (5%). These results indicate organ-specific expression of Gb3 analogs, insights that may lead to a deeper understanding of the pathophysiology of Fabry disease.

Two bacterial glycosphingolipid synthases responsible for the synthesis of glucuronosylceramide and α-galactosylceramide.

Bacterial glycosphingolipids such as glucuronosylceramide and galactosylceramide have been identified as ligands for invariant natural killer T cells and play important roles in host defense. However, the glycosphingolipid synthases required for production of these ceramides have not been well-characterized. Here, we report the identification and characterization of glucuronosylceramide synthase (ceramide UDP-glucuronosyltransferase [Cer-GlcAT]) in Zymomonas mobilis, a Gram-negative bacterium whose cellular membranes contain glucuronosylceramide. On comparing the gene sequences that encode the diacylglycerol GlcAT in bacteria and plants, we found a homologous gene that is widely distributed in the order Sphingomonadales in the Z. mobilis genome. We first cloned the gene and expressed it in Escherichia coli, followed by protein purification using nickel-Sepharose affinity and gel filtration chromatography. Using the highly enriched enzyme, we observed that it has high glycosyltransferase activity with UDP-glucuronic acid and ceramide as sugar donor and acceptor substrate, respectively. Cer-GlcAT deletion resulted in a loss of glucuronosylceramide and increased the levels of ceramide phosphoglycerol, which was expressed in WT cells only at very low levels. Furthermore, we found sequences homologous to Cer-GlcAT in Sphingobium yanoikuyae and Bacteroides fragilis, which have been reported to produce glucuronosylceramide and α-galactosylceramide, respectively. We expressed the two homologs of the cer-glcat gene in E. coli and found that each gene encodes Cer-GlcAT and Cer-galactosyltransferase, respectively. These results contribute to the understanding of the roles of bacterial glycosphingolipids in host-bacteria interactions and the function of bacterial glycosphingolipids in bacterial physiology.

C4-monomethylsterol ß-glucoside and its synthase in Aurantiochytrium limacinum mh0186.

Thraustochytrids, unicellular marine protists, synthesize polyunsaturated fatty acids (PUFAs) and PUFA-containing phospholipids; however, little is known about their glycolipids and their associated metabolism. Here, we report two glycolipids (GL-A, B) and their synthases in Aurantiochytrium limacinum mh0186. Two glycolipids were purified from A. limacinum mh0186, and they were determined by gas chromatography, mass spectrometry and 2D nuclear magnetic resonance to be 3-O-ß-D-glucopyranosyl-stigmasta-5,7,22-triene (GL-A) and 3-O-ß-D-glucopyranosyl-4α-methyl-stigmasta-7,22-diene (GL-B), both of which are sterol ß-glucosides (ß-SGs); the structure of GL-B has not been reported thus far. Seven candidate genes responsible for the synthesis of these ß-SGs were extracted from the draft genome database of A. limacinum using the yeast sterol ß-glucosyltransferase (SGT; EC 2.4.1.173) sequence as a query. Expression analysis using Saccharomyces cerevisiae revealed that two gene products (AlSGT-1 and 2) catalyze the transfer of glucose from uridine diphosphate (UDP)-glucose to sterols, generating sterylglucosides (SGs). Compared to AlSGT-1, AlSGT-2 exhibited wide specificity for sterols and used C4-monomethylsterol to synthesize GL-B. The disruption of alsgt-2 but not alsgt-1 in strain mh0186 resulted in a decrease in the total SG and an almost complete loss of GL-B, indicating that AlSGT-2 is responsible for the synthesis of ß-SGs in A. limacinum mh0186, especially GL-B, which possesses a unique sterol structure.

Lactosylceramide synthases encoded by B4galt5 and 6 genes are pivotal for neuronal generation and myelin formation in mice.

It is uncertain which ß4-galactosyltransferase (ß4GalT; gene name, B4galt), ß4GalT-5 and/or ß4GalT-6, is responsible for the production of lactosylceramide (LacCer) synthase, which functions in the initial step of ganglioside biosynthesis. Here, we generated conditional B4galt5 knockout (B4galt5 cKO) mice, using Nestin-Cre mice, and crossed these with B4galt6 KO mice to generate B4galt5 and 6 double KO (DKO) mice in the central nervous system (CNS). LacCer synthase activity and major brain gangliosides were completely absent in brain homogenates from the DKO mice, although LacCer synthase activity was about half its normal level in B4galt5 cKO mice and B4galt6 KO mice. The DKO mice were born normally but they showed growth retardation and motor deficits at 2 weeks and died by 4 weeks of age. Histological analyses showed that myelin-associated proteins were rarely found localized in axons in the cerebral cortex, and axonal and myelin formation were remarkably impaired in the spinal cords of the DKO mice. Neuronal cells, differentiated from neurospheres that were prepared from the DKO mice, showed impairments in neurite outgrowth and branch formation, which can be explained by the fact that neurospheres from DKO mice could weakly interact with laminin due to lack of gangliosides, such as GM1a. Furthermore, the neurons were immature and perineuronal nets (PNNs) were poorly formed in DKO cerebral cortices. Our results indicate that LacCer synthase is encoded by B4galt5 and 6 genes in the CNS, and that gangliosides are indispensable for neuronal maturation, PNN formation, and axonal and myelin formation.

Regulation of TG accumulation and lipid droplet morphology by the novel TLDP1 in Aurantiochytrium limacinum F26-b.

Thraustochytrids are marine single-cell protists that produce large amounts of PUFAs, such as DHA. They accumulate PUFAs in lipid droplets (LDs), mainly as constituent(s) of triacylglycerol (TG). We identified a novel protein in the LD fraction of Aurantiochytrium limacinum F26-b using 2D-difference gel electrophoresis. The protein clustered with orthologs of thraustochytrids; however, the cluster was evolutionally different from known PAT family proteins or plant LD protein; thus, we named it thraustochytrid-specific LD protein 1 (TLDP1). TLDP1 surrounded LDs when expressed as a GFP-tagged form. Disruption of the tldp1 gene decreased the content of TG and number of LDs per cell; however, irregular and unusually large LDs were generated in tldp1-deficient mutants. Although the level of TG synthesis was unchanged by the disruption of tldp1, the level of TG degradation was higher in tldp1-deficient mutants than in the WT. These phenotypic abnormalities in tldp1-deficient mutants were restored by the expression of tldp1 These results indicate that TLDP1 is a thraustochytrid-specific LD protein and regulates the TG accumulation and LD morphology in A. limacinum F26-b.

Sterylglucoside catabolism in Cryptococcus neoformans with endoglycoceramidase-related protein 2 (EGCrP2), the first steryl-ß-glucosidase identified in fungi.

Cryptococcosis is an infectious disease caused by pathogenic fungi, such as Cryptococcus neoformans and Cryptococcus gattii. The ceramide structure (methyl-d18:2/h18:0) of C. neoformans glucosylceramide (GlcCer) is characteristic and strongly related to its pathogenicity. We recently identified endoglycoceramidase-related protein 1 (EGCrP1) as a glucocerebrosidase in C. neoformans and showed that it was involved in the quality control of GlcCer by eliminating immature GlcCer during the synthesis of GlcCer (Ishibashi, Y., Ikeda, K., Sakaguchi, K., Okino, N., Taguchi, R., and Ito, M. (2012) Quality control of fungus-specific glucosylceramide in Cryptococcus neoformans by endoglycoceramidase-related protein 1 (EGCrP1). J. Biol. Chem. 287, 368-381). We herein identified and characterized EGCrP2, a homologue of EGCrP1, as the enzyme responsible for sterylglucoside catabolism in C. neoformans. In contrast to EGCrP1, which is specific to GlcCer, EGCrP2 hydrolyzed various ß-glucosides, including GlcCer, cholesteryl-ß-glucoside, ergosteryl-ß-glucoside, sitosteryl-ß-glucoside, and para-nitrophenyl-ß-glucoside, but not α-glucosides or ß-galactosides, under acidic conditions. Disruption of the EGCrP2 gene (egcrp2) resulted in the accumulation of a glycolipid, the structure of which was determined following purification to ergosteryl-3ß-glucoside, a major sterylglucoside in fungi, by mass spectrometric and two-dimensional nuclear magnetic resonance analyses. This glycolipid accumulated in vacuoles and EGCrP2 was detected in vacuole-enriched fraction. These results indicated that EGCrP2 was involved in the catabolism of ergosteryl-ß-glucoside in the vacuoles of C. neoformans. Distinct growth arrest, a dysfunction in cell budding, and an abnormal vacuole morphology were detected in the egcrp2-disrupted mutants, suggesting that EGCrP2 may be a promising target for anti-cryptococcal drugs. EGCrP2, classified into glycohydrolase family 5, is the first steryl-ß-glucosidase identified as well as a missing link in sterylglucoside metabolism in fungi.

A label-free impedance-based whole cell assay revealed a new G protein-coupled receptor ligand for mouse microglial cell migration.

We report the usefulness of an impedance-based label-free whole cell assay to identify new ligands for G protein-coupled receptors (GPCRs) involved in microglial cell migration. Authentic GPCR ligands were subjected to the impedance-based cell assay in order to examine the responses of ligands for MG5 mouse microglial cells. Complement component 5 (C5a), adenosine 5'-diphosphate (ADP), uridine 5'-triphosphate (UTP), lysophosphatidic acid (LPA), and lysophosphatidylserine (LysoPS) were found to elicit different cellular impedance patterns, i.e. C5a, ADP, and UTP caused a transient increase in cellular impedance, while LPA and LysoPS decreased it. The responses for C5a and ADP were abolished by pertussis toxin (PTX), but not rho-associated protein kinase inhibitor, Y-27632, indicating that C5a and ADP elicited responses through the Gαi pathway. On the other hand, the response for UTP, LPA or LysoPS was not cancelled by PTX or Y-27632. In a modified Boyden chamber assay, C5a and ADP, but not UTP, LPA, or LysoPS, induced the migration of MG5 cells. These results suggest that PTX-sensitive increase in cellular impedance with the assay is characteristic for ligands of GPCRs involved in microglial cell migration. We found using this assay that 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a new chemoattractant inducing microglial cell migration through the activation of Gαi.

New insight into the structure, reaction mechanism, and biological functions of neutral ceramidase.

Ceramidase (CDase) is an enzyme that hydrolyzes the N-acyl linkage between the sphingoid base and fatty acid of ceramide. These enzymes are classified into three distinct groups, acid (Asah1), neutral (Asah2), and alkaline (Asah3) CDases, based on their primary structure and optimum pH. Acid CDase catabolizes ceramide in lysosomes and is found only in vertebrates. In contrast, the distribution of neutral and alkaline CDases is broad, with both being found in species ranging from lower eukaryotes to mammals; however, only neutral CDase is found in prokaryotes, including some pathogenic bacteria. Neutral CDase is thought to have gained a specific domain (mucin box) in the N-terminal region after the vertebrate split, allowing the enzyme to be stably expressed at the plasma membrane as a type II membrane protein. The X-ray crystal structure of neutral CDase was recently solved, uncovering a unique structure and reaction mechanism for the enzyme. Neutral CDase contains a zinc ion in the active site that functions as a catalytic center, and the hydrolysis of the N-acyl linkage in ceramide proceeds through a mechanism that is similar to that described for zinc-dependent carboxypeptidase. This review describes the structure, reaction mechanism, and biological functions of neutral CDase in association with the molecular evolution, topology, and mechanical conformation. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Ergosteryl-ß-glucosidase (Egh1) involved in sterylglucoside catabolism and vacuole formation in Saccharomyces cerevisiae.

Sterylglucosides (SGs) are composed of a glucose and sterol derivatives, and are distributed in fungi, plants and mammals. We recently identified EGCrP1 and EGCrP2 (endoglycoceramidase-related proteins 1 and 2) as a ß-glucocerebrosidase and steryl-ß-glucosidase, respectively, in Cryptococcus neoformans. We herein describe an EGCrP2 homologue (Egh1; ORF name, Yir007w) involved in SG catabolism in Saccharomyces cerevisiae. The purified recombinant Egh1 hydrolyzed various ß-glucosides including ergosteryl ß-glucoside (EG), cholesteryl ß-glucoside, sitosteryl ß-glucoside, para-nitrophenyl ß-glucoside, 4-methylumberifellyl ß-glucoside and glucosylceramide. The disruption of EGH1 in S. cerevisiae BY4741 (egh1Δ) resulted in the accumulation of EG and fragmentation of vacuoles. The expression of EGH1 in egh1Δ (revertant) reduced the accumulation of EG, and restored the morphology of vacuoles. The accumulation of EG was not detected in EGH1 and UGT51(ATG26) double-disrupted mutants (ugt51Δegh1Δ), indicating that EG was synthesized by Ugt51(Atg26) and degraded by Egh1 in vivo. These results clearly demonstrated that Egh1 is an ergosteryl-ß-glucosidase that is functionally involved in the EG catabolic pathway and vacuole formation in S. cerevisiae.

A novel ether-linked phytol-containing digalactosylglycerolipid in the marine green alga, Ulva pertusa.

Galactosylglycerolipids (GGLs) and chlorophyll are characteristic components of chloroplast in photosynthetic organisms. Although chlorophyll is anchored to the thylakoid membrane by phytol (tetramethylhexadecenol), this isoprenoid alcohol has never been found as a constituent of GGLs. We here described a novel GGL, in which phytol was linked to the glycerol backbone via an ether linkage. This unique GGL was identified as an Alkaline-resistant and Endogalactosylceramidase (EGALC)-sensitive GlycoLipid (AEGL) in the marine green alga, Ulva pertusa. EGALC is an enzyme that is specific to the R-Galα/ß1-6Galß1-structure of galactolipids. The structure of U. pertusa AEGL was determined following its purification to 1-O-phytyl-3-O-Galα1-6Galß1-sn-glycerol by mass spectrometric and nuclear magnetic resonance analyses. AEGLs were ubiquitously distributed in not only green, but also red and brown marine algae; however, they were rarely detected in terrestrial plants, eukaryotic phytoplankton, or cyanobacteria.

Identification of perfluorooctane sulfonate binding protein in the plasma of tiger pufferfish Takifugu rubripes.

It is well known that perfluorooctane sulfonate (PFOS) preferentially accumulates in the plasma of wildlife and humans. Although earlier studies have suggested that this was due to binding of PFOS to a plasma protein, definite characterization of the protein in in vivo exposure studies was not conducted thus far. In this study, we conducted both in vitro and in vivo experiments to identify PFOS binding protein in the plasma of fish. For the in vivo studies, PFOS was administered intraperitoneally to tiger pufferfish, Takifugu rubripes, and the plasma was separated by ammonium sulfate fractionation. High concentrations of PFOS were found in the 65-70 percent ammonium sulfate fraction (190ng/mL). After SDS-PAGE and N-terminal amino acid sequence analysis, the PFOS-binding protein was identified as an apolipoprotein A-I, which was confirmed on the basis of a significant correlation to the PFOS concentration in each fraction. The plasma samples fractionated by ammonium sulfate from untreated pufferfish were subjected to PFOS binding assay by the equilibrium dialysis method. The results further confirmed that the 60-65 percent ammonium sulfate fraction showed a high PFOS-binding ratio, similar to that found from in vivo studies. We demonstrated that PFOS is likely bound to an apolipoprotein A-I in the plasma of tiger pufferfish in in vivo and in vitro studies.

Quality control of fungus-specific glucosylceramide in Cryptococcus neoformans by endoglycoceramidase-related protein 1 (EGCrP1).

A fungus-specific glucosylceramide (GlcCer), which contains a unique sphingoid base possessing two double bonds and a methyl substitution, is essential for pathogenicity in fungi. Although the biosynthetic pathway of the GlcCer has been well elucidated, little is known about GlcCer catabolism because a GlcCer-degrading enzyme (glucocerebrosidase) has yet to be identified in fungi. We found a homologue of endoglycoceramidase tentatively designated endoglycoceramidase-related protein 1 (EGCrP1) in several fungal genomic databases. The recombinant EGCrP1 hydrolyzed GlcCer but not other glycosphingolipids, whereas endoglycoceramidase hydrolyzed oligosaccharide-linked glycosphingolipids but not GlcCer. Disruption of egcrp1 in Cryptococcus neoformans, a typical pathogenic fungus causing cryptococcosis, resulted in the accumulation of fungus-specific GlcCer and immature GlcCer that possess sphingoid bases without a methyl substitution concomitant with a dysfunction of polysaccharide capsule formation. These results indicated that EGCrP1 participates in the catabolism of GlcCer and especially functions to eliminate immature GlcCer in vivo that are generated as by-products due to the broad specificity of GlcCer synthase. We conclude that EGCrP1, a glucocerebrosidase identified for the first time in fungi, controls the quality of GlcCer by eliminating immature GlcCer incorrectly generated in C. neoformans, leading to accurate processing of fungus-specific GlcCer.

Assays and Utilization of Enzymes Involved in Glycolipid Metabolism in Bacteria and Fungi.

Microbial glycosphingolipid (GSL)-degrading enzymes with unique specificity are useful tools for GSL research. On the other hand, some microbial glycolipids, not only GSLs but also steryl glucosides, are closely related to pathogenicity, and, thus, the metabolism of microbial glycolipids is attracting attention as a target for antibiotics. This chapter describes the assays and utilization of microbial enzymes useful for glycolipid research and those involved in pathogenicity or host immune reactions.

Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system.

Endoglycoceramidase (EGCase) is a glycosidase capable of hydrolyzing the ß -glycosidic linkage between the oligosaccharides and ceramides of glycosphingolipids (GSLs). Three molecular species of EGCase differing in specificity were found in the culture fluid of Rhodococcus equi (formerly Rhodococcus sp. M-750) and designated EGCase I, II, and III. This study describes the molecular cloning of EGCase I and characterization of the recombinant enzyme, which was highly expressed in a rhodococcal expression system using Rhodococcus erythropolis. Kinetic analysis revealed the turnover number (k(cat)) (k(cat)) of the recombinant EGCase I to be 22- and 1,200-fold higher than that of EGCase II toward GM1a and Gb3Cer, respectively, although the K(m) of both enzymes was almost the same for these substrates. Comparison of the three-dimensional structure of EGCase I (model) and EGCase II (crystal) indicated that a flexible loop hangs over the catalytic cleft of EGCase II but not EGCase I. Deletion of the loop from EGCase II increased the k(cat) of the mutant enzyme, suggesting that the loop is a critical factor affecting the turnover of substrates and products in the catalytic region. Recombinant EGCase I exhibited broad specificity and good reaction efficiency compared with EGCase II, making EGCase I well-suited to a comprehensive analysis of GSLs.

Analysis of Δ12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304.

Thraustochytrids are known to synthesize PUFAs such as docosahexaenoic acid (DHA). Accumulating evidence suggests the presence of two synthetic pathways of PUFAs in thraustochytrids: the polyketide synthase-like (PUFA synthase) and desaturase/elongase (standard) pathways. It remains unclear whether the latter pathway functions in thraustochytrids. In this study, we report that the standard pathway produces PUFA in Thraustochytrium aureum ATCC 34304. We isolated a gene encoding a putative Δ12-fatty acid desaturase (TauΔ12des) from T. aureum. Yeasts transformed with the tauΔ12des converted endogenous oleic acid (OA) into linoleic acid (LA). The disruption of the tauΔ12des in T. aureum by homologous recombination resulted in the accumulation of OA and a decrease in the levels of LA and its downstream PUFAs. However, the DHA content was increased slightly in tauΔ12des-disruption mutants, suggesting that DHA is primarily produced in T. aureum via the PUFA synthase pathway. The transformation of the tauΔ12des-disruption mutants with a tauΔ12des expression cassette restored the wild-type fatty acid profiles. These data clearly indicate that TauΔ12des functions as Δ12-fatty acid desaturase in the standard pathway of T. aureum and demonstrate that this thraustochytrid produces PUFAs via both the PUFA synthase and the standard pathways.

Purification, molecular cloning, and application of a novel sphingomyelin-binding protein (clamlysin) from the brackishwater clam, Corbicula japonica.

A novel sphingomyelin-binding protein (clamlysin) was purified from the foot muscle of a brackishwater clam, Corbicula japonica. The purified 24.8-kDa protein lysed sheep, horse and rabbit erythrocytes and the hemolytic activity was inhibited by sphingomyelin, but not other phospholipids or glycosphingolipids. The open reading frame of the clamlysin gene encoded a putative 26.9-kDa protein (clamlysin B) which showed high sequence similarity with the actinoporin family. A surface plasmon resonance assay confirmed that clamlysin B specifically bound to sphingomyelin. Furthermore, two cDNA variants of clamlysin, encoding putative 31.4 kDa (clamlysin A) and 11 kDa (clamlysin C) proteins, were isolated. Only the 31.4-kDa variant was found to exhibit sphingomyelin-binding activity. Clamlysin A and B, but not C, shared a sequence (domain II) conserved in all known sphingomyelin-binding proteins. Domain II fused with a glutathione S-transferase bound to sphingomyelin. Horse erythrocytes, mouse melanoma B16 and GM95 cells, and Chinese hamster ovary CHO-K1 cells, but not the same cells treated with bacterial sphingomyelinase, were immunostained with clamlysin B. These results indicate that clamlysin B binds to the sphingomyelin of living cells and thus would be useful as a molecular probe to detect sphingomyelin.