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PubMed2003

Sphingolipid storage induces accumulation of intracellular cholesterol by stimulating SREBP-1 cleavage.

We showed previously that the intracellular transport of sphingolipids (SLs) is altered in SL storage disease fibroblasts, due in part to the secondary accumulation of free cholesterol. In the present study we examined the mechanism of cholesterol elevation in normal human skin fibroblasts induced by treatment with SLs. When cells were incubated with various natural SLs for 44 h, cholesterol levels increased 25-35%, and cholesterol esterification was reduced. Catabolism of the exogenous SLs was not required for elevation of cholesterol because (i) a non-hydrolyzable and a degradable SL analog elevated cellular cholesterol to similar extents, and (ii) incubation of cells with various SL catabolites, including ceramide, had no effect on cholesterol levels. Elevated cholesterol was derived primarily from low density lipoproteins (LDL) and resulted from up-regulation of LDL receptors induced by cleavage of the sterol regulatory element-binding protein-1. Upon SL treatment, cholesterol accumulated with exogenous SLs in late endosomes and lysosomes. These results suggest a model in which excess SLs present in endocytic compartments serve as a "molecular trap" for cholesterol, leading to a reduction in cholesterol at the endoplasmic reticulum, induction of sterol regulatory element-binding protein-1 cleavage, and up-regulation of LDL receptors.

CCAAT-Enhancer-Binding Proteins↗
PubMed2004

HOR7, a multicopy suppressor of the Ca2+-induced growth defect in sphingolipid mannosyltransferase-deficient yeast.

Yeast mutants defective in sphingolipid mannosylation accumulate inositol phosphorylceramide C (IPC-C), which renders cells Ca(2+)-sensitive. A screen for loss of function suppressors of the Ca(2+)-sensitive phenotype previously led to the identification of numerous genes involved in IPC-C synthesis. To better understand the molecular basis of the Ca(2+)-induced growth defect in IPC-C-overaccumulating cells, we searched for genes whose overexpression restored Ca(2+) tolerance in a mutant lacking the IPC mannosyltransferases Csg1p and Csh1p. Here we report the isolation of HOR7 as a multicopy suppressor of the Ca(2+)-sensitive phenotype of Deltacsg1Deltacsh1 cells. HOR7 belongs to a group of hyperosmolarity-responsive genes and encodes a small (59-residue) type I membrane protein that localizes at the plasma membrane. Hor7p is not required for high Ca(2+) or Na(+) tolerance. Instead, we find that Hor7p-overproducing cells display an increased resistance to high salt, sensitivity to low pH, and a reduced uptake of methylammonium, an indicator of the plasma membrane potential. These phenotypes are induced through a mechanism independent of the plasma membrane H(+)-ATPase, Pma1p. Our findings suggest that induction of Hor7p causes a depolarization of the plasma membrane that may counteract a Ca(2+)-induced influx of toxic cations in IPC-C-overaccumulating cells.

Adenosine Triphosphatases↗
PubMed2005

SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism.

The potent sphingolipid metabolite sphingosine 1-phosphate is produced by phosphorylation of sphingosine catalyzed by sphingosine kinase (SphK) types 1 and 2. In contrast to pro-survival SphK1, the putative BH3-only protein SphK2 inhibits cell growth and enhances apoptosis. Here we show that SphK2 catalytic activity also contributes to its ability to induce apoptosis. Overexpressed SphK2 also increased cytosolic free calcium induced by serum starvation. Transfer of calcium to mitochondria was required for SphK2-induced apoptosis, as cell death and cytochrome c release was abrogated by inhibition of the mitochondrial Ca(2+) transporter. Serum starvation increased the proportion of SphK2 in the endoplasmic reticulum and targeting SphK1 to the endoplasmic reticulum converted it from anti-apoptotic to pro-apoptotic. Overexpression of SphK2 increased incorporation of [(3)H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide, whereas SphK1 decreased it. Electrospray ionizationmass spectrometry/mass spectrometry also revealed an opposite effect on ceramide mass levels. Importantly, specific down-regulation of SphK2 reduced conversion of sphingosine to ceramide in the recycling pathway and conversely, down-regulation of SphK1 increased it. Our results demonstrate that SphK1 and SphK2 have opposing roles in the regulation of ceramide biosynthesis and suggest that the location of sphingosine 1-phosphate production dictates its functions.

3T3 Cells↗
PubMed2006

Ceramide regulates atypical PKCzeta/lambda-mediated cell polarity in primitive ectoderm cells. A novel function of sphingolipids in morphogenesis.

In mammals, the primitive ectoderm is an epithelium of polarized cells that differentiates into all embryonic tissues. Our study shows that in primitive ectoderm cells, the sphingolipid ceramide was elevated and co-distributed with the small GTPase Cdc42 and cortical F-actin at the apicolateral cell membrane. Pharmacological or RNA interference-mediated inhibition of ceramide biosynthesis enhanced apoptosis and impaired primitive ectoderm formation in embryoid bodies differentiated from mouse embryonic stem cells. Primitive ectoderm formation was restored by incubation with ceramide or a ceramide analog. Ceramide depletion prevented plasma membrane translocation of PKCzeta/lambda, its interaction with Cdc42, and phosphorylation of GSK-3beta, a substrate of PKCzeta/lambda. Recombinant PKCzeta formed a complex with the polarity protein Par6 and Cdc42 when bound to ceramide containing lipid vesicles. Our data suggest a novel mechanism by which a ceramide-induced, apicolateral polarity complex with PKCzeta/lambda regulates primitive ectoderm cell polarity and morphogenesis.

Actins↗
PubMedDate not supplied

Modulation of entry of enveloped viruses by cholesterol and sphingolipids (Review).

Enveloped animal viruses infect host cells by fusion of viral and target membranes. This crucial fusion event occurs either with the plasma membrane of the host cells at the physiological pH or with the endosomal membranes at low pH and is triggered by specific glycoproteins in the virus envelope. Both lipids and proteins play critical and co-operative roles in the fusion process. Interactions of viral proteins with their receptors direct which membranes fuse and viral fusion proteins then drive the process. These fusion proteins operate on lipid assemblies, whose physical and mechanical properties are equally important to the proper functioning of the process. Lipids contribute to the viral fusion process by virtue of their distinct chemical structure, composition and/or their preferred partitioning into specific microdomains in the plasma membrane called 'rafts'. An involvement of lipid rafts in viral entry and membrane fusion has been examined recently. However, the mechanism(s) by which lipids as dynamic raft components control viral envelope-glycoprotein-triggered fusion is not clear. This paper will review literature findings on the contribution of the two raft-associated lipids, cholesterol and sphingolipids in viral entry.

Animals↗
PubMed2004

A new sphingolipid from the gorgonian Junceella juncea of the Indian Ocean.

A new sphingolipid, (2S,3R,4E)-1,3-dihydroxy-2-[(nonadecanoyl) amino]-octadec-4-ene (1) along with ubiquitous batyl alcohol is isolated from the gorgonian, Junceella juncea Pallas (Gorgonaceae) of the Indian Ocean. The structure of 1 is determined on the basis of 1H- and 13C-NMR, COSY, FABMS, and GC-EIMS experiments.

Animals↗
PubMed1985

Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane.

When monolayer cultures of Chinese hamster lung fibroblasts are briefly incubated at 2 degrees C with the fluorescent sphingolipid analogue, C6-NBD-ceramide (N- [7-(4-nitrobenzo-2-oxa-1,3-diazole)] aminocaproyl sphingosine), fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope occur. During further incubation at 37 degrees C, the Golgi apparatus, and later the plasma membrane, become intensely fluorescent. Within this period, the C6-NBD-ceramide is converted to equal amounts of fluorescent sphingomyelin and glucocerebroside (Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA., 80:2608-2612). In the present study, the intracellular translocation of these metabolites and their subsequent appearance at the plasma membrane were investigated by fluorescence microscopy, the addition of the ionophore monensin, and the technique of "back exchange," in which the amounts and types of fluorescent lipids present at the cell surface are identified after their transfer from the cell surface into recipient vesicles. In control cells, the amount of fluorescent glucocerebroside and sphingomyelin that could be removed from the cell surface by back exchange increased during incubation at 37 degrees C, correlating with the increased fluorescence of the plasma membrane observed by microscopy. In the presence of 10 microM monensin, visible labeling of the plasma membrane was greatly diminished, whereas the Golgi apparatus became highly fluorescent and distended. The ability to remove fluorescent metabolites from the cell surface by back exchange was significantly but reversibly inhibited by monensin. Monensin also increased the total amount of fluorescent sphingomyelin, but not the glucocerebroside found in cells. Subcellular fractions were assayed for their ability to convert radiolabeled and fluorescent ceramides to the corresponding sphingomyelins and glucocerebrosides. The activities of parallel fractions coincided, suggesting that the presence of the NBD moiety did not affect the cellular metabolism of ceramide. Furthermore, the major peak of sphingomyelin- and glucocerebroside-synthesizing activity appeared to coincide with an enriched Golgi fraction. These results strongly suggest that fluorescent sphingomyelin was not synthesized at the plasma membrane as has recently been suggested for endogenous sphingomyelin. Rather, both the sphingomyelin and glucocerebroside analogues were synthesized intracellularly from C6-NBD-ceramide and translocated through the Golgi apparatus to the cell surface.

Animals↗
PubMed1987

Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells.

To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as a probe. This ceramide was readily taken up by filter-grown Madin-Darby canine kidney (MDCK) cells from liposomes at 0 degrees C. After penetration into the cell, the fluorescent probe accumulated in the Golgi area at temperatures between 0 and 20 degrees C. Chemical analysis showed that C6-NBD-ceramide was being converted into C6-NBD-sphingomyelin and C6-NBD-glucosyl-ceramide. An analysis of the fluorescence pattern after 1 h at 20 degrees C by means of a confocal scanning laser fluorescence microscope revealed that the fluorescent marker most likely concentrated in the Golgi complex itself. Little fluorescence was observed at the plasma membrane. Raising the temperature to 37 degrees C for 1 h resulted in intense plasma membrane staining and a loss of fluorescence from the Golgi complex. Addition of BSA to the apical medium cleared the fluorescence from the apical but not from the basolateral plasma membrane domain. The basolateral fluorescence could be depleted only by adding BSA to the basal side of a monolayer of MDCK cells grown on polycarbonate filters. We conclude that the fluorescent sphingomyelin and glucosylceramide were delivered from the Golgi complex to the plasma membrane where they accumulated in the external leaflet of the membrane bilayer. The results also demonstrated that the fatty acyl labeled lipids were unable to pass the tight junctions in either direction. Quantitation of the amount of NBD-lipids delivered to the apical and the basolateral plasma membranes during incubation for 1 h at 37 degrees C showed that the C6-NBD-glucosylceramide was two- to fourfold enriched on the apical as compared to the basolateral side, while C6-NBD-sphingomyelin was about equally distributed. Since the surface area of the apical plasma membrane is much smaller than that of the basolateral membrane, both lipids achieved a higher concentration on the apical surface. Altogether, our results suggest that the NBD-lipids are sorted in MDCK cells in a way similar to their natural counterparts.

Animals↗
PubMed1990

Generation of lipid polarity in intestinal epithelial (Caco-2) cells: sphingolipid synthesis in the Golgi complex and sorting before vesicular traffic to the plasma membrane.

Generation of intestinal epithelial lipid polarity was studied in Caco-2 cells. Confluent monolayers on filters incorporated the exchangeable lipid N-6-NBD-aminocaproyl-sphingosine (C6-NBD-ceramide) from liposomes. The fluorescent ceramide was converted equally to C6-NBD-glucosylceramide and C6-NBD-sphingomyelin, analogues of lipids enriched on the apical and basolateral surface, respectively, of intestinal cells in vivo. Below 16 degrees C, where vesicular traffic is essentially blocked, each fluorescent product accumulated in the Golgi area. At 37 degrees C, 50% had been transported to the cell surface within 0.5 h, as measured by selective extraction of the fluorescent lipids onto BSA in the medium ("back-exchange") at 10 degrees C. Transport to the two surfaces could be assayed separately, as a diffusion barrier existed for both NBD-lipids and BSA. C6-NBD-glucosylceramide was enriched twofold apically, whereas C6-NBD-sphingomyelin was equally distributed over both domains. Polarities did not decrease when 37 degrees C incubations were carried out in the presence of increasing BSA concentrations to trap the fluorescent lipids immediately after their arrival at the cell surface. Within 10 min from the start of synthesis, both products displayed their typical surface polarity. Lipid transcytosis displayed a half time of hours. In conclusion, newly synthesized sphingolipids in Caco-2 cells are sorted before reaching the cell surface. Transcytosis is not required for generating the in vivo lipid polarity.

Animals↗
PubMed2001

N-terminal protein acylation confers localization to cholesterol, sphingolipid-enriched membranes but not to lipid rafts/caveolae.

When variably fatty acylated N-terminal amino acid sequences were appended to a green fluorescent reporter protein (GFP), chimeric GFPs were localized to different membranes in a fatty acylation-dependent manner. To explore the mechanism of localization, the properties of acceptor membranes and their interaction with acylated chimeric GFPs were analyzed in COS-7 cells. Myristoylated GFPs containing a palmitoylated or polybasic region colocalized with cholesterol and ganglioside GM(1), but not with caveolin, at the plasma membrane and endosomes. A dipalmitoylated GFP chimera colocalized with cholesterol and GM(1) at the plasma membrane and with caveolin in the Golgi region. Acylated GFP chimeras did not cofractionate with low-density caveolin-rich lipid rafts prepared with Triton X-100 or detergent-free methods. All GFP chimeras, but not full-length p62(c-yes) and caveolin, were readily solubilized from membranes with various detergents. These data suggest that, although N-terminal acylation can bring GFP to cholesterol and sphingolipid-enriched membranes, protein-protein interactions are required to localize a given protein to detergent-resistant membranes or caveolin-rich membranes. In addition to restricting acceptor membrane localization, N-terminal fatty acylation could represent an efficient means to enrich the concentration of signaling proteins in the vicinity of detergent-resistant membranes and facilitate protein-protein interactions mediating transfer to a detergent-resistant lipid raft core.

Acylation↗
PubMed2006

Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements.

Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.

Animals↗
PubMed1997

Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain.

Sphingolipid-cholesterol rafts are microdomains in biological membranes with liquid-ordered phase properties which are implicated in membrane traffic and signalling events. We have used influenza virus haemagglutinin (HA) as a model protein to analyse the interaction of transmembrane proteins with these microdomains. Here we demonstrate that raft association is an intrinsic property encoded in the protein. Mutant HA molecules with foreign transmembrane domain (TMD) sequences lose their ability to associate with the lipid microdomains, and mutations in the HA TMD reveal a requirement for hydrophobic residues in contact with the exoplasmic leaflet of the membrane. We also provide experimental evidence that cholesterol is critically required for association of proteins with lipid rafts. Our data suggest that the binding to specific membrane domains can be encoded in transmembrane proteins and that this information will be used for polarized sorting and signal transduction processes.

Amino Acid Sequence↗
PubMed2003

The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.

Most soluble lysosomal proteins bind the mannose 6-phosphate receptor (M6P-R) to be sorted to the lysosomes. However, the lysosomes of I-cell disease (ICD) patients, a condition resulting from a mutation in the phosphotransferase that adds mannose 6-phosphate to hydrolases, have near normal levels of several lysosomal proteins, including the sphingolipid activator proteins (SAPs), GM2AP and prosaposin. We tested the hypothesis that SAPs are targeted to the lysosomal compartment via the sortilin receptor. To test this hypothesis, a dominant-negative construct of sortilin and a sortilin small interfering RNA (siRNA) were introduced into COS-7 cells. Our results showed that both the truncated sortilin and the sortilin siRNA block the traffic of GM2AP and prosaposin to the lysosomal compartment. This observation was confirmed by a co-immunoprecipitation, which demonstrated that GM2AP and prosaposin are interactive partners of sortilin. Furthermore, a dominant-negative mutant GGA prevented the trafficking of prosaposin and GM2AP to lysosomes. In conclusion, our results show that the trafficking of SAPs is dependent on sortilin, demonstrating a novel lysosomal trafficking.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

The N-glycosylation defect of cwh8Delta yeast cells causes a distinct defect in sphingolipid biosynthesis.

CWH8/YGR036c of Saccharomyces cerevisiae has been identified as a dolichylpyrophosphate (Dol-PP) phosphatase that removes a phosphate from the Dol-PP generated by the oligosaccharyltransferase (OST), while it adds N-glycans to nascent glycoproteins in the endoplasmic reticulum (ER). Lack of CWH8 was proposed to interrupt the so called dolichol (Dol) cycle by trapping Dol in the form of Dol-PP in the ER lumen. Indeed, cwh8Delta mutants display a severe deficiency in N-glycosylation. We find that cwh8Delta mutants have strongly reduced levels of inositolphosphorylceramide (IPC), whereas its derivative, mannosyl-(inositol-P)2-ceramide (M(IP)2C) is not affected. Microsomes of cwh8Delta contain normal ceramide synthase and IPC synthesis activities. Within a large panel of mutants affecting Dol dependent pathways such as N- or O-glycosylation, or glycosylphosphatidyl inositol (GPI)-anchoring, only the mutants having a deficiency of N-glycan addition show the defect in IPC biosynthesis. By mutating genes required for the addition of N-glycans or by treating cells with tunicamycin (Tm) one can similarly reduce the steady state level of IPC and exactly reproduce the phenotype of cwh8Delta cells. Some potential mechanisms by which the lack of N-glycans could lead to the sphingolipid abnormality were further explored.

Glycolipids↗
PubMed2003

Sphingosine-1-phosphate formation activates phosphatidylinositol-4 kinase in basolateral membranes from kidney cells: crosstalk in cell signaling through sphingolipids and phospholipids.

Sphingosine-1-phosphate (S1P) and phosphatidylinositol-4 phosphate [PtdIns(4)P] are important second messengers in various cellular processes. Here, we show that S1P and PtdIns(4)P are formed in purified basolateral membranes (BLM) derived from kidney proximal tubules, indicating the presence of a plasma membrane associated SPK (BLM-SPK) and phosphatidylinositol-4 kinase (PI-4K). We observed that S1P synthesis is linear with time, dependent on protein concentration, and saturable in the presence of increasing concentrations of sphingosine. Different from the observations on cytosolic SPKs, the formation of S1P by BLM-SPK is Mg(2+)-independent and insensitive to the classical inhibitor of the cytosolic SPKs, DL-threo-dihydrosphingosine. With sphingosine as substrate, the enzyme shows cooperative kinetics (n = 3.4) with a K(0.5) value of 0.12 mM, suggesting that BLM-SPK is different from the previously characterized cytosolic SPK. The formation of PtdIns(4)P markedly inhibits BLM-SPK activity. Conversely, a strong activation of PtdIns(4)P synthesis by the formation of S1P is observed. Taken together, these results indicate that (i) basolateral membranes from kidney cells harbor a SPK activity that potentially regulates renal epithelium function, and (ii) the formation of S1P mediated by SPK enhances PI-4K activity, while PtdIns(4)P in turn inhibits SPK, suggesting an interplay between these lipid signaling molecules. These findings suggest the possibility of crosstalk between sphingolipids and glycerolipids, which might be involved in the regulation of transepithelial fluxes across the BLM of kidney cells.

1-Phosphatidylinositol 4-Kinase↗
PubMed2004

Dual effects of IGFBP-3 on endothelial cell apoptosis and survival: involvement of the sphingolipid signaling pathways.

Insulin-like growth factor binding protein (IGFBP)-3 has both growth-inhibiting and growth-promoting effects at the cellular level. The cytotoxic action of several anticancer drugs is linked to increased ceramide generation through sphingomyelin hydrolysis or de novo biosynthesis. Herein, we investigated the role of IGFBP-3 on apoptosis of human umbilical vein endothelial cells (HUVEC) and its relationship with ceramide levels. We report that IGFBP-3 exerts dual effects on HUVEC, potentiating doxorubicin-induced apoptosis but enhancing survival in serum-starved conditions. Ceramide was increased by IGFBP-3 in the presence of doxorubicin and decreased when IGFBP-3 was added alone to cells cultured in serum-free medium. The protection exerted by the ceramide synthase inhibitor fumonisin B1 over doxorubicin-induced apoptosis was enhanced by IGFBP-3 with concomitant reduction of ceramide levels. IGFBP-3 alone activated sphingosine kinase (SK) and increased SK1 mRNA; the SK inhibitor N,N-dimethylsphingosine (DMS) blocked IGFBP-3 antiapoptotic effect. Moreover, IGFBP-3 increased IGF-I mRNA and dramatically enhanced IGF-I release. IGF-I receptor (IGF-IR) and its downstream signaling pathways Akt and ERK were phosphorylated by IGFBP-3, whereas inhibition of IGF-IR phosphorylation with tyrphostin AG1024 suppressed the antiapopoptic effect of IGFBP-3. Finally, IGFBP-3 increased endothelial cell motility in all experimental conditions. These findings provide evidence that IGFBP-3 differentially regulates endothelial cell apoptosis by involvement of the sphingolipid signaling pathways. Moreover, the survival effect of IGFBP-3 seems to be mediated by the IGF-IR.

Apoptosis↗
PubMed2005

Evidence for a link between sphingolipid metabolism and expression of CD1d and MHC-class II: monocytes from Gaucher disease patients as a model.

Gaucher disease (GD) is an autosomal recessive inherited defect of the lysosomal enzyme glucocerebrosidase (GluCerase) that leads to glucosylceramide (GluCer) accumulation. We previously demonstrated the existence of imbalances in certain lymphocyte populations in GD patients. We now show that GluCerase-deficient monocytes from GD patients or monocytes from healthy subjects treated with conduritol-B-epoxide (CBE), an irreversible inhibitor of GluCerase activity, display high levels of surface expression of the lipid-binding molecule CD1d. GluCerase-deficient monocytes from GD patients also showed increased surface expression of major histocompatibility complex (MHC)-class II, but not of other lysosomal trafficking molecules, such as CD63 and MHC-class I. However, CD1d and MHC-class II mRNA levels were not increased. GluCerase-deficient monocytes from GD patients undergoing enzyme replacement therapy also exhibited increased levels of CD1d and MHC-class II and imbalances in the percentage of CD4+, CD8+, and Valpha24+ T cells. Interestingly, follow-up studies revealed that enzyme replacement therapy induced a decrease in MHC-class II expression and partial correction of the CD4+ T cell imbalances. These results reveal a new link between sphingolipid accumulation in monocytes and the expression of certain MHC molecules that may result in imbalances of regulatory T cell subsets. These immunological anomalies may contribute to the clinical heterogeneity in GD patients.

Adult↗
PubMed2005

Leishmania salvage and remodelling of host sphingolipids in amastigote survival and acidocalcisome biogenesis.

Sphingolipids (SLs) play essential roles in most eukaryotes, but in the trypanosomatid protozoan Leishmania major their functions differ significantly. Previously we showed that null mutants defective in de novo sphingoid base synthesis (spt2-) lacked SLs but grew well and retained lipid rafts while replicating as promastigotes in vitro. However, they experienced catastrophic defects in membrane trafficking on entry into stationary phase, and failed to differentiate to the infective metacyclic form. Here we showed this mutant retained the ability to enter macrophages silently and inhibit activation, although as expected most parasites were destroyed. However, in mouse infections, after a delay rapidly progressive lesions appeared, and purified amastigotes were fully virulent to macrophages and mice. Mass spectrometry of spt2- amastigote lipids revealed the presence of high levels of parasite-specific inositol phosphorylceramides (IPCs) not synthesized by the mammalian hosts. Inhibitor studies showed that salvage occurs at the level of complex SLs, suggesting that parasites carry out 'headgroup' remodelling. Additionally, we describe a new defect of the spt2- promastigotes involving 'empty' acidocalcisomes (ACs), which may point to the origin of this organelle from the lysosome-related organelle/multivesicular body biogenesis pathway. However, ACs in spt2- amastigotes appeared quantitatively and morphologically normal. Thus salvage of SLs and other molecules by intracellular amastigotes play key roles in AC biogenesis and parasite survival in the host.

Animals↗