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Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in humans. At early stages CRC is treated by surgery and at advanced stages combined with chemotherapy. We examined here the potential effect of glucosylceramide synthase (GCS)-inhibition on CRC biology. GCS is the rate-limiting enzyme in the glycosphingolipid (GSL)-biosynthesis pathway and overexpressed in many human tumors. We suppressed GSL-biosynthesis using the GCS inhibitor Genz-123346 (Genz), NB-DNJ (Miglustat) or by genetic targeting of the GCS-encoding gene UDP-glucose-ceramide-glucosyltransferase- ( UGCG ). GCS-inhibition or GSL-depletion led to a marked arrest of the cell cycle in Lovo cells. UGCG silencing strongly also inhibited tumor spheroid growth in Lovo cells and moderately in HCT116 cells. MS/MS analysis demonstrated markedly elevated levels of sphingomyelin (SM) and phosphatidylcholine (PC) that occurred in a Genz-concentration dependent manner. Ultrastructural analysis of Genz-treated cells indicated multi-lamellar lipid storage in vesicular compartments. In mice, Genz lowered the incidence of experimentally induced colorectal tumors and in particular the growth of colorectal adenomas. These results highlight the potential for GCS-based inhibition in the treatment of CRC.

The liver X receptor (LXR) is a key transcriptional regulator of cholesterol, fatty acid, and phospholipid metabolism. Dynamic remodeling of immunometabolic pathways, including lipid metabolism, is a crucial step in T cell activation. Here, we explored the role of LXR-regulated metabolic processes in primary human CD4 + T cells and their role in controlling plasma membrane lipids (glycosphingolipids and cholesterol), which strongly influence T cell immune signaling and function. Crucially, we identified the glycosphingolipid biosynthesis enzyme glucosylceramide synthase as a direct transcriptional LXR target. LXR activation by agonist GW3965 or endogenous oxysterol ligands significantly altered the glycosphingolipid:cholesterol balance in the plasma membrane by increasing glycosphingolipid levels and reducing cholesterol. Consequently, LXR activation lowered plasma membrane lipid order (stability), and an LXR antagonist could block this effect. LXR stimulation also reduced lipid order at the immune synapse and accelerated activation of proximal T cell signaling molecules. Ultimately, LXR activation dampened proinflammatory T cell function. Finally, compared with responder T cells, regulatory T cells had a distinct pattern of LXR target gene expression corresponding to reduced lipid order. This suggests LXR-driven lipid metabolism could contribute to functional specialization of these T cell subsets. Overall, we report a mode of action for LXR in T cells involving the regulation of glycosphingolipid and cholesterol metabolism and demonstrate its relevance in modulating T cell function., Competing Interests: The authors declare no competing interest.

Bardet-Biedl syndrome (BBS) is a pleiotropic autosomal recessive ciliopathy affecting multiple organs. The development of potential disease-modifying therapy for BBS will require concurrent targeting of multi-systemic manifestations. Here, we show for the first time that monosialodihexosylganglioside accumulates in Bbs2-/- cilia, indicating impairment of glycosphingolipid (GSL) metabolism in BBS. Consequently, we tested whether BBS pathology in Bbs2-/- mice can be reversed by targeting the underlying ciliary defect via reduction of GSL metabolism. Inhibition of GSL synthesis with the glucosylceramide synthase inhibitor Genz-667161 decreases the obesity, liver disease, retinal degeneration and olfaction defect in Bbs2-/- mice. These effects are secondary to preservation of ciliary structure and signaling, and stimulation of cellular differentiation. In conclusion, reduction of GSL metabolism resolves the multi-organ pathology of Bbs2-/- mice by directly preserving ciliary structure and function towards a normal phenotype. Since this approach does not rely on the correction of the underlying genetic mutation, it might translate successfully as a treatment for other ciliopathies., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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 Z ymomonas 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., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Okino et al.)

All organisms have stress response systems to protect themselves from various environmental stresses, and regulation of membrane lipids is thought to play an important role in acquirement of stress tolerance. Complex sphingolipids in the yeast Saccharomyces cerevisiae are classified into three types based on differences in the structure of the polar head group, and the compositions and quantities of complex sphingolipids in biomembranes are tightly regulated. In this study, we found that the accumulation of inositol phosphorylceramides (IPCs) due to a defect of mannosylinositol phosphorylceramide biosynthesis (sur1∆ csh1∆), i.e., disruption of the balance of the composition of complex sphingolipids, causes hypersensitivity to low pH conditions (pH 4.0-2.5). Furthermore, screening of suppressor mutations that confer low pH resistance to sur1∆ csh1∆ cells revealed that a change in ergosterol homeostasis at plasma membranes can rescue the hypersensitivity, suggesting the functional relationship between complex sphingolipids and ergosterol under low pH conditions. Under low pH conditions, wild-type yeast cells exhibited decreases in IPC levels, and forced enhancement of the biosynthesis of IPCs causes low pH hypersensitivity. Thus, it was suggested that the accumulation of IPCs is detrimental to yeast under low pH conditions, and downregulation of IPC levels is one of the adaptation mechanisms for low pH conditions.

Multidrug resistance (MDR) in cancer arises from cross-resistance to structurally- and functionally-divergent chemotherapeutic drugs. In particular, MDR is characterized by increased expression and activity of ATP-binding cassette (ABC) superfamily transporters. Sphingolipids are substrates of ABC proteins in cell signaling, membrane biosynthesis, and inflammation, for example, and their products can favor cancer progression. Glucosylceramide (GlcCer) is a ubiquitous glycosphingolipid (GSL) generated by glucosylceramide synthase, a key regulatory enzyme encoded by the UDP-glucose ceramide glucosyltransferase ( UGCG ) gene. Stressed cells increase de novo biosynthesis of ceramides, which return to sub-toxic levels after UGCG mediates incorporation into GlcCer. Given that cancer cells seem to mobilize UGCG and have increased GSL content for ceramide clearance, which ultimately contributes to chemotherapy failure, here we investigated how inhibition of GSL biosynthesis affects the MDR phenotype of chronic myeloid leukemias. We found that MDR is associated with higher UGCG expression and with a complex GSL profile. UGCG inhibition with the ceramide analog d-threo-1-(3,4,-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4) greatly reduced GSL and monosialotetrahexosylganglioside levels, and co-treatment with standard chemotherapeutics sensitized cells to mitochondrial membrane potential loss and apoptosis. ABC subfamily B member 1 (ABCB1) expression was reduced, and ABCC-mediated efflux activity was modulated by competition with nonglycosylated ceramides. Consistently, inhibition of ABCC-mediated transport reduced the efflux of exogenous C 6 -ceramide. Overall, UGCG inhibition impaired the malignant glycophenotype of MDR leukemias, which typically overcomes drug resistance through distinct mechanisms. This work sheds light on the involvement of GSL in chemotherapy failure, and its findings suggest that targeted GSL modulation could help manage MDR leukemias., (© 2020 Salustiano et al.)

Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids interact with each other in the mammalian plasma membranes, forming dynamic microdomains. How their interaction starts in the cells has been unclear. Here, based on a genome-wide CRISPR-Cas9 genetic screen for genes required for GPI side-chain modification by galactose in the Golgi apparatus, we report that β1,3-galactosyltransferase 4 (B3GALT4), the previously characterized GM1 ganglioside synthase, additionally functions in transferring galactose to the N-acetylgalactosamine side-chain of GPI. Furthermore, B3GALT4 requires lactosylceramide for the efficient GPI side-chain galactosylation. Thus, our work demonstrates previously unexpected functional relationships between GPI-anchored proteins and glycosphingolipids in the Golgi. Through the same screening, we also show that GPI biosynthesis in the endoplasmic reticulum (ER) is severely suppressed by ER-associated degradation to prevent GPI accumulation when the transfer of synthesized GPI to proteins is defective. Our data demonstrates cross-talks of GPI biosynthesis with glycosphingolipid biosynthesis and the ER quality control system.

Background: Leishmania spp. are digenetic parasites capable of infecting humans and causing a range of diseases collectively known as leishmaniasis. The main mechanisms involved in the development and permanence of this pathology are linked to evasion of the immune response. Crosstalk between the immune system and particularities of each pathogenic species is associated with diverse disease manifestations. Lipophosphoglycan (LPG), one of the most important molecules present on the surface of Leishmania parasites, is divided into four regions with high molecular variability. Although LPG plays an important role in host-pathogen and vector-parasite interactions, the distribution and phylogenetic relatedness of the genes responsible for its synthesis remain poorly explored. The recent availability of full genomes and transcriptomes of Leishmania parasites offers an opportunity to leverage insight on how LPG-related genes are distributed and expressed by these pathogens., Results: Using a phylogenomics-based framework, we identified a catalog of genes involved in LPG biosynthesis across 22 species of Leishmania from the subgenera Viannia and Leishmania, as well as 5 non-Leishmania trypanosomatids. The evolutionary relationships of these genes across species were also evaluated. Nine genes related to the production of the glycosylphosphatidylinositol (GPI)-anchor were highly conserved among compared species, whereas 22 genes related to the synthesis of the repeat unit presented variable conservation. Extensive gain/loss events were verified, particularly in genes SCG1-4 and SCA1-2. These genes act, respectively, on the synthesis of the side chain attached to phosphoglycans and in the transfer of arabinose residues. Phylogenetic analyses disclosed evolutionary patterns reflective of differences in host specialization, geographic origin and disease manifestation., Conclusions: The multiple gene gain/loss events identified by genomic data mining help to explain some of the observed intra- and interspecies variation in LPG structure. Collectively, our results provide a comprehensive catalog that details how LPG-related genes evolved in the Leishmania parasite specialization process.

Metabolites are small intermediate products of cellular metabolism perturbed in a variety of complex disorders. Identifying genetic markers associated with metabolite concentrations could delineate disease-related metabolic pathways in humans. We tested genetic variants for associations with 136 metabolites in 1954 Chinese from Singapore. At a conservative genome-wide threshold (3.7 × 10-10), we detected 1899 variant-metabolite associations at 16 genetic loci. Three loci (ABCA7, A4GALT, GSTM2) represented novel associations with metabolites, with the strongest association observed between ABCA7 and d18:1/24:1 dihexosylceramide. Among 13 replicated loci, we identified six new variants independent of previously reported metabolite or lipid signals. We observed variant-metabolite associations at two loci (ABCA7, CHCHD2) that have been linked to neurodegenerative diseases. At SGPP1 and SPTLC3 loci, genetic variants showed preferential selectivity for sphingolipids with d16 (rather than d18) sphingosine backbone, including sphingosine-1-phosphate (S1P). Our results provide new genetic associations for metabolites and highlight the role of metabolites as intermediate modulators in disease metabolic pathways., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Fabry disease is caused by α-galactosidase A deficiency. Substrates of this lysosomal enzyme accumulate, resulting in cellular dysfunction. Patients experience neuropathic pain, kidney failure, heart disease, and strokes., Scope of Review: The clinical picture and molecular features of Fabry disease are described, along with updates on disease mechanisms, animal models, and therapies., Major Conclusions: How the accumulation of α-galactosidase A substrates, mainly glycosphingolipids, leads to organ damage is incompletely understood. Enzyme replacement and chaperone therapies are clinically available to patients, while substrate reduction, mRNA-based, and gene therapies are on the horizon. Animal models exist to optimize these therapies and elucidate disease mechanisms for novel treatments., General Significance: Recent newborn screening studies demonstrate that Fabry disease is the most common lysosomal storage disease. As many countries now include Fabry disease in their screening panels, the number of identified patients is expected to increase significantly. Better knowledge of disease pathogenesis is needed to improve treatment options., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Colorectal cancer (CRC) affects both women and men living in societies with a high sedentary lifestyle. Amongst the phenotypic changes exhibited by tumor cells, a wide range of glycosylation has been reported for colon cancer-derived cell lines and CRC tissues. These aberrant modifications affect different aspects of glycosylation, including an increase in core fucosylation and GlcNAc branching on N-glycans, alteration of O-glycans, upregulated sialylation, and O-GlcNAcylation. Although O-GlcNAcylation and complex glycosylations differ in many aspects, sparse evidences report on the interference of O-GlcNAcylation with complex glycosylation. Nevertheless, this relationship is still a matter of debate. Combining different approaches on three human colon cell lines (HT29, HCT116 and CCD841CoN), it is herein reported that silencing O-GlcNAc transferase (OGT, the sole enzyme driving O-GlcNAcylation), only slightly affects overall N- and O-glycosylation patterns. Interestingly, silencing of OGT in HT29 cells upregulates E-cadherin (a major actor of epithelial-to-mesenchymal transition) and changes its glycosylation. On the other hand, OGT silencing perturbs biosynthesis of glycosphingolipids resulting in a decrease in gangliosides and an increase in globosides. Together, these results provide novel insights regarding the selective regulation of complex glycosylations by O-GlcNAcylation in colon cancer cells., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Glycosphingolipids are known to play a role in developing and maintaining the integrity of various organs and tissues. Among glycosphingolipids, there are several reports on the involvement of gangliosides in bone metabolism. However, there have been no reports on the presence or absence of expression of globo-series glycosphingolipids in osteoblasts and osteoclasts, and the involvement of their glycosphingolipids in bone metabolism. In the present study, we investigated the presence or absence of globo-series glycosphingolipids such as Gb3 (globotriaosylceramide), Gb4 (globoside), and Gb5 (galactosyl globoside) in osteoblasts and osteoclasts, and the effects of genetic deletion of Gb3 synthase, which initiates the synthesis of globo-series glycosphingolipids on bone metabolism. Among Gb3, Gb4, and Gb5, only Gb4 was expressed in osteoblasts. However, these glycosphingolipids were not expressed in pre-osteoclasts and osteoclasts. Three-dimensional micro-computed tomography (3D-μCT) analysis revealed that femoral cancellous bone mass in Gb3 synthase-knockout (Gb3S KO) mice was lower than that in wild type (WT) mice. Calcein double labeling also revealed that bone formation in Gb3S KO mice was significantly lower than that in WT mice. Consistent with these results, the deficiency of Gb3 synthase in mice decreased the number of osteoblasts on the bone surface, and suppressed mRNA levels of osteogenic differentiation markers. On the other hand, osteoclast numbers on the bone surface and mRNA levels of osteoclast differentiation markers in Gb3S KO mice did not differ from WT mice. This study demonstrated that deletion of Gb3 synthase in mice decreases bone mass via attenuation of bone formation.

Hereditary sensory and autonomic neuropathy Type 1 (HSAN1) is a rare autosomal dominantly inherited neuropathy, clinically characterized by a loss of distal peripheral sensory and motoneuronal function. Mutations in subunits of serine palmitoyltransferase (SPT) have been linked to the majority of HSAN1 cases. SPTs catalyze the condensation of l-serine with palmitoyl-CoA, the first committed and rate-limiting step in de novo sphingolipid biosynthesis. Despite extensive investigation, the molecular pathogenesis of HSAN1 remains controversial. Here, we established a Caenorhabditis elegans ( C. elegans ) model of HSAN1 by generating a sptl-1(c363g ) mutation, encoding SPTL-1(C121W) and equivalent to human SPTLC1 C133W , at the C. elegans genomic locus through CRISPR. The sptl-1(c363g ) homozygous mutants exhibited the same larval lethality and epithelial polarity defect as observed in sptl-1(RNAi ) animals, suggesting a loss-of-function effect of the SPTL-1(C121W) mutation. sptl-1(c363g) /+ heterozygous mutants displayed sensory dysfunction with concomitant neuronal morphology and axon-dendrite polarity defects, demonstrating that the C. elegans model recapitulates characteristics of the human disease. sptl-1(c363g )-derived neuronal defects were copied in animals with defective sphingolipid biosynthetic enzymes downstream of SPTL-1, including ceramide glucosyltransferases, suggesting that SPTLC1 C133W contributes to the HSAN1 pathogenesis by limiting the production of complex sphingolipids, including glucosylceramide. Overexpression of SPTL-1(C121W) led to similar epithelial and neuronal defects and to reduced levels of complex sphingolipids, specifically glucosylceramide, consistent with a dominant-negative effect of SPTL-1(C121W) that is mediated by loss of this downstream product. Genetic interactions between SPTL-1(C121W) and components of directional trafficking in neurons suggest that the neuronal polarity phenotype could be caused by glycosphingolipid-dependent defects in polarized vesicular trafficking. SIGNIFICANCE STATEMENT The symptoms of inherited metabolic diseases are often attributed to the accumulation of toxic intermediates or byproducts, no matter whether the disease-causing enzyme participates in a biosynthetic or a degradation pathway. By showing that the phenotypes observed in a C. elegans model of HSAN1 disease could be caused by loss of a downstream product (glucosylceramide) rather than the accumulation of a toxic byproduct, our work provides new insights into the origins of the symptoms of inherited metabolic diseases while expanding the repertoire of sphingolipid functions, specifically, of glucosylceramides. These findings not only have their most immediate relevance for neuroprotective treatments for HSAN1, they may also have implications for a much broader range of neurologic conditions., (Copyright © 2019 the authors.)

Alteration of glycosphingolipid (GSL) expression plays key roles in the pathogenesis and pathophysiology of many important human diseases, including cancer, diabetes and glycosphingolipidosis. Inflammatory processes are involved in development and progression of diabetic nephropathy, a major complication of type 2 diabetes mellitus. GSLs are known to play roles in inflammatory responses in various diseases, and levels of renal GSLs are elevated in mouse models of diabetic nephropathy; however, little is known regarding the pathophysiological role of these GSLs in this disease process. We studied proinflammatory activity of GSLs in diabetic nephropathy using spontaneously diabetic mouse strain KK. Mice were fed a high-fat diet (HFD) (60% kcal from fat) or normal diet (ND) (4.6% kcal from fat) for a period of 8 wk. HFD-feeding resulted in quantitative and qualitative changes of renal globo-series GSLs (particularly Gb3Cer), upregulation of TNF-α, and induction of renal inflammation. Gb3Cer/Gb4Cer treatment enhanced inflammatory responses via TLR4 in TLR4/MD-2 complex expressing cells, including HEK293T, mouse bone marrow-derived macrophages (BMDMs) and human monocytes. Our findings suggest that HFD-induced increase of Gb3Cer/Gb4Cer positively modulate TLR4-mediated inflammatory response, and that such GSLs play an important pathophysiological role in diabetic nephropathy., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The globo-series glycosphingolipids (GSLs) SSEA3, SSEA4, and Globo-H specifically expressed on cancer cells are found to correlate with tumor progression and metastasis, but the functional roles of these GSLs and the key enzyme β1,3-galactosyltransferase V (β3GalT5) that converts Gb4 to SSEA3 remain largely unclear. Here we show that the expression of β3GalT5 significantly correlates with tumor progression and poor survival in patients, and the globo-series GSLs in breast cancer cells form a complex in membrane lipid raft with caveolin-1 (CAV1) and focal adhesion kinase (FAK) which then interact with AKT and receptor-interacting protein kinase (RIP), respectively. Knockdown of β3GalT5 disrupts the complex and induces apoptosis through dissociation of RIP from the complex to interact with the Fas death domain (FADD) and trigger the Fas-dependent pathway. This finding provides a link between SSEA3/SSEA4/Globo-H and the FAK/CAV1/AKT/RIP complex in tumor progression and apoptosis and suggests a direction for the treatment of breast cancer, as demonstrated by the combined use of antibodies against Globo-H and SSEA4., Competing Interests: The authors declare no conflict of interest.

The reversible transitions of cancer cells between epithelial and mesenchymal states comprise cellular and molecular processes essential for local tumor growth and respective dissemination. We report here that globoside glycosphingolipid (GSL) glycosyltransferase-encoding genes are elevated in epithelial cells and correlate with characteristic EMT signatures predictive of disease outcome. Depletion of globosides through CRISPR- Cas9 -mediated deletion of the key enzyme A4GALT induces EMT, enhances chemoresistance, and increased CD24 low /CD44 high cells. The cholera toxin-induced mesenchymal-to-epithelial transition occurred only in cells with functional A4GALT. Cells undergoing EMT lost E-cadherin expression through epigenetic silencing at the promoter region of CDH1 However, in ΔA4GALT cells, demethylation was able to rescue E-cadherin-mediated cell-cell adhesion only in the presence of exogenous A4GALT. Overall, our data suggest another class of biomolecules vital for epithelial cancer cells and for maintaining cell integrity and function. Significance: This study highlights the essential role of glycosphingolipids in the maintenance of epithelial cancer cell properties. Cancer Res; 78(11); 2952-65. ©2018 AACR ., (©2018 American Association for Cancer Research.)

A variety of glycoconjugates, including glycosphingolipids (GSLs), expressed in mammalian tissues and cells were isolated and characterized in early biochemical studies. Later studies of virus-transformed fibroblasts demonstrated the association of GSL expression profiles with cell phenotypes. Changes of GSL expression profile were observed during mammalian embryogenesis. Cell surface molecules expressed on embryos in a stage-specific manner appeared to play key roles in regulation of cell-cell interaction and cell sorting during early development. Many mAbs showing stage-specific reactivity with mouse embryos were shown to recognize carbohydrate epitopes. Among various stage-specific embryonic antigens (SSEAs), SSEA-1 was found to react with neolacto-series GSL Le x , while SSEA-3 and SSEA-4 reacted with globo-series Gb5 and monosialyl-Gb5, respectively. GSL expression during mouse early development was shown to shift rapidly from globo-series to neolacto/lacto-series, and then to ganglio-series. We found that multivalent Le x caused decompaction of mouse embryos, indicating a functional role of Le x epitope in the compaction process. Autoaggregation of mouse embryonal carcinoma (EC) F9 cells provided a useful model of the compaction process. We showed that Le x -Le x interaction, a novel type of molecular interavction termed carbohydrate-carbohydrate interaction (CCI), was involved in cell aggregation. Similar shifting of GSL expression profiles from globo-series and neolacto/lacto-series to ganglio-series was observed during differentiation of human EC cells and embryonic stem (ES) cells, reflecting the essential role of cell surface glycoconjugates in early development.

Many studies have suggested that disialogangliosides, GD2 and GD3, are involved in the development of various tumor types. However, the functional relationships between ganglioside expression and cancer development or aggressiveness are not fully described. GD3 is upregulated in approximately half of all invasive ductal breast carcinoma cases, and enhanced expression of GD3 synthase (GD3S, alpha-N-acetylneuraminide alpha-2,8-sialyltransferase) in estrogen receptor-negative breast tumors, was shown to correlate with reduced overall patient survival. We previously found that GD2 and GD3, together with their common upstream glycosyltransferases, GD3S and GD2/GM2 synthase, maintain a stem cell phenotype in breast cancer stem cells (CSCs). In the current study, we demonstrate that GD3S alone can sustain CSC properties and also promote malignant cancer properties. Using MALDI-MS and flow cytometry, we found that breast cancer cell lines, of various subtypes with or without ectopic GD3S-expression, exhibited distinct GD2/GD3 expression profiles. Furthermore, we found that GD3 was associated with EGFR and activated EGFR signaling in both breast CSCs and breast cancer cell lines. In addition, GD3S knockdown enhanced cytotoxicity of the EGFR-inhibitor gefitinib in resistant MDA-MB468 cells, both in vitro and in vivo. Based on this evidence, we propose that GD3S contributes to gefitinib-resistance in EGFR-positive breast cancer cells and may be an effective therapeutic target in drug-resistant breast cancers.

High expectations are held for human-induced pluripotent stem cells (hiPSC) since they are established from autologous tissues thus overcoming the risk of allogeneic immune rejection when used in regenerative medicine. However, little is known regarding the cell-surface carbohydrate antigen profile of hiPSC compared with human embryonic stem cells (hESC). Here, glycosphingolipids were isolated from an adipocyte-derived hiPSC line, and hiPSC and hESC glycosphingolipids were compared by concurrent characterization by binding assays with carbohydrate-recognizing ligands and mass spectrometry. A high similarity between the nonacid glycosphingolipids of hiPSC and hESC was found. The nonacid glycosphingolipids P1 pentaosylceramide, x2 pentaosylceramide and H type 1 heptaosylceramide, not previously described in human pluripotent stem cells (hPSC), were characterized in both hiPSC and hESC. The composition of acid glycosphingolipids differed, with increased levels of GM3 ganglioside, and reduced levels of GD1a/GD1b in hiPSC when compared with hESC. In addition, the hESC glycosphingolipids sulf-globopentaosylceramide and sialyl-globotetraosylceramide were lacking in hiPSC. Neural stem cells differentiating from hiPSC had a reduced expression of sialyl-lactotetra, whereas expression of the GD1a ganglioside was significantly increased. Thus, while sialyl-lactotetra is a marker of undifferentiated hPSC, GD1a is a novel marker of neural differentiation., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Shiga toxin (Stx)-mediated injury to microvascular endothelial cells in the brain significantly contributes to the pathogenesis of the hemolytic-uremic syndrome caused by enterohemorrhagic Escherichia coli (EHEC). Stxs are AB 5 toxins and the B-pentamers of the two major Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) expressed by human endothelial cells. Here we report on comprehensive structural analysis of the different lipoforms of Gb3Cer (Galα4Galβ4Glcβ1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ3Galα4Galβ4Glcβ1Cer, the less effective Stx receptor) of primary human brain microvascular endothelial cells and their association with lipid rafts. Detergent-resistant membranes (DRMs), obtained by sucrose density gradient ultracentrifugation, were used as lipid raft-analogous microdomains of the liquid-ordered phase and nonDRM fractions were employed as equivalents for the liquid-disordered phase of cell membranes. Structures of the prevalent lipoforms of Gb3Cer and Gb4Cer were those with Cer (d18:1, C16:0), Cer (d18:1, C22:0) and Cer (d18:1, C24:1/C24:0) determined by electrospray ionization mass spectrometry that was combined with thin-layer chromatography immunodetection using anti-Gb3Cer and anti-Gb4Cer antibodies as well as Stx1a and Stx2a subtypes. Association of Stx receptor GSLs was determined by co-localization with lipid raft-specific membrane protein flotillin-2 and canonical lipid raft marker sphingomyelin with Cer (d18:1, C16:0) and Cer (d18:1, C24:1/C24:0) in the liquid-ordered phase, whereas lyso-phosphatidylcholine was detectable exclusively in the liquid-disordered phase. Defining the precise microdomain structures of primary endothelial cells may help to unravel the initial mechanisms by which Stxs interact with their target cells and will help to develop novel preventive and therapeutic measures for EHEC-mediated diseases., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Human Gb3/CD77 synthase (α1,4-galactosyltransferase) is the only known glycosyltransferase that changes acceptor specificity because of a point mutation. The enzyme, encoded by A4GALT locus, is responsible for biosynthesis of Gal(α1-4)Gal moiety in Gb3 (CD77, P k antigen) and P1 glycosphingolipids. We showed before that a single nucleotide substitution c.631C > G in the open reading frame of A4GALT, resulting in replacement of glutamine with glutamic acid at position 211 (substitution p. Q211E), broadens the enzyme acceptor specificity, so it can not only attach galactose to another galactose but also to N-acetylgalactosamine. The latter reaction leads to synthesis of NOR antigens, which are glycosphingolipids with terminal Gal(α1-4)GalNAc sequence, never before described in mammals. Because of the apparent importance of position 211 for enzyme activity, we stably transfected the 2102Ep cells with vectors encoding Gb3/CD77 synthase with glutamine substituted by aspartic acid or asparagine, and evaluated the cells by quantitative flow cytometry, high-performance thin-layer chromatography and real-time PCR. We found that cells transfected with vectors encoding Gb3/CD77 synthase with substitutions p. Q211D or p. Q211N did not express P k , P1 and NOR antigens, suggesting complete loss of enzymatic activity. Thus, amino acid residue at position 211 of Gb3/CD77 synthase is critical for specificity and activity of the enzyme involved in formation of P k , P1 and NOR antigens. Altogether, this approach affords a new insight into the mechanism of action of the human Gb3/CD77 synthase., Competing Interests: Authors declare no conflicts of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Background: Leishmaniasis is a neglected tropical disease affecting millions of individuals worldwide. Despite several studies reporting involvement of the innate immune receptor Toll-like receptor 2 (TLR2) in the recognition of surface glycolipids from Leishmania parasites in vitro, the role of TLR2 and its co-receptors during cutaneous leishmaniasis infection in vivo is unknown., Methods: To explore the role of TLR2 and its co-receptors in cutaneous leishmaniasis, mice deficient in either TLR2, 4, 1 or 6, or wild-type (WT) controls, were infected with either Leishmania major promastigotes, L. mexicana promastigotes, L. mexicana amastigotes, or LPG1 -/- L. mexicana promastigotes. For each infection, lesion sizes were monitored and parasite burden was assessed at various time points. To assess immune responses, draining lymph node (DLN) cells were re-stimulated with parasite antigens and the production of cytokines and parasite-specific antibody isotypes in blood was determined by ELISA., Results: Mice deficient in TLR2 and TLR4 presented with larger lesions and higher parasite burdens than WT controls. Mice lacking TLR2 co-receptors TLR1 or TLR6 did not show exacerbated infection, suggesting that TLR2 does not require either co-receptor in the recognition of Leishmania infection. Furthermore, it appears that lipophosphoglycan (LPG) is not the major mediator of TLR2 activation during infection with L. mexicana, as parasites lacking LPG (axenic amastigotes and LPG1 -/- promastigotes) also resulted in exacerbated disease in TLR2 -/- mice. Infected TLR2 -/- mice show a skewed Th2 immune response to Leishmania parasites, as demonstrated by elevated IL-4, IL-13 and IL-10 production by DLN cells from L. mexicana infected mice in response to antigen. Furthermore, L. major infected TLR2 -/- mice have elevated antigen-specific IgG1 antibodies., Conclusions: TLR2 deficiency leads to exacerbation of disease and parasite burden through promotion of Th2 immunity. TLR2 activation in vivo occurs independently of parasite LPG, suggesting other parasite ligands are involved in TLR2 recognition of Leishmania.

Some gangliosides, sialic acid-containing glycosphingolipids, have been considered as tumor-associated antigens. GD1α or a GD1α synthase gene ST6GalNAc5 was reported to be involved in the metastasis of murine lymphomas or human breast cancers, respectively. But expression patterns of 0-series gangliosides GD1α and its precursor GM1b in human cancers have not yet been investigated mainly due to lack of specific antibodies. We established specific monoclonal antibodies (mAbs) reactive with GD1α or GM1b using gangliosides from brain tissues of GM3 synthase (St3gal5)-deficient mice as immunogens. We used GM2/GD2 synthase (B4galnt1)-deficient mice to immunize by liposomes embedded with GD1α or acidic glycolipid fractions from brain of St3gal5-deficient mice. Specificities of established mAbs as analyzed by enzyme-linked immunosorbent assay and thin-layer chromatography-immunostaining were very high among various gangliosides. Increased expression of GD1α and reduced GM1b in the St6galnac5 cDNA-transfected RAW117 cell line also substantiated the specificities of two mAbs. Then, we analyzed expression of GD1α and GM1b, and of relevant glycosyltransferase genes in various human cancer cell lines using generated anti-GD1α mAb 122 or anti-GM1b mAb MR155A-7. A few human cancer cell lines showed significant expression of these gangliosides with reasonable expression of relevant glycosyltransferase genes., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Leishmaniasis is one of the most common infectious diseases transmitted by an obligate intracellular genus Leishmania. As there is no efficient vaccination strategy for leishmaniasis, new immunostimulatory components may enhance protective immune responses against this parasite. Lipophosphoglycan 3 (LPG3) is an essential protein required for LPG assembling. In this study, the ability of recombinant LPG3 (rLPG) and its fragments to activate isolated healthy human T-cells and cytokine secretion was evaluated in vitro. The results showed that rLPG3 and its N-terminal fragment (rNT-LPG3) enhanced expression of CD69 on the surface of T-cells and promoted differentiation of CD4(+) T-lymphocytes toward a T-helper 1 (T(H)1) phenotype, in part, through up-regulation of interferon (IFN)-γ expression in a TLR2-independent manner. These results indicated the protective effects of LPG3 (particularly NT-LPG3 fragment) as a potent immunostimulatory component of leishmania in vaccination against leishmaniasis. Further investigations in in vivo assays are clearly warranted.

The x2 glycosphingolipid is expressed on erythrocytes from individuals of all common blood group phenotypes and elevated on cells of the rare P/P1/P(k)-negative p blood group phenotype. Globoside or P antigen is synthesized by UDP-N-acetylgalactosamine:globotriaosyl-ceramide 3-β-N-acetylgalactosaminyltransferase encoded by B3GALNT1. It is the most abundant non-acid glycosphingolipid on erythrocytes and displays the same terminal disaccharide, GalNAcβ3Gal, as x2. We encountered a patient with mutations in B3GALNT1 causing the rare P-deficient P1 (k) phenotype and whose pretransfusion plasma was unexpectedly incompatible with p erythrocytes. The same phenomenon was also noted in seven other unrelated P-deficient individuals. Thin-layer chromatography, mass spectrometry, and flow cytometry were used to show that the naturally occurring antibodies made by p individuals recognize x2 and sialylated forms of x2, whereas x2 is lacking on P-deficient erythrocytes. Overexpression of B3GALNT1 resulted in synthesis of both P and x2. Knockdown experiments with siRNA against B3GALNT1 diminished x2 levels. We conclude that x2 fulfills blood group criteria and is synthesized by UDP-N-acetylgalactosamine: globotriaosylceramide 3-β-N-acetylgalactosaminyltransferase. Based on this linkage, we proposed that x2 joins P in the GLOB blood group system (ISBT 028) and is renamed PX2 (GLOB2). Thus, in the absence of a functional P synthase, neither P nor PX2 are formed. As a consequence, naturally occurring anti-P and anti-PX2 can be made. Until the clinical significance of anti-PX2 is known, we also recommend that rare P1 (k) or P2 (k) erythrocyte units are preferentially selected for transfusion to P(k) patients because p erythrocytes may pose a risk for hemolytic transfusion reactions due to their elevated PX2 levels., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Glycosphingolipids (GSLs) are essential constituents of cell membranes and lipid rafts and can modulate signal transduction events. The contribution of GSLs in osteoclast (OC) activation and osteolytic bone diseases in malignancies such as the plasma cell dyscrasia multiple myeloma (MM) is not known. Here, we tested the hypothesis that pathological activation of OCs in MM requires de novo GSL synthesis and is further enhanced by myeloma cell-derived GSLs. Glucosylceramide synthase (GCS) inhibitors, including the clinically approved agent N-butyl-deoxynojirimycin (NB-DNJ), prevented OC development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of transcriptional activator NFATc1. GM3 was the prevailing GSL produced by patient-derived myeloma cells and MM cell lines, and exogenous addition of GM3 synergistically enhanced the ability of the pro-osteoclastogenic factors RANKL and insulin-like growth factor 1 (IGF-1) to induce osteoclastogenesis in precursors. In WT mice, administration of GM3 increased OC numbers and activity, an effect that was reversed by treatment with NB-DNJ. In a murine MM model, treatment with NB-DNJ markedly improved osteolytic bone disease symptoms. Together, these data demonstrate that both tumor-derived and de novo synthesized GSLs influence osteoclastogenesis and suggest that NB-DNJ may reduce pathological OC activation and bone destruction associated with MM.

Recent progress in the biological sciences has revealed that a number of extrinsic and intrinsic environmental factors may cause chronic inflammation. When these insults are persistent or intermittently repeated, regardless of extrinsic or intrinsic origins, homeostasis of our bodies would be disturbed and undergo long-term impact. These situations might give rise to chronic inflammation, leading to various diseases as results of accumulative effects of various inflammatory reactions. Complex carbohydrates expressed mainly on the cell surface have been demonstrated to play roles in fine-tuning of various biological processes to maintain homeostasis of cells, organs and our bodies. When abnormal physicochemical insults and harmful pathogens invade, the fine-tuning including modification of the glycosylation patterns is continuously exerted. Therefore, defects in the proper response with proper glycosylation lead to chronic inflammation and subsequent deterioration of individual tissues and organs. Genetic depletion of sialic acid-containing glycolipids, gangliosides resulted in the inflammation of CNS and neurodegeneration. Lactosylceramide was also reported to mediate neuroinflammation, leading to chronic inflammatory diseases. Defects of globoseries glycolipids resulted in the increased sensitivity to LPS toxicity. Thus, possibilities that manipulation of synthesis and expression of glycosphingolipids may be applicable for the disease control are now proposed., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Visceral leishmaniasis is a serious parasitic infection that the development of an effective vaccine is necessary to control the disease. Lipophosphoglycan 3 (LPG3) is essential for the synthesis of glycoconjugates as parasite virulence factors. In this study, we evaluated the immunogenicity of Leishmania infantum LPG3 gene as a DNA vaccine against murine visceral leishmaniasis. For this purpose, BALB/c mice were immunized subcutaneously with the DNA encoding LPG3 either alone or in combination with recombinant heat shock protein 70 (rHSP70). Next, its immunogenicity and protective efficacy were evaluated in the immunized mice. The results showed a mixed Th1/Th2 response following immunization, which was associated with the production of both IFN-γ and IL-10 by splenocytes compared with control groups but did not lead to reduction in the splenic parasite burden. Serum levels of IgG antibody isotypes indicated no significant difference between the LPG3 DNA and the empty vector. In addition, the co-administration of rHSP70 with the DNA vaccine offered no additive protective advantage on experimental infectious challenge. Thus, we propose to strengthen the immunogenic potential of L. infantum LPG3 in prime-boost approach with a powerful adjuvant to elicit a robust parasite-specific protective Th1 response., (© 2014 John Wiley & Sons Ltd.)

The lipophosphoglycan (LPG) of Leishmania major has a major role in the attachment to Phlebotomus papatasi midgut. Here, we investigated the comparative structural features of LPG of L. turanica, another species transmitted by P. papatasi. The mAb WIC 79.3, specific for terminal Gal(β1,3) side-chains, strongly reacted with L. turanica LPG. In contrast, L. turanica LPG was not recognized by arabinose-specific mAb 3F12. In conclusion, LPGs from L. major and L. turanica are similar, with the latter being less arabinosylated than L. major's. The high galactose content in L. turanica LPG is consistent with its predicted recognition by P. papatasi lectin PpGalec., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Antigens belonging to the P1PK, GLOB, and FORS blood group systems and the GLOB blood group collection represent a closely related set of 13 glycosphingolipids (GSLs). They are synthesized by the coordinated action of glycosyltransferases, encoded by at least 7 different loci. Three of these enzymes show either different activity or a different mRNA expression profile due to genetic polymorphisms, resulting in blood group diversity. In recent years, significant progress has been made in understanding the molecular background and biological functions of these GSLs. Their medical significance is often related to the existence of natural antibodies, as they may cause complications after transfusions and during pregnancies. In addition, GSLs belonging to these blood group systems are receptors for several pathogens. This review summarizes the present knowledge about the complicated network of enzymatic interactions leading to synthesis of these GSLs, as well as their clinical implications., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Glycosphingolipid (GSL) storage diseases have been the focus of efforts to develop small molecule therapeutics from design, experimental proof of concept studies, and clinical trials. Two primary alternative strategies that have been pursued include pharmacological chaperones and GSL synthase inhibitors. There are theoretical advantages and disadvantages to each of these approaches. Pharmacological chaperones are specific for an individual glycoside hydrolase and for the specific mutation present, but no candidate chaperone has been demonstrated to be effective for all mutations leading to a given disorder. Synthase inhibitors target single enzymes such as glucosylceramide synthase and inhibit the formation of multiple GSLs. A glycolipid synthase inhibitor could potentially be used to treat multiple diseases, but at the risk of lowering nontargeted cellular GSLs that are important for normal health. The basis for these strategies and specific examples of compounds that have led to clinical trials is the focus of this review., (Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.)

Hypertrophy is central to several heart diseases; however, not much is known about the role of glycosphingolipids (GSLs) in this phenotype. Since GSLs have been accorded several physiological functions, we sought to determine whether these compounds affect cardiac hypertrophy. By using a rat cardiomyoblast cell line, H9c2 cells and cultured primary neonatal rat cardiomyocytes, we have determined the effects of GSLs on hypertrophy. Our study comprises (a) measurement of [(3)H]-leucine incorporation into protein, (b) measurement of cell size and morphology by immunofluorescence microscopy and (c) real-time quantitative mRNA expression assay for atrial natriuretic peptide and brain natriuretic peptide. Phenylephrine (PE), a well-established agonist of cardiac hypertrophy, served as a positive control in these studies. Subsequently, mechanistic studies were performed to explore the involvement of various signaling transduction pathways that may contribute to hypertrophy in these cardiomyocytes. We observed that lactosylceramide specifically exerted a concentration- (50-100 µM) and time (48 h)-dependent increase in hypertrophy in cardiomyocytes but not a library of other structurally related GSLs. Further, in cardiomyocytes, LacCer generated reactive oxygen species, stimulated the phosphorylation of p44 mitogen activated protein kinase and protein kinase-C, and enhanced c-jun and c-fos expression, ultimately leading to hypertrophy. In summary, we report here that LacCer specifically induces hypertrophy in cardiomyocytes via an "oxygen-sensitive signal transduction pathway."

Background: UDP-GlcNAc 2-epimerase/ManNAc 6-kinase (GNE) is a bifunctional enzyme responsible for the first committed steps in the synthesis of sialic acid, a common terminal monosaccharide in both protein and lipid glycosylation. GNE mutations are responsible for a rare autosomal recessive neuromuscular disorder, GNE myopathy (also called hereditary inclusion body myopathy). The connection between the impairment of sialic acid synthesis and muscle pathology in GNE myopathy remains poorly understood., Methods: Glycosphingolipid (GSL) analysis was performed by HPLC in multiple models of GNE myopathy, including patients' fibroblasts and plasma, control fibroblasts with inhibited GNE epimerase activity through a novel imino sugar, and tissues of Gne(M712T/M712T) knock-in mice., Results: Not only neutral GSLs, but also sialylated GSLs, were significantly increased compared to controls in all tested models of GNE myopathy. Treatment of GNE myopathy fibroblasts with N-acetylmannosamine (ManNAc), a sialic acid precursor downstream of GNE epimerase activity, ameliorated the increased total GSL concentrations., Conclusion: GNE myopathy models have increased total GSL concentrations. ManNAc supplementation results in decrease of GSL levels, linking abnormal increase of total GSLs in GNE myopathy to defects in the sialic acid biosynthetic pathway. These data advocate for further exploring GSL concentrations as an informative biomarker, not only for GNE myopathy, but also for other disorders of sialic acid metabolism.

Binding of promastigotes to the sand fly midgut epithelium is regarded as an essential part of the Leishmania life cycle in the vector. Among Leishmania surface molecules putatively involved in attachment to the sand fly midgut, two GPI-anchored molecules are the most prominent: lipophosphoglycan (LPG) and promastigote surface protease gp63. In this work, we examined midgut attachment of Leishmania lines mutated in GPI-anchored molecules and compared results from 2 different techniques: in vivo development in sand flies and in vitro competitive binding assays using fluorescently labelled parasites. In combination with previous studies, our data provide additional support for (1) an LPG-independent parasite-binding mechanism of Leishmania major within the midgut of the permissive vector Phlebotomus perniciosus, and provide strong support for (2) the crucial role of L. major LPG in specific vector Phlebotomus papatasi, and (3) a role for Leishmania amazonensis gp63 in Lutzomyia longipalpis midgut binding. Moreover, our results suggest a critical role for GPI-anchored proteins and gp63 in Leishmania mexicana attachment to L. longipalpis midguts, as the wild type (WT) line accounted for over 99% of bound parasites.

Two different Toll-like receptors (TLRs) have been shown to play a role in host responses to Leishmania infection. TLR-2 is involved in parasite survival in macrophages upon activation by lipophosphoglycan (LPG), a virulence factor expressed by Leishmania. In contrast, activation of TLR-9 has been shown to promote a host-protective response. However, whether there is a relationship between the interaction of LPG and TLR-2, on one hand, with the effect of TLR-9, on the other hand, remains unknown. In this study, we report that in-vitro infection of macrophages with a L. major parasite with high expression levels of LPG results in decreased TLR-9 expression compared to infection with a L. major parasite with lower expression levels of LPG. Addition of anti-LPG as well as anti-TLR-2 antibodies prevents this reduction of TLR-9 expression. Also, the addition of purified LPG to macrophages results in a decrease of TLR-9 expression, which is shown to be mediated by transforming growth factor (TGF)-β and interleukin (IL)-10. Finally, in-vitro treatment of macrophages with anti-LPG and/or anti-TLR-2 antibodies before infection reduces the number of amastigotes in macrophages and co-treatment of mice with anti-TLR-2 antibodies and cytosine-phosphate-guanosine (CpG) reduces footpad swelling and parasite load in the draining lymph nodes, accompanied by an interferon (IFN)-γ-predominant T cell response. Thus, for the first time, we show how interactions between LPG and TLR-2 reduce anti-leishmanial responses via cytokine-mediated decrease of TLR-9 expression., (© 2013 British Society for Immunology.)

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain.

Glycosphingolipids (GSLs) constitute major components of enterocytes and were hypothesized to be potentially important for intestinal epithelial polarization. The enzyme UDP-glucose ceramide glucosyltransferase (Ugcg) catalyzes the initial step of GSL biosynthesis. Newborn and adult mice with enterocyte-specific genetic deletion of the gene Ugcg were generated. In newborn mutants lacking GSLs at day P0, intestinal epithelia were indistinguishable from those in control littermates displaying an intact polarization with regular brush border. However, those mice were not consistently able to absorb nutritional lipids from milk. Between postnatal days 5 and 7, severe defects in intestinal epithelial differentiation occurred accompanied by impaired intestinal uptake of nutrients. Villi of mutant mice became stunted, and enterocytes lacked brush border. The defects observed in mutant mice caused diarrhea, malabsorption, and early death. In this study, we show that GSLs are essential for enterocyte resorptive function but are primarily not for polarization; GSLs are required for intracellular vesicular transport in resorption-active intestine.

Background: Protozoan parasites of the genus Leishmania cause a number of important diseases in humans and undergo a complex life cycle, alternating between a sand fly vector and vertebrate hosts. The parasites have a remarkable capacity to avoid destruction in which surface molecules are determinant for survival. Amongst the many surface molecules of Leishmania, the glycoconjugates are known to play a central role in host-parasite interactions and are the focus of this review., Scope of the Review: The most abundant and best studied glycoconjugates are the Lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs). This review summarizes the main studies on structure and biological functions of these molecules in New World Leishmania species., Major Conclusions: LPG and GIPLs are complex molecules that display inter- and intraspecies polymorphisms. They are key elements for survival inside the vector and to modulate the vertebrate immune response during infection., General Significance: Most of the studies on glycoconjugates focused on Old World Leishmania species. Here, it is reported some of the studies involving New World species and their biological significance on host-parasite interaction. This article is part of a Special Issue entitled Glycoproteomics., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Detection of chromosomal aneuploidies using fetal cells isolated from maternal blood, for prenatal non-invasive genetic investigation, has been a long-sought goal of clinical genetics to replace amniocentesis and chorionic villous sampling to avoid any risk to the fetus. The purpose of this study was to develop a sensitive and specific new assay for diagnosing aneuploidy with circulating fetal cells isolated from maternal blood as previously reported using two novel approaches: (i) simultaneous immunocytochemistry (ICC) evaluation using a monoclonal antibody for i-antigen, followed by fluorescence in situ hybridization (FISH); (ii) dual-probe FISH analysis of interphase nuclei using two differently labeled probes, specific for different loci of chromosomes 21 and 18; in addition, short tandem repeats (STR) analysis on single cells isolated by micromanipulation was applied to confirm the presence of fetal cells in the cell sample enriched from maternal blood. Blood samples were obtained from women carrying trisomic fetuses, and from non-pregnant women and men as controls. Using ICC-FISH approach, a large heterogeneity in immunostaining pattern was observed, which is a source of very subjective signal interpretation. Differently, dual-probe FISH analysis provided for a correct diagnosis of all pregnancies: the mean percentage of trisomic cells was 0.5% (range, 0.36-0.76%), while the mean percentage of trisomic cells in the control group (normal pregnancies or non-pregnant women) was ≤0.20%. The application of the dual-probe FISH protocol on fetal cells isolated from maternal blood enables accurate molecular detection of fetal aneuploidy, thus providing a foundation for development of non-invasive prenatal diagnostic testing., (© 2011 John Wiley & Sons A/S.)

Phosphoprotein associated with glycosphingolipid microdomains 1 (PAG) is an important negative regulator of immune signaling in T lymphocytes. However, newly emerging evidence has indicated that PAG may play important roles in tumor cells. Our previously reported cDNA microarray experiments identified PAG as a gene down-regulated in the high metastatic potential prostate cancer cell line PC-3M-1E8. In this study, we investigated the role of PAG in the proliferation, invasion and metastasis of prostate cancer cells and the underlying mechanisms. We confirmed that the expression of PAG at both the mRNA and protein levels was low in PC-3M-1E8 and DU145 cells compared to low metastatic potential prostate cancer cells PC-3M-2B4. In addition, we demonstrated that the reintroduction of PAG to PC-3M-1E8 and DU145 cells led to reduced proliferation through cell cycle arrest, decreased anchorage-independent growth and reduced invasion ability of tumor cells in vitro. This is the first report demonstrating that PAG inhibits the proliferation and invasion potential of prostate cancer cells via the interaction with RasGAP to recruit RasGAP to the cell membrane, where RasGAP hydrolyzes GTP to GDP, reduces the level of activated Ras, and ultimately suppresses the activation of ERK1/2, cyclin D1 and other effectors of the Ras signaling pathway. Morphologically, we observed that PAG could diminish the formation of pseudopodia on the cell surface and redistribute the intracellular F-actin in PC-3M-1E8 cells, which directly leads to the decreased invasion and metastasis potential of tumor cells. Taken together, these results suggest that PAG acts to inhibit the development and metastasis of prostate cancers and represents a novel therapeutic target for prostate cancer.

NKT cells play an important role in autoimmune diseases, tumor surveillance, and infectious diseases, providing in most cases protection against infection. NKT cells are reactive to CD1d presented glycolipid antigens. They can modulate immune responses by promoting the secretion of type 1, type 2, or immune regulatory cytokines. Pathogen-derived signals to dendritic cells mediated via Toll like Receptors (TLR) can be modulated by activated invariant Natural Killer T (iNKT) cells. The terminal β-(1-4)-galactose residues of glycans can modulate host responsiveness in a T helper type-1 direction via IFN-γ and TLRs. We have attempted to develop a defined immunotherapeutic, based on the cooperative action of a TLR ligand and iNKT cell using a mouse model of visceral leishmaniasis. We evaluated the anti-Leishmania immune responses and the protective efficacy of the β-(1-4)-galactose terminal NKT cell ligand glycosphingophospholipid (GSPL) antigen of L. donovani parasites. Our results suggest that TLR4 can function as an upstream sensor for GSPL and provoke intracellular inflammatory signaling necessary for parasite killing. Treatment with GSPL was able to induce a strong effective T cell response that contributed to effective control of acute parasite burden and led to undetectable parasite persistence in the infected animals. These studies for the first time demonstrate the interactions between a TLR ligand and iNKT cell activation in visceral leishmaniasis immunotherapeutic.

Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.

Brain tissue is characterized by its high glycosphingolipid content, particularly those containing sialic acid (gangliosides). As a result of this observation, brain tissue was a focus for studies leading to the characterization of the enzymes participating in ganglioside biosynthesis, and their participation in driving the compositional changes that occur in glycolipid expression during brain development. Later on, this focus shifted to the study of cellular aspects of the synthesis, which lead to the identification of the site of synthesis in the neuronal soma and their axonal transport toward the periphery. In this review article, we will focus in subcellular aspects of the biosynthesis of glycosphingolipid oligosaccharides, particularly the mechanisms underlying the trafficking of glycosphingolipid glycosyltransferases from the endoplasmic reticulum to the Golgi, those that promote their retention in the Golgi and those that participate in their topological organization as part of the complex membrane bound machinery for the synthesis of glycosphingolipids., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Recent studies have shown that histone proteins can act as antimicrobial peptides in host defense against extracellular bacteria, fungi, and Leishmania promastigotes. In this study, we used human recombinant histone proteins to further study their leishmaniacidal effects and the underlying mechanisms. We found that the histones H2A and H2B (but not H1(0)) could directly and efficiently kill promastigotes of Leishmania amazonensis, L. major, L. braziliensis, and L. mexicana in a treatment dose-dependent manner. Scanning electron microscopy revealed surface disruption of histone-treated promastigotes. More importantly, the preexposure of promastigotes to histone proteins markedly decreased the infectivity of promastigotes to murine macrophages (Mφs) in vitro. However, axenic and lesion-derived amastigotes of L. amazonensis and L. mexicana were relatively resistant to histone treatment, which correlated with the low levels of intracellular H2A in treated amastigotes. To understand the mechanisms underlying these differential responses, we investigated the role of promastigote surface molecules in histone-mediated killing. Compared with the corresponding controls, transgenic L. amazonensis promastigotes expressing lower levels of surface gp63 proteins were more susceptible to histone H2A, while L. major and L. mexicana promastigotes with targeted deletion of the lipophosphoglycan 2 (lpg2) gene (but not the lpg1 gene) were more resistant to histone H2A. We discuss the influence of promastigote major surface molecules in the leishmaniacidal effect of histone proteins. This study provides new information on host innate immunity to different developmental stages of Leishmania parasites.

Interspecies variations in lipophosphoglycan (LPG) have been the focus of intense study over the years due its role in specificity during sand fly-Leishmania interaction. This cell surface glycoconjugate is highly polymorphic among species with variations in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO(4) backbone of repeat units. However, the degree of intraspecies polymorphism in LPG of Leishmania infantum (syn. Leishmania chagasi) is not known. In this study, intraspecific variation in the repeat units of LPG was evaluated in 16 strains of L. infantum from Brazil, France, Algeria and Tunisia. The structural polymorphism in the L. infantum LPG repeat units was relatively slight and consisted of three types: type I does not have side chains; type II has one β-glucose residue that branches off the disaccharide-phosphate repeat units and type III has up to three glucose residues (oligo-glucosylated). The significance of these modifications was investigated during in vivo interaction of L. infantum with Lutzomyia longipalpis, and in vitro interaction of the parasites and respective LPGs with murine macrophages. There were no consequential differences in the parasite densities in sand fly midguts infected with Leishmania strains exhibiting type I, II and III LPGs. However, higher nitric oxide production was observed in macrophages exposed to glucosylated type II LPG., (Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

A fragment about 2.2 kb located at upstream of lipophosphonoglycan 1 (LPG1) gene of Leishmania donovani isolate from hilly foci of China was obtained by PCR. The nucleotide sequence of the fragment was determined by sequencing. The sequence of the LPG1 gene and its downstream fragment (about 2 kb) was determined by using genome walking method. The above two fragment splicing result showed that the nucleotide sequence of the LPG1 gene with the noncoding region was 4 121 bp (GenBank accession number: HMO27899). The BLAST results showed that the LPG1 gene of L. donovani isolate from hilly foci of China had 72%-78% nucleotide identity compared to that of other Leishmania spp. in GenBank.

Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by motor, psychiatric, and cognitive abnormalities. In this present study, we tested whether abnormal motor behavior in a mouse model of HD, the R6/1 transgenic (Tg) mice, was associated with changes in cerebellar lipid composition and gene expression. We report altered motor behavior, which was associated with abnormal expression of glycosyltransferase genes in the cerebellum of R6/1 Tg mice. Cerebellar wet weight and total ganglioside concentration was significantly lower in R6/1 Tg mice than in wild-type (Wt) mice. Furthermore, the Purkinje cell-enriched ganglioside LD1 and the granule cell-enriched ganglioside GD1a were significantly lower in R6/1 Tg mice than in Wt mice. The myelin-enriched lipid sulfatides was also reduced in the cerebellum of R6/1 Tg mice. In contrast to the R6/1 Tg mice, total cerebellar ganglioside concentration did not differ between HD and control subjects. However, expression of several cerebellar glycosyltransferases genes was significantly less in HD subjects than in control subjects. Our findings indicate that the R6/1 Tg mice have severe cerebellar glycosphingolipid (GSL) abnormalities that may account, in part, for their abnormal motor behavior. Although the cerebellar lipid abnormalities found in the R6/1 Tg mice were not found in these HD subjects, the R6/1 Tg mice may be useful for evaluating the role of GSLs in cerebellar development., (© 2010 The Authors. Journal of Neurochemistry © 2010 International Society for Neurochemistry.)

Rapid advances in genotyping and sequencing technology have dramatically accelerated the discovery of genes underlying human disease. Elucidating the function of such genes and understanding their role in pathogenesis, however, remain challenging. Here, we introduce a genomic strategy to characterize such genes functionally, and we apply it to LRPPRC, a poorly studied gene that is mutated in Leigh syndrome, French-Canadian type (LSFC). We utilize RNA interference to engineer an allelic series of cellular models in which LRPPRC has been stably silenced to different levels of knockdown efficiency. We then combine genome-wide expression profiling with gene set enrichment analysis to identify cellular responses that correlate with the loss of LRPPRC. Using this strategy, we discovered a specific role for LRPPRC in the expression of all mitochondrial DNA-encoded mRNAs, but not the rRNAs, providing mechanistic insights into the enzymatic defects observed in the disease. Our analysis shows that nuclear genes encoding mitochondrial proteins are not collectively affected by the loss of LRPPRC. We do observe altered expression of genes related to hexose metabolism, prostaglandin synthesis, and glycosphingolipid biology that may either play an adaptive role in cell survival or contribute to pathogenesis. The combination of genetic perturbation, genomic profiling, and pathway analysis represents a generic strategy for understanding disease pathogenesis.

T cell receptor (TCR) contact with self ligands keeps T cells alive and is shown here to cause naive CD8(+), but not CD4(+), T cells to be hypersensitive to certain gamma(c) cytokines, notably interleukin (IL)-2, IL-15, and IL-7. Hypersensitivity of CD8(+) T cells to IL-2 was dependent on a low-level TCR signal, associated with high expression of CD5 and GM1, a marker for lipid rafts, and was abolished by disruption of lipid rafts. By contrast, CD4(+) T cells expressed low amounts of GM1 and were unresponsive to IL-2. Physiologically, sensitivity to IL-7 and IL-15 maintains survival of resting CD8(+) T cells, whereas sensitivity to IL-2 may be irrelevant for normal homeostasis but crucial for the immune response. Thus, TCR contact with antigen upregulates GM1 and amplifies responsiveness of naive CD8(+) T cells to IL-2, thereby making the cells highly sensitive to exogenous IL-2 from CD4(+) T helper cells., (Copyright 2010 Elsevier Inc. All rights reserved.)

GDP-mannose:inositol-phosphorylceramide (MIPC)-derived glycosphingolipids are important pathogen-associated molecular patterns (PAMP) of Candida albicans and according to recently published data also of Aspergillus fumigatus. MIPC transferases are essential for the synthesis of MIPC, but have so far been studied only in Saccharomyces cerevisiae and C. albicans. Here, we have identified MitA as the only MIPC transferase in A. fumigatus. The DeltamitA mutant lacks MIPC and MIPC-derived glycosphingolipids and accumulates the precursor IPC. The mutant grows normally, shows no defects in cell wall or membrane organization and a normal resistance to different stressors. It is, however, sensitive to high Ca(2+) concentrations, especially during germination. Germination of DeltamitA mutant conidia is also decelerated under normal growth conditions, but neither the virulence of this mutant in a systemic model of infection nor its ability to trigger a cytokine response in macrophages is impaired, arguing against a role of MIPC-derived glycosphingolipids as important A. fumigatus PAMPs., (Copyright 2009 Elsevier Inc. All rights reserved.)