Animal Models for the Study of Gaucher Disease.

In Gaucher disease (GD), a relatively common sphingolipidosis, the mutant lysosomal enzyme acid ß-glucocerebrosidase (GCase), encoded by the GBA1 gene, fails to properly hydrolyze the sphingolipid glucosylceramide (GlcCer) in lysosomes, particularly of tissue macrophages. As a result, GlcCer accumulates, which, to a certain extent, is converted to its deacylated form, glucosylsphingosine (GlcSph), by lysosomal acid ceramidase. The inability of mutant GCase to degrade GlcSph further promotes its accumulation. The amount of mutant GCase in lysosomes depends on the amount of mutant ER enzyme that shuttles to them. In the case of many mutant GCase forms, the enzyme is largely misfolded in the ER. Only a fraction correctly folds and is subsequently trafficked to the lysosomes, while the rest of the misfolded mutant GCase protein undergoes ER-associated degradation (ERAD). The retention of misfolded mutant GCase in the ER induces ER stress, which evokes a stress response known as the unfolded protein response (UPR). GD is remarkably heterogeneous in clinical manifestation, including the variant without CNS involvement (type 1), and acute and subacute neuronopathic variants (types 2 and 3). The present review discusses animal models developed to study the molecular and cellular mechanisms underlying GD.

Direct trafficking pathways from the Golgi apparatus to the plasma membrane.

In eukaryotic cells, protein sorting is a highly regulated mechanism important for many physiological events. After synthesis in the endoplasmic reticulum and trafficking to the Golgi apparatus, proteins sort to many different cellular destinations including the endolysosomal system and the extracellular space. Secreted proteins need to be delivered directly to the cell surface. Sorting of secreted proteins from the Golgi apparatus has been a topic of interest for over thirty years, yet there is still no clear understanding of the machinery that forms the post-Golgi carriers. Most evidence points to these post-Golgi carriers being tubular pleomorphic structures that bud from the trans-face of the Golgi. In this review, we present the background studies and highlight the key components of this pathway, we then discuss the machinery implicated in the formation of these carriers, their translocation across the cytosol, and their fusion at the plasma membrane.

ATP-binding cassette transporters mediate differential biosynthesis of glycosphingolipid species.

The cytosolic-oriented glucosylceramide (GlcCer) synthase is enigmatic, requiring nascent GlcCer translocation to the luminal Golgi membrane to access glycosphingolipid (GSL) anabolic glycosyltransferases. The mechanism by which GlcCer is flipped remains unclear. To investigate the role of GlcCer-binding partners in this process, we previously made cleavable, biotinylated, photoreactive GlcCer analogs in which the reactive nitrene was closely apposed to the GlcCer head group, while maintaining a C16-acyl chain. GlcCer-binding protein specificity was validated for both photoprobes. Using one probe, XLB, here we identified ATP-binding cassette (ABC) transporters ABCA3, ABCB4, and ABCB10 as unfractionated microsomal GlcCer-binding proteins in DU-145 prostate tumor cells. siRNA knockdown (KD) of these transporters differentially blocked GSL synthesis assessed in toto and via metabolic labeling. KD of ABCA3 reduced acid/neutral GSL levels, but increased those of LacCer, while KD of ABCB4 preferentially reduced neutral GSL levels, and KD of ABCB10 reduced levels of both neutral and acidic GSLs. Depletion of ABCA12, implicated in GlcCer transport, preferentially decreased neutral GSL levels, while ABCB1 KD preferentially reduced gangliosides, but increased neutral GSL Gb3. These results imply that multiple ABC transporters may provide distinct but overlapping GlcCer and LacCer pools within the Golgi lumen for anabolism of different GSL series by metabolic channeling. Differential ABC family member usage may fine-tune GSL biosynthesis depending on cell/tissue type. We conclude that ABC transporters provide a new tool for the regulation of GSL biosynthesis and serve as potential targets to reduce selected GSL species/subsets in diseases in which GSLs are dysregulated.

Pisum sativum Defensin 1 Eradicates Mouse Metastatic Lung Nodules from B16F10 Melanoma Cells.

Psd1 is a pea plant defensin which can be actively expressed in Pichia pastoris and shows broad antifungal activity. This activity is dependent on fungal membrane glucosylceramide (GlcCer), which is also important for its internalization, nuclear localization, and endoreduplication. Certain cancer cells present a lipid metabolism imbalance resulting in the overexpression of GlcCer in their membrane. In this work, in vitroassays using B16F10 cells showed that labeled fluorescein isothiocyanate FITC-Psd1 internalized into live cultured cells and targeted the nucleus, which underwent fragmentation, exhibiting approximately 60% of cells in the sub-G0/G1 stage. This phenomenon was dependent on GlcCer, and the participation of cyclin-F was suggested. In a murine lung metastatic melanoma model, intravenous injection of Psd1 together with B16F10 cells drastically reduced the number of nodules at concentrations above 0.5 mg/kg. Additionally, the administration of 1 mg/kg Psd1 decreased the number of lung inflammatory cells to near zero without weight loss, unlike animals that received melanoma cells only. It is worth noting that 1 mg/kg Psd1 alone did not provoke inflammation in lung tissue or weight or vital signal losses over 21 days, inferring no whole animal cytotoxicity. These results suggest that Psd1 could be a promising prototype for human lung anti-metastatic melanoma therapy.

Glucose availability and glycolytic metabolism dictate glycosphingolipid levels.

Cancer therapeutics has seen an emergence and re-emergence of two metabolic fields in recent years, those of bioactive sphingolipids and glycolytic metabolism. Anaerobic glycolysis and its implications in cancer have been at the forefront of cancer research for over 90 years. More recently, the role of sphingolipids in cancer cell metabolism has gained recognition, notably ceramide's essential role in programmed cell death and the role of the glucosylceramide synthase (GCS) in chemotherapeutic resistance. Despite this knowledge, a direct link between these two fields has yet to be definitively drawn. Herein, we show that in a model of highly glycolytic cells, generation of the glycosphingolipid (GSL) glucosylceramide (GlcCer) by GCS was elevated in response to increased glucose availability, while glucose deprivation diminished GSL levels. This effect was likely substrate dependent, independent of both GCS levels and activity. Conversely, leukemia cells with elevated GSLs showed a significant change in GCS activity, but no change in glucose uptake or GCS expression. In a leukemia cell line with elevated GlcCer, treatment with inhibitors of glycolysis or the pentose phosphate pathway (PPP) significantly decreased GlcCer levels. When combined with pre-clinical inhibitor ABT-263, this effect was augmented and production of pro-apoptotic sphingolipid ceramide increased. Taken together, we have shown that there exists a definitive link between glucose metabolism and GSL production, laying the groundwork for connecting two distinct yet essential metabolic fields in cancer research. Furthermore, we have proposed a novel combination therapeutic option targeting two metabolic vulnerabilities for the treatment of leukemia.

Surfactins modulate the lateral organization of fluorescent membrane polar lipids: a new tool to study drug:membrane interaction and assessment of the role of cholesterol and drug acyl chain length.

The lipopeptide surfactin exhibits promising antimicrobial activities which are hampered by haemolytic toxicity. Rational design of new surfactin molecules, based on a better understanding of membrane:surfactin interaction, is thus crucial. We here performed bioimaging of lateral membrane lipid heterogeneity in adherent living human red blood cells (RBCs), as a new relevant bioassay, and explored its potential to better understand membrane:surfactin interactions. RBCs show (sub)micrometric membrane domains upon insertion of BODIPY analogs of glucosylceramide (GlcCer), sphingomyelin (SM) and phosphatidylcholine (PC). These domains exhibit increasing sensitivity to cholesterol depletion by methyl-ß-cyclodextrin. At concentrations well below critical micellar concentration, natural cyclic surfactin increased the formation of PC and SM, but not GlcCer, domains, suggesting preferential interaction with lipid assemblies with the highest vulnerability to methyl-ß-cyclodextrin. Surfactin not only reversed disappearance of SM domains upon cholesterol depletion but further increased PC domain abundance over control RBCs, indicating that surfactin can substitute cholesterol to promote micrometric domains. Surfactin sensitized excimer formation from PC and SM domains, suggesting increased lipid recruitment and/or diffusion within domains. Comparison of surfactin congeners differing by geometry, charge and acyl chain length indicated a strong dependence on acyl chain length. Thus, bioimaging of micrometric lipid domains is a visual powerful tool, revealing that intrinsic lipid domain organization, cholesterol abundance and drug acyl chain length are key parameters for membrane:surfactin interaction. Implications for surfactin preferential location in domains or at their boundaries are discussed and may be useful for rational design of better surfactin molecules.

New insights on glucosylated lipids: metabolism and functions.

Ceramide, cholesterol, and phosphatidic acid are major basic structures for cell membrane lipids. These lipids are modified with glucose to generate glucosylceramide (GlcCer), cholesterylglucoside (ChlGlc), and phosphatidylglucoside (PtdGlc), respectively. Glucosylation dramatically changes the functional properties of lipids. For instance, ceramide acts as a strong tumor suppressor that causes apoptosis and cell cycle arrest, while GlcCer has an opposite effect, downregulating ceramide activities. All glucosylated lipids are enriched in lipid rafts or microdomains and play fundamental roles in a variety of cellular processes. In this review, we discuss the biological functions and metabolism of these three glucosylated lipids.

A review on Gaucher disease: therapeutic potential of ß-glucocerebrosidase-targeted mRNA/saRNA approach.

Gaucher disease (GD), a rare hereditary lysosomal storage disorder, occurs due to a deficiency in the enzyme ß-glucocerebrosidase (GCase). This deficiency leads to the buildup of substrate glucosylceramide (GlcCer) in macrophages, eventually resulting in various complications. Among its three types, GD2 is particularly severe with neurological involvements. Current treatments, such as enzyme replacement therapy (ERT), are not effective for GD2 and GD3 due to their inability to cross the blood-brain barrier (BBB). Other treatment approaches, such as gene or chaperone therapies are still in experimental stages. Additionally, GD treatments are costly and can have certain side effects. The successful use of messenger RNA (mRNA)-based vaccines for COVID-19 in 2020 has sparked interest in nucleic acid-based therapies. Remarkably, mRNA technology also offers a novel approach for protein replacement purposes. Additionally, self-amplifying RNA (saRNA) technology shows promise, potentially producing more protein at lower doses. This review aims to explore the potential of a cost-effective mRNA/saRNA-based approach for GD therapy. The use of GCase-mRNA/saRNA as a protein replacement therapy could offer a new and promising direction for improving the quality of life and extending the lifespan of individuals with GD.

Cholesterol glucosylation is catalyzed by transglucosylation reaction of ß-glucosidase 1.

Cholesteryl glucoside (ß-ChlGlc), a monoglucosylated derivative of cholesterol, is involved in the regulation of heat shock responses. ß-ChlGlc, which is rapidly induced in response to heat shock, activates heat shock transcription factor 1 (HSF1) leading to the expression of heat shock protein 70 (HSP70) in human fibroblasts. Identification and biochemical characterization of the enzyme responsible for ß-ChlGlc formation is important for a complete understanding of the molecular mechanisms leading to HSP70-induction following heat shock. Recently, we demonstrated that ß-ChlGlc synthesis is not dependent on UDP-Glucose but glucosylceramide (GlcCer) in animal tissue and human fibroblasts. In this study, we examined the possibility of glucocerebrosidase, a GlcCer-degrading glycosidase, acting as ß-ChlGlc-synthesizing enzyme. Overexpression of ß-glucosidase 1 (GBA1, lysosomal acid ß-glucocerebrosidase) led to an increase in cholesterol glucosylation activity in human fibroblasts. Using a cell line generated from type 2 Gaucher disease patients with severe defects in GBA1 activity, we found that cholesterol glucosylation activity was very low in the cells and the overexpression of GBA1 rescued the activity. In addition, purified recombinant GBA1 exhibits conduritol B-epoxide-sensitive cholesterol glucosylation activity. The optimum pH and temperature for cholesterol glucosylation by GBA1 were at about 5.3 and 43 °C, respectively. Short chain C8:0-GlcCer was the most effective donor for cholesterol glucosylation activity among GlcCer containing saturated fatty acid (C8:0 to C18:0) tested. GlcCer containing mono-unsaturated fatty acid was more preferred substrate for cholesterol glucosylation when compared with GlcCer containing same chain length of saturated fatty acid. These results demonstrate, for the first time, a novel function of GBA1 as a ß-ChlGlc-synthesizing enzyme. Therefore, our results also reveal a new pathway for glycolipid metabolism in mammals.

Defining the glucosylceramide population of C. elegans.

Glucosylceramides (GlcCer) are lipids that impact signaling pathways, serve as critical components of cellular membranes, and act as precursors for hundreds of other complex glycolipid species. Abnormal GlcCer metabolism is linked to many diseases, including cancers, diabetes, Gaucher disease, neurological disorders, and skin disorders. A key hurdle to fully understanding the role of GlcCer in disease is the development of methods to accurately detect and quantify these lipid species in a model organism. This will allow for the dissection of the role of this pool in vivo with a focus on all the individual types of GlcCer. In this review, we will discuss the analysis of the GlcCer population specifically in the nematode Caenorhabditis elegans, focusing on the mass spectrometry-based methods available for GlcCer quantification. We will also consider the combination of these approaches with genetic interrogation of GlcCer metabolic genes to define the biological role of these unique lipids. Furthermore, we will explore the implications and obstacles for future research.

Ligand Activation of the Aryl Hydrocarbon Receptor Upregulates Epidermal Uridine Diphosphate Glucose Ceramide Glucosyltransferase and Glucosylceramides.

Ligand activation of the aryl hydrocarbon receptor (AHR) accelerates keratinocyte differentiation and the formation of the epidermal permeability barrier. Several classes of lipids, including ceramides, are critical to the epidermal permeability barrier. In normal human epidermal keratinocytes, the AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, increased RNA levels of ceramide metabolism and transport genes: uridine diphosphate glucose ceramide glucosyltransferase (UGCG), ABCA12, GBA1, and SMPD1. Levels of abundant skin ceramides were also increased by 2,3,7,8-tetrachlorodibenzo-p-dioxin. These included the metabolites synthesized by UGCG, glucosylceramides, and acyl glucosylceramides. Chromatin immunoprecipitation-sequence analysis and luciferase reporter assays identified UGCG as a direct AHR target. The AHR antagonist, GNF351, inhibited the 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated RNA and transcriptional increases. Tapinarof, an AHR ligand approved for the treatment of psoriasis, increased UGCG RNA, protein, and its lipid metabolites hexosylceramides as well as increased the RNA expression of ABCA12, GBA1, and SMPD1. In Ahr-null mice, Ugcg RNA and hexosylceramides were lower than those in the wild type. These results indicate that the AHR regulates the expression of UGCG, a ceramide-metabolizing enzyme required for ceramide trafficking, keratinocyte differentiation, and epidermal permeability barrier formation.

Screening for potential undiagnosed Gaucher disease patients: Utilisation of the Gaucher earlier diagnosis consensus point-scoring system (GED-C PSS) in conjunction with electronic health record data, tissue specimens, and small nucleotide polymorphism (SNP) genotype data available in Finnish biobanks.

Background: Autosomal recessive Gaucher disease (GD) is likely underdiagnosed in many countries. Because the number of diagnosed GD patients in Finland is relatively low, and the true prevalence is currently not known, it was hypothesized that undiagnosed GD patients may exist in Finland. Our previous study demonstrated the applicability of Gaucher Earlier Diagnosis Consensus point-scoring system (GED-C PSS; Mehta et al., 2019) and Finnish biobank data and specimens in the automated point scoring of large populations. An indicative point-score range for Finnish GD patients was determined, but undiagnosed patients were not identified partly due to high number of high-score subjects in combination with a lack of suitable samples for diagnostics in the assessed biobank population. The current study extended the screening to another biobank and evaluated the feasibility of utilising the automated GED-C PSS in conjunction with small nucleotide polymorphism (SNP) chip genotype data from the FinnGen study of biobank sample donors in the identification of undiagnosed GD patients in Finland. Furthermore, the applicability of FFPE tissues and DNA restoration in the next-generation sequencing (NGS) of the GBA gene were tested. Methods: Previously diagnosed Finnish GD patients eligible to the study, and up to 45,100 sample donors in Helsinki Biobank (HBB) were point scored. The GED-C point scoring, adjusted to local data, was automated, but also partly manually verified for GD patients. The SNP chip genotype data for rare GBA variants was visually assessed. FFPE tissues of GD patients were obtained from HBB and Biobank Borealis of Northern Finland (BB). Results: Three previously diagnosed GD patients and one patient previously treated for GD-related features were included. A genetic diagnosis was confirmed for the patient treated for GD-related features. The GED-C point score of the GD patients was 12.5-22.5 in the current study. The score in eight Finnish GD patients of the previous and the current study is thus 6-22.5 points per patient. In the automated point scoring of the HBB subpopulation (N ≈ 45,100), the overall scores ranged from 0 to 17.5, with 0.77% (346/45,100) of the subjects having ≥10 points. The analysis of SNP chip genotype data was able to identify the diagnosed GD patients, but potential undiagnosed patients with the GED-C score and/or the GBA genotype indicative of GD were not discovered. Restoration of the FFPE tissue DNA improved the quality of the GBA NGS, and pathogenic GBA variants were confirmed in five out of six unrestored and in all four restored FFPE DNA samples. Discussion: These findings imply that the prevalence of diagnosed patients (~1:325,000) may indeed correspond the true prevalence of GD in Finland. The SNP chip genotype data is a valuable tool that complements the screening with the GED-C PSS, especially if the genotyping pipeline is tuned for rare variants. These proof-of-concept biobank tools can be adapted to other rare genetic diseases.

TRIP12 ubiquitination of glucocerebrosidase contributes to neurodegeneration in Parkinson's disease.

Impairment in glucocerebrosidase (GCase) is strongly associated with the development of Parkinson's disease (PD), yet the regulators responsible for its impairment remain elusive. In this paper, we identify the E3 ligase Thyroid Hormone Receptor Interacting Protein 12 (TRIP12) as a key regulator of GCase. TRIP12 interacts with and ubiquitinates GCase at lysine 293 to control its degradation via ubiquitin proteasomal degradation. Ubiquitinated GCase by TRIP12 leads to its functional impairment through premature degradation and subsequent accumulation of α-synuclein. TRIP12 overexpression causes mitochondrial dysfunction, which is ameliorated by GCase overexpression. Further, conditional TRIP12 knockout in vitro and knockdown in vivo promotes the expression of GCase, which blocks α-synuclein preformed fibrils (α-syn PFFs)-provoked dopaminergic neurodegeneration. Moreover, TRIP12 accumulates in human PD brain and α-synuclein-based mouse models. The identification of TRIP12 as a regulator of GCase provides a new perspective on the molecular mechanisms underlying dysfunctional GCase-driven neurodegeneration in PD.

The Gaucher earlier diagnosis consensus point-scoring system (GED-C PSS): Evaluation of a prototype in Finnish Gaucher disease patients and feasibility of screening retrospective electronic health record data for the recognition of potential undiagnosed patients in Finland.

BACKGROUND: Gaucher disease (GD) is a rare inherited multiorgan disorder, yet a diagnosis can be significantly delayed due to a broad spectrum of symptoms and lack of disease awareness. Recently, the prototype of a GD point-scoring system (PSS) was established by the Gaucher Earlier Diagnosis Consensus (GED-C) initiative, and more recently, validated in Gaucher patients in UK. In our study, the original GED-C PSS was tested in Finnish GD patients. Furthermore, the feasibility of point scoring large electronic health record (EHR) data set by data mining to identify potential undiagnosed GD cases was evaluated. METHODS: This biobank study was conducted in collaboration with two Finnish biobanks. Five previously diagnosed Finnish GD patients and ~ 170,000 adult biobank subjects were included in the study. The original PSS was locally adjusted due to data availability issues and applied to the Finnish EHR data representing special health care recordings. RESULTS: All GD patients had high levels of the biomarker lyso-Gb1 and deleterious GBA mutations. One patient was a compound heterozygote with a novel variant, potentially pathogenic mutation. Finnish EHR data allowed the retrospective assessment of 27-30 of the 32 original GED-C signs/co-variables. Total point scores of GD patients were high but variable, 6-18.5 points per patient (based on the available data on 28-29 signs/co-variables per patient). All GD patients had been recorded with anaemia while only three patients had a record of splenomegaly. 0.72% of biobank subjects were assigned at least 6 points but none of these potential "GD suspects" had a point score as high as 18.5. Splenomegaly had been recorded for 0.25% of biobank subjects and was associated with variable point score distribution and co-occurring ICD-10 diagnoses. DISCUSSION: This study provides an indicative GED-C PSS score range for confirmed GD patients, also representing potential mild cases, and demonstrates the feasibility of scoring Finnish EHR data by data mining in order to screen for undiagnosed GD patients. Further prioritisation of the "GD suspects" with more developed algorithms and data-mining approaches is needed. FUNDING: This study was funded by Shire (now part of Takeda).

Glucosylceramides From Lomentospora prolificans Induce a Differential Production of Cytokines and Increases the Microbicidal Activity of Macrophages.

Lomentospora prolificans is an emerging opportunistic fungus with a high resistance to antifungal agents and it can cause localized infections in immunocompetent patients and disseminated infections with a high mortality rate in immunosuppressed patients. Glucosylceramides (GlcCer) are synthetized in the majority of known fungal pathogens. They are bioactive molecules presenting different functions, such as involvement in fungal growth and morphological transitions in several fungi. The elucidation of the primary structure of the fungal surface glycoconjugates could contribute for the understanding of the mechanisms of pathogenicity. In this work, GlcCer species were isolated from mycelium and conidia forms of L. prolificans and their chemical structures were elucidated by mass spectrometry (ESI-MS). GlcCer purified from both forms presented a major species at m/z 750 that corresponds to N-2-hydroxyhexadecanoyl-1-ß-D-glucopyranosyl-9-methyl-4,8-sphingadienine. Monoclonal antibodies against GlcCer could recognize L. prolificans GlcCer species from mycelium and conidia, suggesting a conserved epitope in fungal GlcCer. In addition, in vivo assays showed that purified GlcCer species from both forms was able to induce a high secretion of pro-inflammatory cytokines by splenocytes. GlcCer species also promote the recruitment of polymorphonuclear, eosinophils, small peritoneal macrophage (SPM) and mononuclear cells to the peritoneal cavity. GlcCer species were also able to induce the oxidative burst by peritoneal macrophages with NO and superoxide radicals production, and to increase the killing of L. prolificans conidia by peritoneal macrophages. These results indicate that GlcCer species from L. prolificans are a potent immune response activator.

Drosophila melanogaster Mutated in its GBA1b Ortholog Recapitulates Neuronopathic Gaucher Disease.

Gaucher disease (GD) results from mutations in the GBA1 gene, which encodes lysosomal glucocerebrosidase (GCase). The large number of mutations known to date in the gene lead to a heterogeneous disorder, which is divided into a non-neuronopathic, type 1 GD, and two neurological, type 2 and type 3, forms. We studied the two fly GBA1 orthologs, GBA1a and GBA1b. Each contains a Minos element insertion, which truncates its coding sequence. In the GBA1am/m flies, which express a mutant protein, missing 33 C-terminal amino acids, there was no decrease in GCase activity or substrate accumulation. However, GBA1bm/m mutant flies presented a significant decrease in GCase activity with concomitant substrate accumulation, which included C14:1 glucosylceramide and C14:0 glucosylsphingosine. GBA1bm/m mutant flies showed activation of the Unfolded Protein Response (UPR) and presented inflammation and neuroinflammation that culminated in development of a neuronopathic disease. Treatment with ambroxol did not rescue GCase activity or reduce substrate accumulation; however, it ameliorated UPR, inflammation and neuroinflammation, and increased life span. Our results highlight the resemblance between the phenotype of the GBA1bm/m mutant fly and neuronopathic GD and underlie its relevance in further GD studies as well as a model to test possible therapeutic modalities.

Psd2 pea defensin shows a preference for mimetic membrane rafts enriched with glucosylceramide and ergosterol.

Psd2 is a pea defensin with 47 amino acid residues that inhibits the growth of fungal species by an uncharacterized mechanism. In this work, Psd2 interactions with model membranes mimicking the lipid compositions of different organisms were evaluated. Protein-lipid overlay assays indicated that Psd2 recognizes Fusarium solani glucosylceramide (GlcCerF.solani) and ergosterol (Erg) in addition to phosphatidylcholine (POPC) and some phosphatidylinositol species, such as PtdIns (3)P, (5)P and (3,5)P2, suggesting that these lipids may play important roles as Psd2 targets. Assays using lipid vesicles were also performed to study the behaviour and dynamics that occur after peptide-membrane interactions. Surface plasmon resonance analysis showed that Psd2 has a higher affinity for pure POPC and POPC-based vesicles containing GlcCer and Erg at a 70:30 proportion than for vesicles containing cholesterol (Chol). Partition experiments by fluorescence spectroscopy showed a decrease in Trp42 quantum yield of Psd2 in the presence of GlcCerF.solani and Erg, individually or in simultaneously enriched membranes. The partition coefficient (Kp) obtained indicated a Psd2 partition preference for this vesicles, confirmed by quenching assays using acrylamide and 5/16-doxyl-stearic acid. Furthermore, we showed that the presence of C8C9 double bonds and a methyl group at position C9 of the sphingoid base backbone of GlcCer was relevant to Psd2 activity against Aspergillus nidulans. These results are consistent with the selectivity of Psd2 against fungi and its lack of toxicity in human erythrocytes. Psd2 represents a promising natural compound for the treatment of fungal infections.

Konjac ceramide (kCer) regulates keratinocyte migration by Sema3A-like repulsion mechanism.

Previously, we proposed the following mechanism for konjac ceramide (kCer)-mediated neurite outgrowth inhibition: kCer binds to Nrp as a Sema3A agonist, resulting in Nrp1/PlexA complex formation and activation of the Sema3A signaling pathway to induce phosphorylation of CRMP2 and microtubule depolymerization. The Sema3A/Nrp1 signaling pathway is known to be also expressed in normal human keratinocytes. To determine whether kCer can function in human keratinocytes as it does in neurites, that is, if it can bind to Nrp1 in place of Sema3A, we studied the effect of kCer on HaCaT cell migration activity. Using a trans-well chamber assay, we compared the effects of Sema3A and kCer on serum-derived cell migration activity. kCer showed Sema3A-like suppression of cell migration activity and induction of cellular Cofilin phosphorylation. In addition, kCer and Sema3A inhibited histamine (His)-enhanced migration of immature HaCaT cells. We have demonstrated that kCer does not interact with histaime receptors H1R or H4R directly, but we speculate that kCer may transduce a signal downstream of the His signaling pathway.

Mass spectrometric quantification of plasma glycosphingolipids in human GM3 ganglioside deficiency.

BACKGROUND: Among Amish communities of North America, biallelic mutations of ST3GAL5 (c.694C > T) eliminate synthesis of GM3 and its derivative downstream a- and b-series gangliosides. Systemic ganglioside deficiency is associated with infantile onset psychomotor retardation, slow brain growth, intractable epilepsy, deafness, and cortical visual impairment. We developed a robust quantitative assay to simultaneously characterize glycan and ceramide moieties of plasma glycosphingolipids (GSLs) among ST3GAL5 c.694C > T homozygotes (n = 8), their heterozygous siblings (n = 24), and wild type control (n = 19) individuals. METHODS: Following extraction and saponification of total plasma lipids, GSLs were purified on a tC18 cartridge column, permethylated, and subjected to nanospray ionization mass spectrometry utilizing neutral loss scanning and data-dependent acquisition. Plasma GSLs were quantified against appropriate synthetic standards. RESULTS: Our method demonstrated linearity from 5 to 250 µl of plasma. Recovery of synthetic GSLs spiked into plasma was 99-104% with no matrix interference. Quantitative plasma GSL profiles discriminated among ST3GAL5 genotypes: GM3 and GD3 were undetectable in ST3GAL5 c.694C > T homozygotes, who had markedly elevated lactosylceramide (19.17 ±â€¯4.20 nmol/ml) relative to heterozygous siblings (9.62 ±â€¯2.46 nmol/ml) and wild type controls (6.55 ±â€¯2.16 nmol/ml). Children with systemic ganglioside deficiency had a distinctive shift in ceramide composition toward higher mass species. CONCLUSIONS: Our quantitative glycolipidomics method discriminates among ST3GAL5 c.694C > T genotypes, can reveal subtle structural heterogeneity, and represents a useful new strategy to diagnose and monitor GSL disorders in humans.

Effect of Torula Yeast (Candida utilis)-Derived Glucosylceramide on Skin Dryness and Other Skin Conditions in Winter.

Glucosylceramide (GlcCer) is present in foods such as barley, corn, and wheat flour. GlcCer derived from different foods has differences in its physiological effects, depending on the sphingoid backbone and constituent fatty acids. In this study, we investigated the moisturizing and skin conditioning effects of GlcCer derived from torula yeast (Candida utilis) in healthy human subjects. The participants were randomly distributed in a crossover, double-blind comparative manner. Seventeen volunteers were orally administered both 1.8 mg/d of GlcCer derived from torula yeast and a placebo for 4 wk. Before and after oral administration, transepidermal water loss (TEWL) was measured and the objective skin condition observation and a questionnaire on skin condition were conducted. The primary endpoint was TEWL; secondary endpoints included the objective and subjective skin conditions. The change in TEWL over the study period on the forearm was -0.97±0.48 and -1.26±0.46 g/m2•h in the placebo and GlcCer groups, respectively, with significantly lower (p=0.01) TEWL observed in the GlcCer group. Brown spots increased in the placebo group but significantly decreased in the GlcCer group (p=0.04). Although chapped skin worsened in the placebo group, it significantly improved in the GlcCer group (p=0.04). The use of torula yeast-derived GlcCer as a functional cosmeceutical food is a viable option to ameliorate skin conditions, including improvement in skin barrier function, reduction of brown spots, and fixation of chapped skin.