Racemization of the substrate and product by serine palmitoyltransferase from Sphingobacterium multivorum yields two enantiomers of the product from d-serine.

Serine palmitoyltransferase (SPT) catalyzes the pyridoxal-5'-phosphate (PLP)-dependent decarboxylative condensation of l-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (KDS). Although SPT was shown to synthesize corresponding products from amino acids other than l-serine, it is still arguable whether SPT catalyzes the reaction with d-serine, which is a question of biological importance. Using high substrate and enzyme concentrations, KDS was detected after the incubation of SPT from Sphingobacterium multivorum with d-serine and palmitoyl-CoA. Furthermore, the KDS comprised equal amounts of 2S and 2R isomers. 1H-NMR study showed a slow hydrogen-deuterium exchange at Cα of serine mediated by SPT. We further confirmed that SPT catalyzed the racemization of serine. The rate of the KDS formation from d-serine was comparable to those for the α-hydrogen exchange and the racemization reaction. The structure of the d-serine-soaked crystal (1.65 Å resolution) showed a distinct electron density of the PLP-l-serine aldimine, interpreted as the racemized product trapped in the active site. The structure of the α-methyl-d-serine-soaked crystal (1.70 Å resolution) showed the PLP-α-methyl-d-serine aldimine, mimicking the d-serine-SPT complex prior to racemization. Based on these enzymological and structural analyses, the synthesis of KDS from d-serine was explained as the result of the slow racemization to l-serine, followed by the reaction with palmitoyl-CoA, and SPT would not catalyze the direct condensation between d-serine and palmitoyl-CoA. It was also shown that the S. multivorum SPT catalyzed the racemization of the product KDS, which would explain the presence of (2R)-KDS in the reaction products.

GPRC5C regulates the composition of cilia in the olfactory system.

BACKGROUND: Olfactory sensory neurons detect odourants via multiple long cilia that protrude from their dendritic endings. The G protein-coupled receptor GPRC5C was identified as part of the olfactory ciliary membrane proteome, but its function and localization is unknown. RESULTS: High-resolution confocal and electron microscopy revealed that GPRC5C is located at the base of sensory cilia in olfactory neurons, but not in primary cilia of immature neurons or stem cells. Additionally, GPRC5C localization in sensory cilia parallels cilia formation and follows the formation of the basal body. In closer examination, GPRC5C was found in the ciliary transition zone. GPRC5C deficiency altered the structure of sensory cilia and increased ciliary layer thickness. However, primary cilia were unaffected. Olfactory sensory neurons from Gprc5c-deficient mice exhibited altered localization of olfactory signalling cascade proteins, and of ciliary phosphatidylinositol-4,5-bisphosphat. Sensory neurons also exhibited increased neuronal activity as well as altered mitochondrial morphology, and knockout mice had an improved ability to detect food pellets based on smell. CONCLUSIONS: Our study shows that GPRC5C regulates olfactory cilia composition and length, thereby controlling odour perception.

Ceramides Increase Fatty Acid Utilization in Intestinal Progenitors to Enhance Stemness and Increase Tumor Risk.

BACKGROUND & AIMS: Cancers of the alimentary tract, including esophageal adenocarcinomas, colorectal cancers, and cancers of the gastric cardia, are common comorbidities of obesity. Prolonged, excessive delivery of macronutrients to the cells lining the gut can increase one's risk for these cancers by inducing imbalances in the rate of intestinal stem cell proliferation vs differentiation, which can produce polyps and other aberrant growths. We investigated whether ceramides, which are sphingolipids that serve as a signal of nutritional excess, alter stem cell behaviors to influence cancer risk. METHODS: We profiled sphingolipids and sphingolipid-synthesizing enzymes in human adenomas and tumors. Thereafter, we manipulated expression of sphingolipid-producing enzymes, including serine palmitoyltransferase (SPT), in intestinal progenitors of mice, cultured organoids, and Drosophila to discern whether sphingolipids altered stem cell proliferation and metabolism. RESULTS: SPT, which diverts dietary fatty acids and amino acids into the biosynthetic pathway that produces ceramides and other sphingolipids, is a critical modulator of intestinal stem cell homeostasis. SPT and other enzymes in the sphingolipid biosynthesis pathway are up-regulated in human intestinal adenomas. They produce ceramides, which serve as prostemness signals that stimulate peroxisome-proliferator activated receptor-α and induce fatty acid binding protein-1. These actions lead to increased lipid utilization and enhanced proliferation of intestinal progenitors. CONCLUSIONS: Ceramides serve as critical links between dietary macronutrients, epithelial regeneration, and cancer risk.

Lysophosphatidylglucoside/GPR55 signaling promotes foam cell formation in human M2c macrophages.

Atherosclerosis is a major cause of cerebral and cardiovascular diseases. Intravascular plaques, a well-known pathological finding of atherosclerosis, have a necrotic core composed of macrophages and dead cells. Intraplaque macrophages, which are classified into various subtypes, play key roles in maintenance of normal cellular microenvironment. Excessive uptake of oxidized low-density lipoprotein causes conversion of macrophages to foam cells, and consequent progression/exacerbation of atherosclerosis. G-protein-coupled receptor 55 (GPR55) signaling has been reported to associate with atherosclerosis progression. We demonstrated recently that lysophosphatidylglucoside (lysoPtdGlc) is a specific ligand of GPR55, although in general physiological ligands of GPR55 are poorly understood. Phosphatidylglucoside is expressed on human monocytes and can be converted to lysoPtdGlc. In the present study, we examined possible involvement of lysoPtdGlc/GPR55 signaling in foam cell formation. In monocyte-derived M2c macrophages, lysoPtdGlc/GPR55 signaling inhibited translocation of ATP binding cassette subfamily A member 1 to plasma membrane, and cholesterol efflux. Such inhibitory effect was reversed by GPR55 antagonist ML193. LysoPtdGlc/GPR55 signaling in M2c macrophages was involved in excessive lipid accumulation, thereby promoting foam cell formation. Our findings suggest that lysoPtdGlc/GPR55 signaling is a potential therapeutic target for inhibition of atherosclerosis progression.

GPR55 contributes to neutrophil recruitment and mechanical pain induction after spinal cord compression in mice.

Pain transmission and processing in the nervous system are modulated by various biologically active substances, including lysophospholipids, through direct and indirect actions on the somatosensory pathway. Lysophosphatidylglucoside (LysoPtdGlc) was recently identified as a structurally unique lysophospholipid that exerts biological actions via the G protein-coupled receptor GPR55. Here, we demonstrated that GPR55-knockout (KO) mice show impaired induction of mechanical pain hypersensitivity in a model of spinal cord compression (SCC) without the same change in the models of peripheral tissue inflammation and peripheral nerve injury. Among these models, only SCC recruited peripheral inflammatory cells (neutrophils, monocytes/macrophages, and CD3+ T-cells) in the spinal dorsal horn (SDH), and GPR55-KO blunted these recruitments. Neutrophils were the first cells recruited to the SDH, and their depletion suppressed the induction of SCC-induced mechanical hypersensitivity and inflammatory responses in compressed SDH. Furthermore, we found that PtdGlc was present in the SDH and that intrathecal administration of an inhibitor of secretory phospholipase A2 (an enzyme required for producing LysoPtdGlc from PtdGlc) reduced neutrophil recruitment to compressed SDH and suppressed pain induction. Finally, by screening compounds from a chemical library, we identified auranofin as a clinically used drug with an inhibitory effect on mouse and human GPR55. Systemically administered auranofin to mice with SCC effectively suppressed spinal neutrophil infiltration and pain hypersensitivity. These results suggest that GPR55 signaling contributes to the induction of inflammatory responses and chronic pain after SCC via the recruitment of neutrophils and may provide a new target for reducing pain induction after spinal cord compression, such as spinal canal stenosis.

Protecting-group-free glycosylation of phosphatidic acid in aqueous media.

The glycosylation of unprotected carbohydrates has emerged as an area of significant interest because it obviates the need for long reaction sequences involving protecting-group manipulations. Herein, we report the one-pot synthesis of anomeric glycosyl phosphates through the condensation of unprotected carbohydrates with phospholipid derivatives while retaining high stereo- and regioselective control. The anomeric center was activated using 2-chloro-1,3-dimethylimidazolinium chloride to facilitate condensation with glycerol-3-phosphate derivatives in an aqueous solution. A water/propionitrile mixture provided superior stereoselectivity while maintaining good yields. Under these optimized conditions, the condensation of stable isotope-labeled glucose with phosphatidic acid provided efficient access to labeled glycophospholipids as an internal standard for mass spectrometry.

Synthesis of O-Linked Glycoconjugates in the Nervous System.

Glycoproteins carrying O-linked N-acetylgalactosamine, N-acetylglucosamine, mannose, fucose, glucose, and xylose are found in the nervous system. Lipids are glycosylated by distinct glycosylation enzymes as well. Membrane lipid, ceramide, is modified by the addition of either glucose or galactose to form glycosphingolipid, galactosylceramide, or glucosylceramide. Recent careful analyses by MS have identified glucosylated lipids of cholesterol and phosphatidic acid. These O-linked carbohydrate residues are found primarily on the outer surface of the plasma membrane or in the extracellular space. Their expression is cell or tissue specific and developmentally regulated. Due to their structural diversity, they play important roles in a variety of biological processes such as membrane transport, metabolic stress responses, cell-cell interactions and so on. Discoveries of human diseases associated with glycosylation enzyme deficits have proved modification of lipids and proteins with carbohydrates play critical roles in human health and disease in the nervous systems.

A pilot study assessing sphingolipids and glycolipids dysmetabolism in idiopathic normal pressure hydrocephalus.

Idiopathic normal pressure hydrocephalus usually exhibits triad of symptoms including gait disturbance, urinary incontinence, and dementia with ventriculomegaly. Currently, its pathogenesis remains to be fully elucidated. To provide a better understanding of this order, we examined whether dysmetabolism of sphingolipids as major lipid components in the brain present in cerebrospinal fluid (CSF) of the patients. Here, we measured various sphingolipidsincluding ceramide and sphingomyelin and glycolipids by electrospray ionization-tandem mass spectrometry in the cerebrospinal fluid of 19 consecutive idiopathic normal pressure hydrocephalus patients, 49 Parkinson's disease patients, and 17 neurologically normal controls. The data showed that there was a significant and specific reduction of all galactosylceramide subspecies levels in idiopathic normal pressure hydrocephalus patients compared with other groups, whereas ceramide and sphingomyelin levels as well as other neutral glycolipids such as glucosylceramide and lactosylceramide were similar in both disease states. Multiple regression analysis of sex and age did not show any correlation with galactosylceramide levels. We also examined whether MMSE scores are correlated with sphingolipid levels in iNPH patients. A specific subspecies of sphingomyelin (d18:1/18:0) only exhibited a statistically significant negative correlation (p = 0.0473, R = -0.4604) with MMSE scores but no other sphingolipids in iNPH patients. These data strongly suggest that myelin-rich galactosylceramide metabolism is severely impaired in idiopathic normal pressure hydrocephalus patients and might serve as the basis of biomarker for this disorder.

Selective involvement of UGGT variant: UGGT2 in protecting mouse embryonic fibroblasts from saturated lipid-induced ER stress.

Secretory proteins and lipids are biosynthesized in the endoplasmic reticulum (ER). The "protein quality control" system (PQC) monitors glycoprotein folding and supports the elimination of terminally misfolded polypeptides. A key component of the PQC system is Uridine diphosphate glucose:glycoprotein glucosyltransferase 1 (UGGT1). UGGT1 re-glucosylates unfolded glycoproteins, to enable the re-entry in the protein-folding cycle and impede the aggregation of misfolded glycoproteins. In contrast, a complementary "lipid quality control" (LQC) system that maintains lipid homeostasis remains elusive. Here, we demonstrate that cytotoxic phosphatidic acid derivatives with saturated fatty acyl chains are one of the physiological substrates of UGGT2, an isoform of UGGT1. UGGT2 produces lipid raft-resident phosphatidylglucoside regulating autophagy. Under the disruption of lipid metabolism and hypoxic conditions, UGGT2 inhibits PERK-ATF4-CHOP-mediated apoptosis in mouse embryonic fibroblasts. Moreover, the susceptibility of UGGT2 KO mice to high-fat diet-induced obesity is elevated. We propose that UGGT2 is an ER-localized LQC component that mitigates saturated lipid-associated ER stress via lipid glucosylation.

Hepatocyte-Specific Phgdh-Deficient Mice Culminate in Mild Obesity, Insulin Resistance, and Enhanced Vulnerability to Protein Starvation.

l-Serine (Ser) is synthesized de novo from 3-phosphoglycerate via the phosphorylated pathway committed by phosphoglycerate dehydrogenase (Phgdh). A previous study reported that feeding a protein-free diet increased the enzymatic activity of Phgdh in the liver and enhanced Ser synthesis in the rat liver. However, the nutritional and physiological functions of Ser synthesis in the liver remain unclear. To clarify the physiological significance of de novo Ser synthesis in the liver, we generated liver hepatocyte-specific Phgdh KO (LKO) mice using an albumin-Cre driver. The LKO mice exhibited a significant gain in body weight compared to Floxed controls at 23 weeks of age and impaired systemic glucose metabolism, which was accompanied by diminished insulin/IGF signaling. Although LKO mice had no apparent defects in steatosis, the molecular signatures of inflammation and stress responses were evident in the liver of LKO mice. Moreover, LKO mice were more vulnerable to protein starvation than the Floxed mice. These observations demonstrate that Phgdh-dependent de novo Ser synthesis in liver hepatocytes contributes to the maintenance of systemic glucose tolerance, suppression of inflammatory response, and resistance to protein starvation.

Lysophosphatidylglucoside is a GPR55 -mediated chemotactic molecule for human monocytes and macrophages.

Neutrophils undergo spontaneous apoptosis within 24-48 h after leaving bone marrow. Apoptotic neutrophils are subsequently phagocytosed and cleared by macrophages, thereby maintaining neutrophil homeostasis. Previous studies have demonstrated involvement of lysophosphatidylglucoside (lysoPtdGlc), a degradation product of PtdGlc, in modality-specific repulsive guidance of spinal sensory axons, via its specific receptor GPR55. In the present study, using human monocytic cell line THP-1 as a model, we demonstrated that lysoPtdGlc induces monocyte/macrophage migration with typical bell-haped curve and a peak at concentration 10-9 M. Lysophosphatidylinositol (lysoPtdIns), a known GPR55 ligand, induced migration at higher concentration (10-7 M). LysoPtdGlc-treated cells had a polarized shape, whereas lysoPtdIns-treated cells had a spherical shape. In EZ-TAXIScan (chemotaxis) assay, lysoPtdGlc induced chemotactic migration activity of THP-1 cells, while lysoPtdIns induced random migration activity. GPR55 antagonist ML193 inhibited lysoPtdGlc-induced THP-1 cell migration, whereas lysoPtdIns-induced migration was inhibited by CB2-receptor inverse agonist. SiRNA experiments showed that GPR55 mediated lysoPtdGlc-induced migration, while lysoPtdIns-induced migration was mediated by CB2 receptor. Our findings, taken together, suggest that lysoPtdGlc functions as a chemotactic molecule for human monocytes/macrophages via GPR55 receptor, while lysoPtdIns induces random migration activity via CB2 receptor.

Impact of Gba2 on neuronopathic Gaucher's disease and α-synuclein accumulation in medaka (Oryzias latipes).

Homozygous mutations in the lysosomal glucocerebrosidase gene, GBA1, cause Gaucher's disease (GD), while heterozygous mutations in GBA1 are a strong risk factor for Parkinson's disease (PD), whose pathological hallmark is intraneuronal α-synuclein (asyn) aggregates. We previously reported that gba1 knockout (KO) medaka exhibited glucosylceramide accumulation and neuronopathic GD phenotypes, including short lifespan, the dopaminergic and noradrenergic neuronal cell loss, microglial activation, and swimming abnormality, with asyn accumulation in the brains. A recent study reported that deletion of GBA2, non-lysosomal glucocerebrosidase, in a non-neuronopathic GD mouse model rescued its phenotypes. In the present study, we generated gba2 KO medaka and examined the effect of Gba2 deletion on the phenotypes of gba1 KO medaka. The Gba2 deletion in gba1 KO medaka resulted in the exacerbation of glucosylceramide accumulation and no improvement in neuronopathic GD pathological changes, asyn accumulation, or swimming abnormalities. Meanwhile, though gba2 KO medaka did not show any apparent phenotypes, biochemical analysis revealed asyn accumulation in the brains. gba2 KO medaka showed a trend towards an increase in sphingolipids in the brains, which is one of the possible causes of asyn accumulation. In conclusion, this study demonstrated that the deletion of Gba2 does not rescue the pathological changes or behavioral abnormalities of gba1 KO medaka, and GBA2 represents a novel factor affecting asyn accumulation in the brains.

[Pathogenic and Compensatory Mechanisms in Epidermis of Sphingomyelin Synthase 2-Deficient Mice].

Sphingomyelin (SM) is a constituent of cellular membranes, while ceramides (Cer) produced from SM on plasma membranes serve as a lipid mediator that regulates cell proliferation, differentiation, and apoptosis. In the skin, SM also is a precursor of Cer, an important constituent of epidermal permeability barrier. We investigated the role of epidermal SM synthase (SMS)2, an isoform of SMS, which modulates SM and Cer levels on plasma membranes. Although SMS2-knockout (SMS2-KO) mice were not neonatal lethal, an ichthyotic phenotype with epidermal hyperplasia and hyperkeratosis was evident at birth, which persisted until 2 weeks of age. These mice showed abnormal lamellar body morphology and secretion, and abnormal extracellular lamellar membranes in the stratum corneum. These abnormalities were no longer evident by 4 weeks of age in SMS2-KO mice. Our study suggests that (1) exposure to a dry terrestrial environment initiates compensatory responses, thereby normalizing epidermal ichthyotic abnormalities and (2) that a nonlethal gene abnormality can cause an ichthyotic skin phenotype.

Total Synthesis and Structural Characterization of Caveolin-1.

Caveolin-1, which is an essential protein for caveola formation, was chemically synthesized. It is composed of 177 amino acid residues, is triply palmitoylated at the C-terminal region, and is inserted into the lipid bilayer to form a V-shaped structure in the middle of the polypeptide chain. The entire sequence was divided into five peptide segments, each of which was synthesized by the solid-phase method. To improve the solubility of the C-terminal region, O-acyl isopeptide structures were incorporated. After ligation by the thioester method and the introduction of the palmitoyl groups, all the protecting groups were removed and the isopeptide structures were converted into the native peptide bond. Finally, the obtained polypeptide was successfully inserted into bicelles, thus showing the success of the synthesis.

Effect of selectively knocking down key metabolic genes in Müller glia on photoreceptor health.

The importance of Müller glia for retinal homeostasis suggests that they may have vulnerabilities that lead to retinal disease. Here, we studied the effect of selectively knocking down key metabolic genes in Müller glia on photoreceptor health. Immunostaining indicated that murine Müller glia expressed insulin receptor (IR), hexokinase 2 (HK2) and phosphoglycerate dehydrogenase (PHGDH) but very little pyruvate dehydrogenase E1 alpha 1 (PDH-E1α) and lactate dehydrogenase A (LDH-A). We crossed Müller glial cell-CreER (MC-CreER) mice with transgenic mice carrying a floxed IR, HK2, PDH-E1α, LDH-A, or PHGDH gene to study the effect of selectively knocking down key metabolic genes in Müller glia cells on retinal health. Selectively knocking down IR, HK2, or PHGDH led to photoreceptor degeneration and reduced electroretinographic responses. Supplementing exogenous l-serine prevented photoreceptor degeneration and improved retinal function in MC-PHGDH knockdown mice. We unexpectedly found that the levels of retinal serine and glycine were not reduced but, on the contrary, highly increased in MC-PHGDH knockdown mice. Moreover, dietary serine supplementation, while rescuing the retinal phenotypes caused by genetic deletion of PHGDH in Müller glial cells, restored retinal serine and glycine homeostasis probably through regulation of serine transport. No retinal abnormalities were observed in MC-CreER mice crossed with PDH-E1α- or LDH-A-floxed mice despite Cre expression. Our findings suggest that Müller glia do not complete glycolysis but use glucose to produce serine to support photoreceptors. Supplementation with exogenous serine is effective in preventing photoreceptor degeneration caused by PHGDH deficiency in Müller glia.

Galabiosylceramide is present in human cerebrospinal fluid.

Cerebrospinal fluid (CSF) contains glycosphingolipids, including lactosylceramide (LacCer, Galß(1,4)Glcß-ceramide). LacCer and its structural isomer, galabiosylceramide (Gb2, Galα(1,4)Galß-ceramide), are classified as ceramide dihexosides (CDH). Gb2 is degraded by α-galactosidase A (GLA) in lysosomes, and genetic GLA deficiency causes Fabry disease, an X-linked lysosomal storage disorder. In patients with Fabry disease, Gb2 accumulates in organs throughout the body. While Gb2 has been reported to be in the liver, kidney, and urine of healthy individuals, its presence in CSF has not been reported, either in patients with Fabry disease or healthy controls. Here, we isolated CDH fractions from CSF of patients with idiopathic normal pressure hydrocephalus. Purified CDH fractions showed positive reaction with Shiga toxin, which specifically binds to the Galα(1,4)Galß structure. The isolated CDH fractions were analyzed by hydrophilic interaction chromatography (HILIC)-electrospray ionization tandem mass spectrometry (ESI-MS/MS). HILIC-ESI-MS/MS separated LacCer and Gb2 and revealed the presence of Gb2 and LacCer in the fractions. We also found Gb2 in CSF from neurologically normal control subjects. This is the first report to show Gb2 exists in human CSF.

Ceramides are necessary and sufficient for diet-induced impairment of thermogenic adipocytes.

OBJECTIVE: Aging and weight gain lead to a decline in brown and beige adipocyte functionality that exacerbates obesity and insulin resistance. We sought to determine whether sphingolipids, such as ceramides, a class of lipid metabolites that accumulate in aging and overnutrition, are sufficient or necessary for the metabolic impairment of these thermogenic adipocytes. METHODS: We generated new mouse models allowing for the conditional ablation of genes required for ceramide synthesis (i.e., serine palmitoyltransferase subunit 2, Sptlc2) or degradation (i.e., acid ceramidase 1, Asah1) from mature, thermogenic adipocytes (i.e., from cells expressing uncoupling protein-1). Mice underwent a comprehensive suite of phenotyping protocols to assess energy expenditure and glucose and lipid homeostasis. Complementary studies were conducted in primary brown adipocytes to dissect the mechanisms controlling ceramide synthesis or action. RESULTS: Depletion of Sptlc2 increased energy expenditure, improved glucose homeostasis, and prevented diet-induced obesity. Conversely, depletion of Asah1 led to ceramide accumulation, diminution of energy expenditure, and exacerbation of insulin resistance and obesity. Mechanistically, ceramides slowed lipolysis, inhibited glucose uptake, and decreased mitochondrial respiration. Moreover, ß-adrenergic receptor agonists, which activate thermogenesis in brown adipocytes, decreased transcription of enzymes required for ceramide synthesis. CONCLUSIONS: These studies support our hypothesis that ceramides are necessary and sufficient for the impairment in thermogenic adipocyte function that accompanies obesity. Moreover, they suggest that implementation of therapeutic strategies to block ceramide synthesis in thermogenic adipocytes may serve as a means of improving adipose health and combating obesity and cardiometabolic disease.

Cerebrospinal Fluid Profiles in Parkinson's Disease: No Accumulation of Glucosylceramide, but Significant Downregulation of Active Complement C5 Fragment.

BACKGROUND: As mutations in glucocerebrosidase 1 (GBA1) are a major risk factor for Parkinson's disease (PD), decreased GBA1 activity might play an important role in the pathogenesis of the disease. However, there are currently no reports on glucosylceramide levels in the cerebrospinal fluid (CSF) in PD. OBJECTIVE: We investigated whether glucosylceramide accumulation and abnormal immune status in the brain are associated with PD. METHODS: We measured glucosylceramide by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) as well as levels of the active fragment of complement C5, C5a, in the CSF of 33 PD, 15 amyotrophic lateral sclerosis (ALS) and 22 neurologically normal control (NNC) subjects. Serum C5a levels in all PD and ALS cases and in a limited number of NNC subjects (n = 8) were also measured. RESULTS: C5a levels in CSF were significantly downregulated in PD compared with NNC. Moreover, CSF C5a/serum C5a ratio showed pronounced perturbations in PD and ALS patients. LC-ESI-MS/MS revealed a statistically significant accumulation of a specific subspecies of glucosylceramide (d18 : 1/C23 : 0 acyl chain fatty acid) in ALS, but not in PD. Interestingly, CSF glucosylceramide (d18 : 1/C23 : 0) exhibited a significant correlation with CSF C5a levels in PD, but not ALS. No correlation was observed between C5a levels or glucosylceramide subspecies content and disease duration, levodopa equivalent daily dose or Hoehn & Yahr staging in PD. CONCLUSION: Our findings demonstrate complement dysregulation without glucosylceramide accumulation in PD CSF. Furthermore, we found an association between a specific glucosylceramide subspecies and immune status in PD.

ß-Glucosylation of cholesterol reduces sterol-sphingomyelin interactions.

Cholesteryl-ß-D-glucoside (ChoGlc) is a mammalian glycolipid that is expressed in brain tissue. The effects of glucosylation on the ordering and lipid interactions of cholesterol (Cho) were examined in membranes composed of N-stearoyl sphingomyelin (SSM), which is abundant in the brain, and to investigate the possible molecular mechanism involved in these interactions. Differential scanning calorimetry revealed that ChoGlc was miscible with SSM in a similar extent of Cho. Solid-state 2H NMR of deuterated SSM and fluorescent anisotropy using 1,6-diphenylhexatriene demonstrated that the glucosylation of Cho significantly reduced the effect of the sterol tetracyclic core on the ordering of SSM chains. The orientation of the sterol core was further examined by solid-state NMR analysis of deuterated and fluorinated ChoGlc analogues. ChoGlc had a smaller tilt angle between the long molecular axis (C3-C17) and the membrane normal than Cho in SSM bilayers, and the fluctuations in the tilt angle were largely unaffected by temperature-dependent mobility changes of SSM acyl chains. This orientation of the sterol core of ChoGlc leads to reduce sterol-SSM interactions. The MD simulation results suggested that the Glc moiety perturbs the SSM-sterol interactions, which reduces the umbrella effect of the phosphocholine headgroup because the hydrophilic glucose moiety resides at the same depth as an SSM amide group. These differences between ChoGlc and Cho also weaken the SSM-ChoGlc interactions. Thus, the distribution and localization of Cho and ChoGlc possibly control the stability of sphingomyelin-based domains that transiently occur at specific locations in biological membranes.