Effect of Cholesterol glycosylation on membrane organization We examined the effect of 3-O-glucosylation of Chol on the ordering and lipid interactions of Chol in artificial membrane composed of brain major lipid, N-stearoyl sphingomyelin (C18:0-SM) in detail using solid-state NMR and physicochemical techniques (collaboration with Dr. S. Hanashima and Prof. M. Murata). Glucosylation of cholesterol weakens the interaction with C18:0-SM, resulting in the release of GlcChol from the lipid rafts (Figure 3) 7). Molecular dynamics (MD) simulation suggested that GlcChol has reduced umbrella effect from the phosphocholine headgroup of C18:0-SM due to the Glc head group, probably affecting the distribution and localization of the sterol core in biological membranes. Since GlcChol is enriched in extracellular vesicles (EVs/exosomes), possibly GlcChol is localized in a raft-like membrane domain (Figure 2C). Since no specific probe to GlcChol is available, there is no experimental evidence to show membrane distribution and localization of GlcChol at this moment. Also, it is of interest that whether or not ceramide generation from GlcCer is involved in EVs formation and secretion from cell membranes since ceramide produced from the hydrolysis of SM by neutral sphingomyelinase plays an important role in the fate of exosomes.In the process of analyzing GSLs in CSF, we found a glycolipid that is similar in structure to lactosylceramide (LacCer) but shows a different elution time from LacCer by HILIC-LC-MS analysis, and its structure is determined to be Gb2 (Galα1-4Gal1-1’Cer) 9). Gb2 production is dependent on the expression of both UGT8 (GalCer synthase) and A4GALT, and Gb2 in CSF is possibly derived from pia mater and choroid plexus. Since Gb2 has Shiga toxin receptor activity, this is an important finding in relation to STGEC-HUS (Shiga toxin-producing enterohemorrhagic E. coli associated hemolytic uremic syndrome).Detection of glycosylated lipids in cerebrospinal fluidIt has been reported that GSLs are present in human cerebrospinal fluid (CSF) and useful markers for the genetic disorders of GSLs metabolism such as Gaucher and Fabry diseases. It is also suggested that abnormal GSL metabolism is associated with aging associated neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). To understand whether the metabolites of cholesterol are involved in PD pathology we have examined human CSF as an alternative to pathological tissues of the human brains and confirmed that both GlcChol and GalChol are indeed present, although the pathological significance is not evident. Interestingly, we noticed a statistically significant accumulation of a specific subspecies of GlcCer (d18:1/C23:0) in ALS, but not in PD 8).Glycolipids generated by metabolic crosstalk between sphingolipid and sterolYoshio HirabayashiFigure 3. Orientation of GlcChol in sphingomyelin rich membranes Based on the characteristics of the ceramide structure, sphingolipids and cholesterol are thought to form a hydrogen bonding network across the hydroxyl group at position 3 of cholesterol in the membrane. The large size of the hydrophilic head group on sphingomyelin (phosphocholine) shields the 3-hydroxy group of Chol from water molecules (umbrella effect) which makes it possible to carry our trans-glycosylation. Glycosylation of Chol perturbs the SM-Chol interaction.82
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