The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading β-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa. Furthermore, the natural occurrence of 1-O-cholesteryl-β-D-glucopyranoside (GlcChol) in mouse tissues and human plasma is demonstrated using LC-MS/MS and 13C6-labeled GlcChol as internal standard. In cells, the inhibition of GBA increases GlcChol, whereas inhibition of GBA2 decreases glucosylated sterol. Similarly, in GBA2-deficient mice, GlcChol is reduced. Depletion of GlcCer by inhibition of GlcCer synthase decreases GlcChol in cells and likewise in plasma of inhibitor-treated Gaucher disease patients. In tissues of mice with Niemann-Pick type C disease, a condition characterized by intralysosomal accumulation of cholesterol, marked elevations in GlcChol occur as well. When lysosomal accumulation of cholesterol is induced in cultured cells, GlcChol is formed via lysosomal GBA. This illustrates that reversible transglucosylation reactions are highly dependent on local availability of suitable acceptors. In conclusion, mammalian tissues contain GlcChol formed by transglucosylation through β-glucosidases using GlcCer as donor. Our findings reveal a novel metabolic function for GlcCer.
IntroductionHematopoietic stem cells (HSCs) are self-renewing multipotent cells that give rise to all hematopoietic lineages throughout life. Their stem cell characteristics are maintained and regulated in a special microenvironment termed the niche. 1 In adult bone marrow (BM), 2 types of HSC niches have been identified: the osteoblastic niche and the vascular niche. 2 In the osteoblastic niche, osteoblasts and surrounding stromal components in the endosteum region regulate long-term HSCs (LT-HSCs) via cell adhesion and cytokinemediated signaling. [3][4][5][6][7] The vascular niche consists of sinusoidal endothelial cells (ECs) and reticular cells that secrete CXCL12, which is a chemokine that promotes HSC maintenance. 8,9 HSCs maintain cell-cycle quiescence via the interaction with these niches. In contrast with the adult stage, the fetal liver (FL) is a main hematopoietic organ during development in mid-late gestation, where HSCs are rapidly self-renewed to secure an HSC pool that is sufficient for postnatal life. 10 Unlike what is observed for adult BM, the microenvironment of FL and the molecular mechanism that underlies HSC expansion and maintenance are largely unknown.Endothelial protein C receptor (EPCR, Procr) is a type I transmembrane glycoprotein 11 expressed mainly in ECs of larger blood vessels, liver sinusoids, monocytes, leukocytes, and several tumor cells. [12][13][14][15][16] The major role of EPCR is anticoagulation. EPCR binds to protein C with high affinity and augments the activation of protein C by thrombin-thrombomodulin (TM) complex. 17 Released activated protein C (APC) binds to cofactor protein S and degrades the coagulation factors Va and VIIIa. Another important functional aspect of EPCR is its cytoprotective action. EPCR-bound APC induces signaling that is mediated by the protease-activated receptor 1 (Par-1). 18 This APC/EPCR/Par-1 pathway exerts multiple activities, which include antiapoptosis, 19,20 anti-inflammatory action, 21 and anti-endothelial barrier activation. 22 The expression of Procr in HSCs was first observed during the exploration of the nonhematopoietic gene profile using DNA microarray technology. 23 Recent studies showed that EPCR can be used as a tool to identify LT-HSCs, or to purify signaling lymphocytic activation molecule (CD48 Ϫ CD150 ϩ ) HSCs. 24,25 However, whether EPCR has a functional role in HSC maintenance or regulation remains to be elucidated.Achieving ex vivo expansion of HSCs is clinically important for securing a stable and readily available source of HSCs for transplantation. FL-derived HSCs are a good candidate for this purpose because of their distinct capabilities in the in utero treatment of severe immunologic, hematologic, and metabolic diseases. 26 The understanding of the biology of developing FLderived HSCs and the identification of the HSC niche in FL and of the molecular mechanism that governs HSC maintenance are crucial not only for the treatment of these difficult cases, but also for the development of novel therapeutic protocols for H...
Gaucher disease is caused by an inherited deficiency of the enzyme glucosylceramidase. Due to the lack of a fully functional enzyme, there is progressive build-up of the lipid component glucosylceramide. Insufficient glucosylceramidase activity results in hepatosplenomegaly, cytopenias, and bone disease in patients. Gene therapy represents a future therapeutic option for patients unresponsive to enzyme replacement therapy and lacking a suitable bone marrow donor. By proof-of-principle experiments, we have previously demonstrated a reversal of symptoms in a murine disease model of type 1 Gaucher disease, using gammaretroviral vectors harboring strong viral promoters to drive glucosidase β-acid (GBA) gene expression. To investigate whether safer vectors can correct the enzyme deficiency, we utilized self-inactivating lentiviral vectors (SIN LVs) with the GBA gene under the control of human phosphoglycerate kinase (PGK) and CD68 promoter, respectively. Here, we report prevention of, as well as reversal of, manifest disease symptoms after lentiviral gene transfer. Glucosylceramidase activity above levels required for clearance of glucosylceramide from tissues resulted in reversal of splenomegaly, reduced Gaucher cell infiltration and a restoration of hematological parameters. These findings support the use of SIN-LVs with cellular promoters in future clinical gene therapy protocols for type 1 Gaucher disease.
Sudden infant death syndrome (SIDS) is the most frequent manner of post-perinatal death among infants. One of the suggested causes of the syndrome is inherited cardiac diseases, mainly channelopathies, that can trigger arrhythmias and sudden death. The purpose of this study was to investigate cases of sudden unexpected death in infancy (SUDI) for potential causative variants in 100 cardiac-associated genes. We investigated 47 SUDI cases of which 38 had previously been screened for variants in RYR2, KCNQ1, KCNH2 and SCN5A. Using the Haloplex Target Enrichment System (Agilent) and next-generation sequencing (NGS), the coding regions of 100 genes associated with inherited channelopathies and cardiomyopathies were captured and sequenced on the Illumina MiSeq platform. Sixteen (34%) of the SUDI cases had variants with likely functional effects, based on conservation, computational prediction and allele frequency, in one or more of the genes screened. The possible effects of the variants were not verified with family or functional studies. Eight (17%) of the SUDI cases had variants in genes affecting ion channel functions. The remaining eight cases had variants in genes associated with cardiomyopathies. In total, one third of the SUDI victims in a forensic setting had variants with likely functional effect that presumably contributed to the cause of death. The results support the assumption that channelopathies are important causes of SUDI. Thus, analysis of genes associated with cardiac diseases in SUDI victims is important in the forensic setting and a valuable supplement to the clinical investigation in all cases of sudden death.
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