Saskia Scheij

Elevated plasma glucosylsphingosine in Gaucher disease: relation to phenotype, storage cell markers, and therapeutic response.

Gaucher disease, caused by a deficiency of the lysosomal enzyme glucocerebrosidase, leads to prominent glucosylceramide accumulation in lysosomes of tissue macrophages (Gaucher cells). Here we show glucosylsphingosine, the deacylated form of glucosylceramide, to be markedly increased in plasma of symptomatic nonneuronopathic (type 1) Gaucher patients (n = 64, median = 230.7 nM, range 15.6-1035.2 nM; normal (n = 28): median 1.3 nM, range 0.8-2.7 nM). The method developed for mass spectrometric quantification of plasma glucosylsphingosine is sensitive and robust. Plasma glucosylsphingosine levels correlate with established plasma markers of Gaucher cells, chitotriosidase (ρ = 0.66) and CCL18 (ρ = 0.40). Treatment of Gaucher disease patients by supplementing macrophages with mannose-receptor targeted recombinant glucocerebrosidase results in glucosylsphingosine reduction, similar to protein markers of Gaucher cells. Since macrophages prominently accumulate the lysoglycosphingolipid on glucocerebrosidase inactivation, Gaucher cells seem a major source of the elevated plasma glucosylsphingosine. Our findings show that plasma glucosylsphingosine can qualify as a biomarker for type 1 Gaucher disease, but that further investigations are warranted regarding its relationship with clinical manifestations of Gaucher disease.

Novel Activity‐Based Probes for Broad‐Spectrum Profiling of Retaining β‐Exoglucosidases In Situ and In Vivo

A high-end label: Cyclophellitol aziridine-type activity-based probes allow for ultra-sensitive visualization of mammalian β-glucosidases (GBA1, GBA2, GBA3, and LPH) as well as several non-mammalian β-glucosidases (see picture). These probes offer new ways to study β-exoglucosidases, and configurational isomers of the cyclophellitol aziridine core may give activity-based probes targeting other retaining glycosidase families.

Unexpected effects of fasting on murine lipid homeostasis – Transcriptomic and lipid profiling

BACKGROUND & AIMS Starvation induces massive perturbations in metabolic pathways involved in energy metabolism, but its effect on the metabolism of lipids, particularly cholesterol, is little understood. METHODS A comparative genomic analysis of the gut and the liver in response to fasting was performed, with intestinal perfusion and lipid profiling of the plasma, bile, liver, intestinal tissue, perfusate, and faeces in FVB mice. RESULTS The expression profiles suggested increased cholesterol trafficking in the liver and decreased trafficking in the small intestine. Plasma cholesterol concentrations significantly increased, and triglycerides decreased in fasting. Surprisingly, in prolonged fasting, the biliary bile salt and lipid output rates increased, with increased hepatic and intestinal lipid turnover, and enhanced trans-intestinal cholesterol excretion. In contrast, faecal sterol loss declined sharply. To investigate whether the increased biliary phospholipid secretion could nourish the intestinal epithelium, we studied the histology of the small intestines upon fasting in multidrug resistant protein 2 deficient mice with scarce biliary phospholipids. Their adaptive biliary response to fasting was lost, while the shortage of biliary phospholipids strongly induced apoptosis and proliferation in the small intestine and increased the number of mucin-producing cells. CONCLUSION Even with no dietary fat, lipid levels remain remarkably constant in the murine liver and intestines during prolonged fasting. The biliary system, always assumed to be coupled to the postprandial response, shows a paradoxical increase in activity. We hypothesise that biliary lipids are mobilised to supply the enterocytes with luminal fuel and to stabilise transport systems in the intestine for ensuring a rapid recovery when the food supply resumes.

TLR- and NOD2-dependent regulation of human phagocyte-specific chitotriosidase

Human chitotriosidase is specifically expressed by phagocytes, has anti‐fungal activity towards chitin‐containing fungi in vitro and in vivo, and is part of innate immunity. We studied the effect of toll‐like receptor (TLR)‐ and nucleotide‐binding oligomerization domain (NOD)‐2 triggering on chitotriosidase expression and release by phagocytes. We find that TLR, but not NOD2 activation, regulates chitotriosidase release by neutrophils. Furthermore, both TLR and NOD2 activation resulted in diminished induction by monocytes. Lastly, NOD2 activation, but not TLR stimulation, induces chitinase expression in macrophages. We conclude that phagocyte‐specific regulation is important for efficient eradication of chitin‐containing pathogens.

Lysosomal glycosphingolipid catabolism by acid ceramidase: formation of glycosphingoid bases during deficiency of glycosidases

Glycosphingoid bases are elevated in inherited lysosomal storage disorders with deficient activity of glycosphingolipid catabolizing glycosidases. We investigated the molecular basis of the formation of glucosylsphingosine and globotriaosylsphingosine during deficiency of glucocerebrosidase (Gaucher disease) and α‐galactosidase A (Fabry disease). Independent genetic and pharmacological evidence is presented pointing to an active role of acid ceramidase in both processes through deacylation of lysosomal glycosphingolipids. The potential pathophysiological relevance of elevated glycosphingoid bases generated through this alternative metabolism in patients suffering from lysosomal glycosidase defects is discussed.

Reduction of glycosphingolipid biosynthesis stimulates biliary lipid secretion in mice

Recent reports indicate that glycosphingolipids play an important role in regulation of carbohydrate metabolism. We have shown that the iminosugar N‐(5′‐adamantane‐1′‐yl‐methoxy)‐pentyl‐1‐deoxynojirimycin (AMP‐DNM), an inhibitor of the enzyme glucosylceramide synthase, is a potent enhancer of insulin signaling in rodent models for insulin resistance and type 2 diabetes. In this study, we determined whether AMP‐DNM also affects lipid homeostasis and, in particular, the reverse cholesterol transport pathway. Treatment of C57BL/6J mice with AMP‐DNM for 5 weeks decreased plasma levels of triglycerides and cholesterol by 35%, whereas neutral sterol excretion increased twofold. Secretion of biliary lipid also increased twofold, which resulted in a similar rise in bile flow. This effect was not due to altered expression levels or kinetics of the various export pumps involved in bile formation. However, the bile salt pool size increased and the expression of Cyp7A1 was up‐regulated. In vitro experiments using HepG2 hepatoma cell line revealed this to be due to inhibition of fibroblast growth factor‐19 (FGF19)‐mediated suppression of Cyp7A1 via the FGF receptor. Conclusion: Pharmacological modulation of glycosphingolipid metabolism showed surprising effects on lipid homeostasis in C57BL/6J mice. Upon administration of 100 mg AMP‐DNM/kg body weight/day, plasma cholesterol and triglyceride levels decreased, biliary lipid secretion doubled and also the endpoint of reverse cholesterol transport, neutral sterol excretion, doubled. (HEPATOLOGY 2008.)

Potent and Selective Activity-Based Probes for GH27 Human Retaining α-Galactosidases

Lysosomal degradation of glycosphingolipids is mediated by the consecutive action of several glycosidases. Malfunctioning of one of these hydrolases can lead to a lysosomal storage disorder such as Fabry disease, which is caused by a deficiency in α-galactosidase A. Herein we describe the development of potent and selective activity-based probes that target retaining α-galactosidases. The fluorescently labeled aziridine-based probes 3 and 4 inhibit the two human retaining α-galactosidases αGal A and αGal B covalently and with high affinity. Moreover, they enable the visualization of the endogenous activity of both α-galactosidases in cell extracts, thereby providing a means to study the presence and location of active enzyme levels in different cell types, such as healthy cells versus those derived from Fabry patients.

Identification and development of biphenyl substituted iminosugars as improved dual glucosylceramide synthase/neutral glucosylceramidase inhibitors.

This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of D-gluco and L-ido-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring N-pentyloxymethylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (D-gluco and L-ido) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted L-ido-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their D-gluco-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutics.

N-Azidoacetylmannosamine-mediated chemical tagging of gangliosides

Peracetylated N-α-azidoacetylmannosamine (Ac4ManNAz) is metabolized by cells to CMP-azidosialic acid. It has been demonstrated previously that in this way azidosialic acid-containing glycoproteins are formed that can be labeled on the cell surface by a modified Staudinger ligation. Here, we first demonstrate that the same procedure also results in the formation of azidosialic acid-containing gangliosides. Deoxymannojirimycin, an inhibitor of N-glycan processing in proteins, decreases the total cell surface labeling in Jurkat cells by ∼25%. Inhibition of ganglioside biosynthesis with N-[5-(adamantan-1-yl-methoxy)-pentyl]1-deoxynojirimycin reduces cell surface labeling by ∼75%. In conclusion, exposure of cells to Ac4ManNAz allows in vivo chemical tagging of gangliosides.

Action myoclonus-renal failure syndrome: diagnostic applications of activity-based probes and lipid analysis

Lysosomal integral membrane protein-2 (LIMP2) mediates trafficking of glucocerebrosidase (GBA) to lysosomes. Deficiency of LIMP2 causes action myoclonus-renal failure syndrome (AMRF). LIMP2-deficient fibroblasts virtually lack GBA like the cells of patients with Gaucher disease (GD), a lysosomal storage disorder caused by mutations in the GBA gene. While GD is characterized by the presence of glucosylceramide-laden macrophages, AMRF patients do not show these. We studied the fate of GBA in relation to LIMP2 deficiency by employing recently designed activity-based probes labeling active GBA molecules. We demonstrate that GBA is almost absent in lysosomes of AMRF fibroblasts. However, white blood cells contain considerable amounts of residual enzyme. Consequently, AMRF patients do not acquire lipid-laden macrophages and do not show increased plasma levels of macrophage markers, such as chitotriosidase, in contrast to GD patients. We next investigated the consequences of LIMP2 deficiency with respect to plasma glycosphingolipid levels. Plasma glucosylceramide concentration was normal in the AMRF patients investigated as well as in LIMP2-deficient mice. However, a marked increase in the sphingoid base, glucosylsphingosine, was observed in AMRF patients and LIMP2-deficient mice. Our results suggest that combined measurements of chitotriosidase and glucosylsphingosine can be used for convenient differential laboratory diagnosis of GD and AMRF.