Conduritol B Epoxide Research Articles

Klotho-Related Protein KLrP: Structure and Functions.

Klotho (KL) family proteins share one or two glycoside hydrolase (GH) motifs homologous to GH family 1. However, the biological significance of GH motifs in KL family proteins remains elusive. We describe here that KL-related protein (KLrP), which is composed of a single GH motif, is a cytosolic β-glucocerebrosidase (GCase, EC 3.2.1.145). We detected a neutral conduritol B epoxide (CBE)-insensitive glucosylceramide (GlcCer)-degrading activity in the cytosol fractions of human fibroblasts, rat brains, and zebrafish embryos. KL family proteins emerged as a potent candidate for the neutral GCase using a bioinformatics approach. Recombinant human KLrP, but not α-KL, β-KL, or KLPH, exhibited GCase activity with a neutral pH optimum in the presence of CBE. We solved the crystal structures of KLrP and a KLrP mutant (E165Q) in complex with glucose, which indicate that KLrP forms a (β/α)8TIM barrel structure with the double-displacement mechanism of the retaining β-glycosidase. Furthermore, knockdown of endogenous KLrP in CHOP cells using small interfering RNA (siRNA) decreased the CBE-insensitive neutral GCase activity and increased the cellular levels of GlcCer, which suggests that KLrP is involved in a novel GlcCer catabolism pathway. A KLrP D106N mutant was discovered in patients with severe Gaucher disease; however, this mutation did not affect the GCase activity of KLrP.

Glucocerebrosidase deficiency and mitochondrial impairment in experimental Parkinson disease

Gaucher disease is an autosomal recessive disease, caused by a lack or functional deficiency of the lysosomal enzyme, glucocerebrosidase (GCase). Recently, mutations in the glucocerebrosidase gene (GBA) have been associated with Parkinson's disease (PD) and GBA mutations are now considered the most important genetic vulnerability factor for PD. In this study, we have investigated (i) in vivo whether inhibition of the enzyme glucosylceramide synthase by miglustat may protect C57Bl/6 mice against subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication and (ii) in vitro whether a decrease of GCase activity may render dopaminergic neurons susceptible to MPP+ (1-methyl-4-phenylpyridinium) or alpha-synuclein (α-Syn) toxicity and amenable to miglustat treatment. We could demonstrate that reduction of glucocerebroside by inhibition of glucosylceramide synthase partially protects mice against MPTP-induced toxicity. Conversely, we could show that inhibition of GCase activity with conduritol-B-epoxide (CBE) enhances both α-Syn and MPP+ induced toxicity in vitro. However, only CBE-induced enhancement of MPP+ toxicity could be reversed by miglustat. Moreover, we were unable to reveal any alterations of complex I activity or cell respiration upon treatment with either CBE or miglustat. Our findings suggest that the reduction of GCase activity rather than an accumulation of glucocerebroside increases aSyn toxicity.

Glucosylceramide induced complement activation triggers inflammation in Gaucher disease (CCR5P.212)

Abstract Gaucher disease (GD) happens due to mutations in GBA1, which causes a deficiency of acid β-glucosidase critical for degradation of glucosylceramide (GC) into glucose and ceramide. Macrophages (Mɸs) were mainly having this defect and associated with eminent expression of cytokines, chemokines, and recruitment of activated immune cells in GD. The mechanism by which GC triggers such inflammation is still wanted. Excess of GC storage in dendritic and T cells has been reported to be the reason for their activation and production of inflammatory cytokines in Gba1 mouse model (D409V/null; 9V/null). Here, we have identified elevated expression of C5a and C5aR on 9V/null mice APCs. We therefore, hypothesized that GC induced C5a elicits immune inflammation in GD. To confirm this, conduritol B epoxide (CBE), induced chemical model of GD was developed in WT and C5aR-/- mice. We found that C5aR-/- mice were protected from CBE-induced GD development, which was associated with prolonged survival, marked reduction of GC, DCs and T cells positivity for CD40, CD80, CD86, CD40L, CD69, cytokines-chemokines secretion, immune cells recruitment, and decreased loss of red blood cells (RBCs) and platelets. Pharmacological blocking of C5aR in 9V/null mice showed marked reduction in several of these GD manifestations. Our data suggest that GC induced C5a contributes in disease development and targeting C5a signaling may have therapeutic potential for GD.

Multiple pathogenic proteins implicated in neuronopathic Gaucher disease mice

Gaucher disease, a prevalent lysosomal storage disease (LSD), is caused by insufficient activity of acid β-glucosidase (GCase) and the resultant glucosylceramide (GC)/glucosylsphingosine (GS) accumulation in visceral organs (Type 1) and the central nervous system (Types 2 and 3). Recent clinical and genetic studies implicate a pathogenic link between Gaucher and neurodegenerative diseases. The aggregation and inclusion bodies of α-synuclein with ubiquitin are present in the brains of Gaucher disease patients and mouse models. Indirect evidence of β-amyloid pathology promoting α-synuclein fibrillation supports these pathogenic proteins as a common feature in neurodegenerative diseases. Here, multiple proteins are implicated in the pathogenesis of chronic neuronopathic Gaucher disease (nGD). Immunohistochemical and biochemical analyses showed significant amounts of β-amyloid and amyloid precursor protein (APP) aggregates in the cortex, hippocampus, stratum and substantia nigra of the nGD mice. APP aggregates were in neuronal cells and colocalized with α-synuclein signals. A majority of APP co-localized with the mitochondrial markers TOM40 and Cox IV; a small portion co-localized with the autophagy proteins, P62/LC3, and the lysosomal marker, LAMP1. In cultured wild-type brain cortical neural cells, the GCase-irreversible inhibitor, conduritol B epoxide (CBE), reproduced the APP/α-synuclein aggregation and the accumulation of GC/GS. Ultrastructural studies showed numerous larger-sized and electron-dense mitochondria in nGD cerebral cortical neural cells. Significant reductions of mitochondrial adenosine triphosphate production and oxygen consumption (28-40%) were detected in nGD brains and in CBE-treated neural cells. These studies implicate defective GCase function and GC/GS accumulation as risk factors for mitochondrial dysfunction and the multi-proteinopathies (α-synuclein-, APP- and Aβ-aggregates) in nGD.

β-Glucosidase 2 (GBA2) Activity and Imino Sugar Pharmacology

β-Glucosidase 2 (GBA2) is an enzyme that cleaves the membrane lipid glucosylceramide into glucose and ceramide. The GBA2 gene is mutated in genetic neurological diseases (hereditary spastic paraplegia and cerebellar ataxia). Pharmacologically, GBA2 is reversibly inhibited by alkylated imino sugars that are in clinical use or are being developed for this purpose. We have addressed the ambiguity surrounding one of the defining characteristics of GBA2, which is its sensitivity to inhibition by conduritol B epoxide (CBE). We found that CBE inhibited GBA2, in vitro and in live cells, in a time-dependent fashion, which is typical for mechanism-based enzyme inactivators. Compared with the well characterized impact of CBE on the lysosomal glucosylceramide-degrading enzyme (glucocerebrosidase, GBA), CBE inactivated GBA2 less efficiently, due to a lower affinity for this enzyme (higher KI) and a lower rate of enzyme inactivation (k(inact)). In contrast to CBE, N-butyldeoxygalactonojirimycin exclusively inhibited GBA2. Accordingly, we propose to redefine GBA2 activity as the β-glucosidase that is sensitive to inhibition by N-butyldeoxygalactonojirimycin. Revised as such, GBA2 activity 1) was optimal at pH 5.5-6.0; 2) accounted for a much higher proportion of detergent-independent membrane-associated β-glucosidase activity; 3) was more variable among mouse tissues and neuroblastoma and monocyte cell lines; and 4) was more sensitive to inhibition by N-butyldeoxynojirimycin (miglustat, Zavesca®), in comparison with earlier studies. Our evaluation of GBA2 makes it possible to assess its activity more accurately, which will be helpful in analyzing its physiological roles and involvement in disease and in the pharmacological profiling of monosaccharide mimetics.

Glucosylceramide modulates endolysosomal pH in Gaucher disease

GlcCer accumulation causes Gaucher disease where GlcCer breakdown is inhibited due to a hereditary deficiency in glucocerebrosidase. Glycolipids are endocytosed and targeted to the Golgi apparatus in normal cells but in Gaucher disease they are mistargeted to lysosomes. To better understand the role of GlcCer in endocytic sorting RAW macrophages were treated with Conduritol B-epoxide to inhibit GlcCer breakdown. Lipid analysis found increases in GlcCer led to accumulation of both triacylglycerol and cholesterol consistent with increased lysosomal pH. Ratio imaging of macrophages using both acridine orange and lysosensor yellow/blue to measure endolysosomal pH revealed increases in Conduritol B-epoxide treated RAW macrophages and Gaucher patient lymphoblasts. Increased endolysosomal pH was restricted to Gaucher lymphoblasts as no significant increases in pH were seen in Fabry, Krabbe, Tay–Sachs and GM1-gangliosidosis lymphoblasts. Substrate reduction therapy utilises inhibitors of GlcCer synthase to reduce storage in Gaucher disease. The addition of inhibitors of GlcCer synthesis to RAW macrophages also led to increases in cholesterol and triacylglycerol and an endolysosomal pH increase of up to 1 pH unit. GlcCer modulation appears specific since glucosylsphingosine but not galactosylsphingosine reversed the effects of GlcCer depletion. Although no acute effects on glycolipid trafficking were observed using bafilomycin A the results are consistent with a multistep model whereby increases in pH lead to altered trafficking via cholesterol accumulation. GlcCer modulates endolysosomal pH in lymphocytes suggesting an important role in normal lysosomes which may be disrupted in Gaucher disease.

Functional analyses of the digestive β-glucosidase of Formosan subterranean termites (Coptotermes formosanus)

The research was to elucidate the function of the β-glucosidase of Formosan subterranean termites in vitro and in vivo. The gene transcript was detected predominantly in the salivary gland tissue, relative to the midgut and the hindgut of the foraging worker caste, indicating salivary glands were the major expression sites of the β-glucosidase. Using recombinant β-glucosidase produced in Escherichia coli, the enzyme showed higher affinity and activity toward cellobiose and cellotriose than other substrates tested. In assessing impacts of specific inhibitors, we found that the β-glucosidase could be irreversibly inactivated by conduritol B epoxide (CBE) but not gluconolactone. Termite feeding assays showed that the CBE treatment reduced the glucose supply in the midgut and resulted in the body weight loss while no effect was observed for the gluconolactone treatment. These findings highlighted that the β-glucosidase is one of the critical cellulases responsible for cellulose degradation and glucose production; inactivation of these digestive enzymes by specific inhibitors may starve the termite.

Glucocerebrosidase deficiency dramatically impairs human bone marrow haematopoiesis in an in vitro model of Gaucher disease

One of the cardinal symptoms of type 1 Gaucher Disease (GD) is cytopenia, usually explained by bone marrow (BM) infiltration by Gaucher cells and hypersplenism. However, some cases of cytopenia in splenectomized or treated patients suggest possible other mechanisms. To evaluate intra-cellular glucocerebrosidase (GlcC) activity in immature progenitors and to prove the conduritol B epoxide (CBE)-induced inhibition of the enzyme, we used an adapted flow cytometric technique before assessing the direct effect of GlcC deficiency in functional assays. Among haematopoietic cells from healthy donors, monocytes showed the highest GlcC activity but immature CD34(+) and mesenchymal cells also had significant GlcC activity. CBE greatly inhibited the enzyme activity of all cell categories. GlcC-deficient CD34(+) cells showed impaired ability to proliferate and differentiate in the expansion assay and had lower frequency of erythroid burst-forming units, granulocyte colony-forming units (CFU) and macrophage CFU progenitors, but the effect of GlcC deficiency on megakaryocyte CFU lineage was not significant. GlcC deficiency strongly impaired primitive haematopoiesis in long-term culture. Furthermore, GlcC deficiency progressively impaired proliferation of mesenchymal progenitors. These data suggest an intrinsic effect of GlcC deficiency on BM immature cells that supplements the pathophysiology of GD and opens new perspectives of therapeutic approach.

Uncoupling between CD1d upregulation induced by retinoic acid and conduritol-B-epoxide and iNKT cell responsiveness

Gaucher disease (GD) is associated with upregulation of CD1d and MHC-class II expression by monocytes. While the physiological impact of CD1d upregulation remains uncertain, it has been proposed that MHC-class II upregulation is associated with inflammation. Hereby, we show that the decrease in MHC-class II expression seen in GD patients under therapy correlates positively with chitotriosidase activity, a marker of inflamed macrophages. We also show that retinoic acid (RA) and the β-glucocerebrosidase inhibitor conduritol-B-epoxide (CBE) lead to upregulation of CD1d expression by THP-1 cells, which correlated with an increase in mRNA expression. In vitro co-culture experiments showed that RA treated THP-1 cells were more stimulatory for CD4 + than for CD8 + T cells, as determined by CFSE loss, in comparison to untreated THP-1 cells. Interestingly, even though addition of exogenous isoglobotrihexosylceramide (iGb3), a physiological CD1d ligand, augmented the percentage of dividing CD4 + T cells, we could not detect a significant expansion of CD4 +Vα24 + invariant Natural Killer T (iNKT) cells. In contrast, addition of α-galactosylceramide (α-GC) induced expansion of Vα24 + iNKT cells as determined by using α-GC-loaded human CD1d dimers. These results strengthen the existence of a cross-talk between monocyte lipid accumulation, inflammation and changes in cell surface CD1d and MHC-class II in monocytes, which may result in inappropriate recognition events by immune cells and perpetuate chronic inflammation.

Alpha-synuclein-glucocerebrosidase interactions in pharmacological Gaucher models: A biological link between Gaucher disease and parkinsonism

A growing body of experimental and clinical literature indicates an association between Gaucher disease and parkinsonism, raising the possibility that convergent mechanisms may contribute to neurodegeneration in these disorders. The aim of this study was to determine whether there is a relationship between alpha-synuclein (α-syn), a key protein in Parkinson's disease pathogenesis, and abnormalities in glucocerebroside (GC) catabolism that lead to the development of Gaucher disease. We inhibited glucocerebrosidase (GCase) with conduritol B epoxide (CBE) in neuroblastoma cells and mice to test whether a biological link exists between GCase activity and α-syn. After CBE exposure, enhanced α-syn protein was detected in differentiated cells challenged with CBE as compared to vehicle, with no change in α-syn mRNA. In the mouse model, after one injection of CBE, elevated nigral α-syn levels were also detected. Analyses by Western blot and confocal microscopy revealed that normal α-syn distribution was perturbed after CBE exposure with its accumulation apparent within nigral cell bodies as well as astroglia. These findings raise the possibility that α-syn may contribute to the cascade of events that promote neuronal dysfunction in Gaucher disease and are the first to implicate this protein as a plausible biological intersection between Gaucher disease and parkinsonism using a pharmacological model.

A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral β-glucocerebrosidases

The activity of lysosomal acid β-glucocerebrosidase (AGC, EC 3.2.1.45), which hydrolyzes the O-glycosidic linkage between d-glucose and ceramide of glucosylceramide (GlcCer), is a marker for the diagnosis of Gaucher disease because the disease is caused by dysfunction of AGC due to mutations in the gene. The activity of AGC is potently inhibited by conduritol B epoxide (CBE), whereas CBE-insensitive nonlysosomal neutral β-glucocerebrosidase (NGC) activities have been found in various vertebrates, including humans. We report here a new reliable method to determine AGC as well as NGC activities using normal-phase high-performance liquid chromatography (HPLC) and NBD (4-nitrobenzo-2-oxa-1,3-diazole)- or BODIPY (4,4-difluoro-4-bora-3a,4a-diaza- s-indacene)-labeled GlcCer as a substrate. The reaction products of the enzymes, C6-NBD-ceramide and C12-BODIPY-ceramide, were clearly separated from the corresponding substrates on a normal-phase column within 5 min using a different solvent system. Reaction products could be detected quantitatively at concentrations ranging from 50 fmol to 50 pmol for C6-NBD-ceramide and from 10 fmol to 5 pmol for C12-BODIPY-ceramide. V max/ K m values of human fibroblast AGC for fluorescent GlcCer were much higher than those for 4-methylumbelliferyl-β- d-glucoside (4MU-Glc), which is used prevalently for Gaucher disease diagnosis. As a result, AGC activity was detected quantitatively using fluorescent GlcCer, but not 4MU-Glc, using 5 μl of human serum or 1 × 10 4 cultured human fibroblasts. The current method clearly showed the decrease of AGC activities in fibroblasts and serum from the patient with Gaucher disease compared with normal individuals, suggesting that the method is applicable for the diagnosis of Gaucher disease. Furthermore, this method was found to be useful for measuring the activities of nonlysosomal NGC of various cells and tissues in the presence of CBE.

Changes in macrophage morphology in a Gaucher disease model are dependent on CTP:phosphocholine cytidylyltransferase α

We have recently shown that phosphatidylcholine (PC) metabolism is altered in a macrophage model of Gaucher disease. We now demonstrate that treatment of macrophages with conduritol-B-epoxide (CBE), a glucocerebrosidase inhibitor, results in elevated activity of CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme in the pathway of PC biosynthesis. Furthermore, we provide evidence for a role for CCT in Gaucher macrophage growth by using macrophages derived from a genetically modified mouse which lacks a specific CCT isoform, CCTα, in macrophages. Upon CBE-treatment, macrophage size, analyzed by microscopy and by FACS, was significantly increased in macrophages from control mice, but did not increase, or increased to a much lower extent, in CCTα−/− macrophages. Together, these results suggest that the increase in PC biosynthesis is mediated via CCTα, and suggests a possible role for macrophage CCTα in Gaucher disease pathology.

Identification of the Non-lysosomal Glucosylceramidase as β-Glucosidase 2

The primary catabolic pathway for glucosylceramide is catalyzed by the lysosomal enzyme glucocerebrosidase that is defective in Gaucher disease patients. A distinct non-lysosomal glucosylceramidase has been described but its identity remained enigmatic for years. We here report that the non-lysosomal glucosylceramidase is identical to the earlier described bile acid beta-glucosidase, being beta-glucosidase 2 (GBA2). Expressed GBA2 is identical to the native non-lysosomal glucosylceramidase in various enzymatic features such as substrate specificity and inhibitor sensitivity. Expression of GBA2 coincides with increased non-lysosomal glucosylceramidase activity, and GBA2-targeted RNA interference reduces endogenous non-lysosomal glucosylceramidase activity in cells. GBA2 is found to be located at or close to the cell surface, and its activity is linked to sphingomyelin generation. Hydrophobic deoxynojirimycins are extremely potent inhibitors for GBA2. In mice pharmacological inhibition of GBA2 activity is associated with impaired spermatogenesis, a phenomenon also very recently reported for GBA2 knock-out mice (Yildiz, Y., Matern, H., Thompson, B., Allegood, J. C., Warren, R. L., Ramirez, D. M., Hammer, R. E., Hamra, F. K., Matern, S., and Russell, D. W. (2006) J. Clin. Invest. 116, 2985-2994). In conclusion, GBA2 plays a role in cellular glucosylceramide metabolism.

Recombinant glucocerebrosidase uptake by Gaucher disease human osteoblast culture model

Bone lesions are a major cause of morbidity in Gaucher disease (GD) type I. Enzyme replacement therapy (ERT) has been successful in treating many symptoms of type I GD but skeletal response lags behind. Local exogenous glucocerebrosidase supplementation in bone lesions via a drug delivery system may overcome this limitation. Although local enzyme supplementation aims to target lipid-engorged macrophages (Gaucher Cells) in bone compartment, enzyme uptake by osteoblasts is not excluded. To investigate the ability of human osteoblasts to internalize recombinant glucocerebrosidase (rGCR), we have used an artificial GD human osteoblasts cell culture system. MG63 human osteoblasts were treated with conduritol B epoxide (CBE) to induce complete and prolonged inhibition of endogenous glucocerebrosidase activity of cells. rGCR uptake by glucocerebrosidase-inactivated osteoblasts was examined using 125I-radiolabelling, Western blot analysis and measurement of glucocerebrosidase activity. Analysis of radiolabeled enzyme uptake by CBE treated osteoblasts showed 67.9% of internalized protein in cell extract. Enzyme internalization was also observed by Western blot analysis where the amount of mature form of glucocerebrosidase protein recognized by the glucocerebrosidase antibody was increased following the administrations of rGCR. Moreover, enzymatic activity measurement showed 23.9% of glucocerebrosidase activity of control cells. The rGCR internalization by MG63 osteoblast seems to be partially mediated by mannose receptors. These data provide evidence that MG63 human osteoblasts are able to internalize rGCR.

X-ray Structure of Human Acid-β-Glucosidase Covalently Bound to Conduritol-B-Epoxide: IMPLICATIONS FOR GAUCHER DISEASE

Gaucher disease is an inherited metabolic disorder caused by mutations in the lysosomal enzyme acid-beta-glucosidase (GlcCerase). We recently determined the x-ray structure of GlcCerase to 2.0 A resolution (Dvir, H., Harel, M., McCarthy, A. A., Toker, L., Silman, I., Futerman, A. H., and Sussman, J. L. (2003) EMBO Rep.4, 704-709) and have now solved the structure of Glc-Cerase conjugated with an irreversible inhibitor, conduritol-B-epoxide (CBE). The crystal structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu340 is the catalytic nucleophile. However, only one of two alternative conformations of a pair of flexible loops (residues 345-349 and 394-399) located at the entrance to the active site in native GlcCerase is observed in the GlcCerase-CBE structure, a conformation in which the active site is accessible to CBE. Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. This possibility is supported by a cluster of mutations in loop 394-399 that cause Gaucher disease by reducing catalytic activity. Moreover, in silico mutational analysis demonstrates that all these mutations stabilize the conformation that limits access to the active site, thus providing a mechanistic explanation of how mutations in this loop result in Gaucher disease.

Evidence for a link between sphingolipid metabolism and expression of CD1d and MHC‐class II: monocytes from Gaucher disease patients as a model

Gaucher disease (GD) is an autosomal recessive inherited defect of the lysosomal enzyme glucocerebrosidase (GluCerase) that leads to glucosylceramide (GluCer) accumulation. We previously demonstrated the existence of imbalances in certain lymphocyte populations in GD patients. We now show that GluCerase-deficient monocytes from GD patients or monocytes from healthy subjects treated with conduritol-B-epoxide (CBE), an irreversible inhibitor of GluCerase activity, display high levels of surface expression of the lipid-binding molecule CD1d. GluCerase-deficient monocytes from GD patients also showed increased surface expression of major histocompatibility complex (MHC)-class II, but not of other lysosomal trafficking molecules, such as CD63 and MHC-class I. However, CD1d and MHC-class II mRNA levels were not increased. GluCerase-deficient monocytes from GD patients undergoing enzyme replacement therapy also exhibited increased levels of CD1d and MHC-class II and imbalances in the percentage of CD4+, CD8+, and Valpha24+ T cells. Interestingly, follow-up studies revealed that enzyme replacement therapy induced a decrease in MHC-class II expression and partial correction of the CD4+ T cell imbalances. These results reveal a new link between sphingolipid accumulation in monocytes and the expression of certain MHC molecules that may result in imbalances of regulatory T cell subsets. These immunological anomalies may contribute to the clinical heterogeneity in GD patients.

New aminocyclitols as modulators of glucosylceramide metabolism

A series of 13 aminocyclitol derivatives belonging to two different families is described. Their configuration is governed by the regio- and stereocontrolled epoxide opening of a suitably protected conduritol-B epoxide. Studies on several glycosyl processing enzymes indicate that some of them are good inhibitors of glucosylceramide hydrolase. A rationale to account for preliminary structure-activity relationships is provided.

Correlation between enzyme activity and substrate storage in a cell culture model system for Gaucher disease

Gaucher disease, the most common sphingolipidosis, is caused by a decreased activity of glucosylceramide beta-glucosidase, resulting in the accumulation of glucosylceramide in macrophage-derived cells known as Gaucher cells. Much of the storage material is thought to originate from the turnover of cell membranes, such as phagocytosed red and white blood cells. In this study, an in vitro model of Gaucher disease was developed by treating the murine macrophage cell line J774 with a specific inhibitor of glucosylceramide beta-glucosidase, conduritol B-epoxide, and feeding red blood cell ghosts, in order to mimic the disease state. It was found in this model system that glucosylceramide beta-glucosidase activity could be reduced to about 11-15% of the normal control level before increased storage of glucosylceramide occurred. This in vitro system allows insight into the correlation between enzyme activity and lipid storage as predicted by the theory of residual enzyme activity that was proposed by Conzelmann and Sandhoff.

Glucosylceramide modulates membrane traffic along the endocytic pathway

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus, but are mistargeted to lysosomes in numerous sphingolipidoses. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Gaucher disease is a hereditary deficiency in glucocerebrosidase leading to glucosylceramide accumulation; however, Gaucher fibroblasts exhibited normal Golgi transport of lactosylceramide. To better understand the effects of glycosphingolipid accumulation on intracellular trafficking and the use of imino sugar inhibitors, we studied sphingolipid endocytosis in fibroblast and macrophage models for Gaucher disease. Treatment of fibroblasts or RAW macrophages with conduritol B epoxide, an inhibitor of lysosomal glucocerebrosidase, resulted in a change in the endocytic targeting of lactosylceramide from the Golgi to the lysosomes. Co-treatment of macrophages with conduritol B-epoxide and 12-25 microM N-butyldeoxygalactonojirimycin, an inhibitor of glycosphingolipid biosynthesis, prevented the mistargeting of lactosylceramide to the lysosomes and restored trafficking to the Golgi. Surprisingly, higher doses (>25 microM) of NB-DGJ induced targeting of lactosylceramide to the lysosomes, even in the absence of conduritol B-epoxide. These data demonstrate that both increases and decreases in glucosylceramide levels can dramatically alter the endocytic targeting of lactosylceramide and suggest a role for glucosylceramide in regulation of membrane transport.

Enzymatic resolution of (±)-conduritol-B, a key intermediate for the synthesis of glycosidase inhibitors

Lipases from porcine pancreas, Candida cylindracea and Mucor miehei (adsorbed on support, Lipozyme ® IM) catalysed in t-butylmethylether the alcoholysis of rac-conduritol-B peracetate, (±)- 1, by n-butanol to give enantiopure (2 S,3 S)-diacetoxy-(1 R,4 R)-dihydroxycyclohex-5-ene, (−)- 3, and (1 S,2 R,3 R,4 S)-tetraacetoxy-cyclohex-5-ene, (+)- 1. The enantioforms (+)- and (−)-conduritol-B, obtained after chemical hydrolysis of (−)- 3 and (+)- 1, respectively, may be employed to prepare both the enantiomers of conduritol-B epoxide and cyclophellitol, powerful inhibitors of glycosidases.