HEPES activates a MiT/TFE-dependent lysosomal-autophagic gene network in cultured cells: A call for caution.

In recent years, the lysosome has emerged as a highly dynamic, transcriptionally regulated organelle that is integral to nutrient-sensing and metabolic rewiring. This is coordinated by a lysosome-to-nucleus signaling nexus in which MTORC1 controls the subcellular distribution of the microphthalmia-transcription factor E (MiT/TFE) family of "master lysosomal regulators". Yet, despite the importance of the lysosome in cellular metabolism, the impact of traditional in vitro culture media on lysosomal dynamics and/or MiT/TFE localization has not been fully appreciated. Here, we identify HEPES, a chemical buffering agent that is broadly applied in cell culture, as a potent inducer of lysosome biogenesis. Supplementation of HEPES to cell growth media is sufficient to decouple the MiT/TFE family members-TFEB, TFE3 and MITF-from regulatory mechanisms that control their cytosolic retention. Increased MiT/TFE nuclear import in turn drives the expression of a global network of lysosomal-autophagic and innate host-immune response genes, altering lysosomal dynamics, proteolytic capacity, autophagic flux, and inflammatory signaling. In addition, siRNA-mediated MiT/TFE knockdown effectively blunted HEPES-induced lysosome biogenesis and gene expression profiles. Mechanistically, we show that MiT/TFE activation in response to HEPES requires its macropinocytic ingestion and aberrant lysosomal storage/pH, but is independent of MTORC1 signaling. Altogether, our data underscore the cautionary use of chemical buffering agents in cell culture media due to their potentially confounding effects on experimental results.

Elevation of glycoprotein nonmetastatic melanoma protein B in type 1 Gaucher disease patients and mouse models.

Gaucher disease is caused by inherited deficiency of lysosomal glucocerebrosidase. Proteome analysis of laser-dissected splenic Gaucher cells revealed increased amounts of glycoprotein nonmetastatic melanoma protein B (gpNMB). Plasma gpNMB was also elevated, correlating with chitotriosidase and CCL18, which are established markers for human Gaucher cells. In Gaucher mice, gpNMB is also produced by Gaucher cells. Correction of glucocerebrosidase deficiency in mice by gene transfer or pharmacological substrate reduction reverses gpNMB abnormalities. In conclusion, gpNMB acts as a marker for glucosylceramide-laden macrophages in man and mouse and gpNMB should be considered as candidate biomarker for Gaucher disease in treatment monitoring.

Synthesis and Evaluation of Hybrid Structures Composed of Two Glucosylceramide Synthase Inhibitors.

Glucosylceramide metabolism and the enzymes involved have attracted significant interest in medicinal chemistry, because aberrations in the levels of glycolipids that are derived from glucosylceramide are causative in a range of human diseases including lysosomal storage disorders, type 2 diabetes, and neurodegenerative diseases. Selective modulation of one of the glycoprocessing enzymes involved in glucosylceramide metabolism-glucosylceramide synthase (GCS), acid glucosylceramidase (GBA1), or neutral glucosylceramidase (GBA2)-is therefore an attractive research objective. In this study we took two established GCS inhibitors, one based on deoxynojirimycin and the other a ceramide analogue, and merged characteristic features to obtain hybrid compounds. The resulting 39-compound library does not contain new GCS inhibitors; however, a potent (200 nm) GBA1 inhibitor was identified that has little activity toward GBA2 and might therefore serve as a lead for further biomedical development as a selective GBA1 modulator.

Mass spectrometric quantification of glucosylsphingosine in plasma and urine of type 1 Gaucher patients using an isotope standard.

Deficiency of glucocerebrosidase (GBA) leads to Gaucher disease (GD), an inherited disorder characterised by storage of glucosylceramide (GlcCer) in lysosomes of tissue macrophages. Recently, we reported marked increases of deacylated GlcCer, named glucosylsphingosine (GlcSph), in plasma of GD patients. To improve quantification, [5-9] (13)C5-GlcSph was synthesised for use as internal standard with quantitative LC-ESI-MS/MS. The method was validated using plasma of 55 GD patients and 20 controls. Intra-assay variation was 1.8% and inter-assay variation was 4.9% for GlcSph (m/z 462.3). Plasma GlcSph levels with the old and new methods closely correlate (r=0.968, slope=1.038). Next, we analysed GlcSph in 24h urine samples of 30 GD patients prior to therapy. GlcSph was detected in the patient samples (median 1.20nM, range 0.11-8.92nM), but was below the limit of quantification in normal urine. Enzyme replacement therapy led to a decrease of urinary GlcSph of GD patients, coinciding with reductions in plasma GlcSph and markers of Gaucher cells (chitotriosidase and CCL18). In analogy to globotriaosylsphingsone in urine of Fabry disease patients, additional isoforms of GlcSph differing in structure of the sphingosine moiety were identified in GD urine samples. In conclusion, GlcSph can be sensitively detected by LC-ESI-MS/MS with an internal isotope standard. Abnormalities in urinary GlcSph are a hallmark of Gaucher disease allowing biochemical confirmation of diagnosis.

In vitro and in vivo comparative and competitive activity-based protein profiling of GH29 α-l-fucosidases.

GH29 α-l-fucosidases catalyze the hydrolysis of α-l-fucosidic linkages. Deficiency in human lysosomal α-l-fucosidase (FUCA1) leads to the recessively inherited disorder, fucosidosis. Herein we describe the development of fucopyranose-configured cyclophellitol aziridines as activity-based probes (ABPs) for selective in vitro and in vivo labeling of GH29 α-l-fucosidases from bacteria, mice and man. Crystallographic analysis on bacterial α-l-fucosidase confirms that the ABPs act by covalent modification of the active site nucleophile. Competitive activity-based protein profiling identified l-fuconojirimycin as the single GH29 α-l-fucosidase inhibitor from eight configurational isomers.

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.

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.

Exploring functional cyclophellitol analogues as human retaining beta-glucosidase inhibitors.

The natural product, cyclophellitol and its aziridine analogue are potent mechanism-based retaining ß-glucosidase inhibitors. In this paper we explore the inhibitory potency of a number of cyclophellitol analogues against the three human retaining ß-glucosidases, GBA, GBA2 and GBA3. We demonstrate that N-alkyl cyclophellitol aziridine is at least equally potent in inhibiting the enzymes evaluated as its N-acyl congener, whereas the N-sulfonyl analogue is a considerably weaker inhibitor. Our results complement the literature on the inhibitory potency of cyclophellitol analogues and hold promise for the future design of more effective activity-based retaining glycosidase probes with respect to probe stability in physiological media.

Long-term effect of antibodies against infused alpha-galactosidase A in Fabry disease on plasma and urinary (lyso)Gb3 reduction and treatment outcome.

INTRODUCTION: Enzyme replacement therapy (ERT) with alpha-Galactosidase A (aGal A) may cause antibody (AB) formation against aGal A in males with Fabry disease (FD). Anti agalsidase ABs negatively influence globotriaosylceramide (Gb3) reduction. We investigated the impact of agalsidase AB on Gb3 and lysoGb3 and clinical outcome in Fabry patients on ERT. METHODS: Adult male and female patients on ERT for at least one year were included. Urinary Gb3 was measured by HPLC, plasma lysoGb3 by LC-ESI-MS/MS and AB with a neutralization assay. RESULTS: Of the 59 patients evaluable patients, 0/30 females and 17/29 males developed anti-agalsidase antibodies (AB+). Only 3/17 males had transient (low) titers (tolerized). All AB+ patients developed antibodies during the first year of treatment. Change of agalsidase preparation (or dose) did not induce antibody formation. AB+ males had significant less decline in plasma lysoGb3 compared to AB- males (p = 0.04). Urinary Gb3 levels decreased markedly in AB- but remained comparable to baseline in AB+ males (p<0.01). (Lyso)Gb3 reduction in plasma and urine on ERT was correlated with LVmass reduction in females and development white matter lesions and stroke. CONCLUSION: In male patients antibodies against aGal A remained present up to 10 years of ERT. The presence of these antibodies is associated with a less robust decrease in plasma lysoGb3 and a profound negative effect on urinary Gb3 reduction, which may reflect worse treatment outcome.

Activity-based profiling of retaining ß-glucosidases: a comparative study.

Activity-based protein profiling (ABPP) is a versatile strategy to report on enzyme activity in vitro, in situ, and in vivo. The development and use of ABPP tools and techniques has met with considerable success in monitoring physiological processes involving esterases and proteases. Activity-based profiling of glycosidases, on the other hand, has proven more difficult, and to date no broad-spectrum glycosidase activity-based probes (ABPs) have been reported. In a comparative study, we investigated both 2-deoxy-2-fluoroglycosides and cyclitol epoxides for their utility as a starting point towards retaining ß-glucosidase ABP. We also investigated the merits of direct labeling and two-step bio-orthogonal labeling in reporting on glucosidase activity under various conditions. Our results demonstrate that 1) in general cyclitol epoxides are the superior glucosidase ABPs, 2) that direct labeling is the more efficient approach but it hinges on the ability of the glucosidase to be accommodated in the active site of the reporter (BODIPY) entity, and 3) that two-step bio-orthogonal labeling can be achieved on isolated enzymes but translating this protocol to cell extracts requires more investigation.

Identification of potent and selective glucosylceramide synthase inhibitors from a library of N-alkylated iminosugars.

Glucosylceramide synthase (GCS) is an important target for clinical drug development for the treatment of lysosomal storage disorders and a promising target for combating type 2 diabetes. Iminosugars are useful leads for the development of GCS inhibitors; however, the effective iminosugar type GCS inhibitors reported have some unwanted cross-reactivity toward other glyco-processing enzymes. In particular, iminosugar type GCS inhibitors often also inhibit to some extent human acid glucosylceramidase (GBA1) and the nonlysosomal glucosylceramidase (GBA2), the two enzymes known to process glucosylceramide. Of these, GBA1 itself is a potential drug target for the treatment of the lysosomal storage disorder, Gaucher disease, and selective GBA1 inhibitors are sought after as potential chemical chaperones. The physiological importance of GBA2 in glucosylceramide processing in relation to disease states is less clear, and here, selective inhibitors can be of use as chemical knockout entities. In this communication, we report our identification of a highly potent and selective N-alkylated l-ido-configured iminosugar. In particular, the selectivity of 27 for GCS over GBA1 is striking.

Assessment of partially deoxygenated deoxynojirimycin derivatives as glucosylceramide synthase inhibitors.

Glucosylceramide synthase (GCS) is an approved drug target for the treatment of Gaucher disease and is considered as a valid target for combating other human pathologies, including type 2 diabetes. The clinical drug N-butyldeoxynojirimycin (Zavesca) is thought to inhibit through mimicry of its substrate, ceramide. In this work we demonstrate that, in contrast to what is proposed in this model, the C2-hydroxyl of the deoxynojirimycin core is important for GCS inhibition. Here we show that C6-OH appears of less important, which may set guidelines for the development of GCS inhibitors that have less affinity (in comparison with Zavesca) for other glycoprocessing enzymes, in particular those hydrolases that act on glucosylceramide.

Ultrasensitive in situ visualization of active glucocerebrosidase molecules.

Deficiency of glucocerebrosidase (GBA) underlies Gaucher disease, a common lysosomal storage disorder. Carriership for Gaucher disease has recently been identified as major risk for parkinsonism. Presently, no method exists to visualize active GBA molecules in situ. We here report the design, synthesis and application of two fluorescent activity-based probes allowing highly specific labeling of active GBA molecules in vitro and in cultured cells and mice in vivo. Detection of in vitro labeled recombinant GBA on slab gels after electrophoresis is in the low attomolar range. Using cell or tissue lysates, we obtained exclusive labeling of GBA molecules. We present evidence from fluorescence-activated cell sorting analysis, fluorescence microscopy and pulse-chase experiments of highly efficient labeling of GBA molecules in intact cells as well as tissues of mice. In addition, we illustrate the use of the fluorescent probes to study inhibitors and tentative chaperones in living cells.

Nanomolar affinity, iminosugar-based chemical probes for specific labeling of lysosomal glucocerebrosidase.

Three different photoprobes were synthesized to label beta-glucosidases; one probe was based on glucose, two probes on the iminosugar deoxynojirimycin. The affinity of the probes for three different beta-glucosidases was determined. Furthermore, their labeling efficiencies, binding specificities through competition with deoxynojirimycin, and binding specificities in the presence of cell lysate, were evaluated. Especially one showed very high affinity towards non-lysosomal glucoceramidase (IC(50)=20 nM).

Synthesis and evaluation of D-gluco-pyranocyclopropyl amines as potential glucosidase inhibitors.

In the recent past sugar-derived cyclopropylamines were proposed as structurally new glycosidase inhibitors. In this Letter we report our efforts in the synthesis of a set of alpha-glucose configured oxabicyclo[4.1.0] heptanes, based on this hypothesis, bearing an amine substituent on the propyl ring and reveal that their inhibitory potential towards a range of mammalian glucosidases is modest.

Common G102S polymorphism in chitotriosidase differentially affects activity towards 4-methylumbelliferyl substrates.

Chitotriosidase (CHIT1) is a chitinase that is secreted by activated macrophages. Plasma chitotriosidase activity reflects the presence of lipid-laden macrophages in patients with Gaucher disease. CHIT1 activity can be conveniently measured using fluorogenic 4-methylumbelliferyl (4MU)-chitotrioside or 4MU-chitobioside as the substrate, however, nonsaturating concentrations have to be used because of apparent substrate inhibition. Saturating substrate concentrations can, however, be used with the newly designed substrate 4MU-deoxychitobioside. We studied the impact of a known polymorphism, G102S, on the catalytic properties of CHIT1. The G102S allele was found to be common in type I Gaucher disease patients in the Netherlands ( approximately 24% of alleles). The catalytic efficiency of recombinant Ser102 CHIT1 was approximately 70% that of wild-type Gly102 CHIT1 when measured with 4MU-chitotrioside at a nonsaturating concentration. However, the activity was normal with 4MU-deoxychitobioside as the substrate at saturating concentrations, consistent with predictions from molecular dynamics simulations. In conclusion, interpretation of CHIT1 activity measurements with 4MU-chitotrioside with respect to CHIT1 protein concentrations depends on the presence of Ser102 CHIT1 in an individual, complicating estimation of the body burden of storage macrophages. Use of the superior 4MU-deoxychitobioside substrate avoids such complications because activity towards this substrate under saturating conditions is not affected by the G102S substitution.

Treatment of Fabry disease with different dosing regimens of agalsidase: effects on antibody formation and GL-3.

Two different enzyme preparations are used for the treatment of Fabry disease patients, agalsidase alpha (Replagal, Shire) and agalsidase beta (Fabrazyme, Genzyme). Therapeutic efficacy of both products has been variable probably due to differences in gender, severity, age and other patient characteristics. We studied the occurrence of alpha-Gal A antibodies and their effect on urinary and plasma globotriaosylceramide (GL-3), plasma chitotriosidase and clinical outcome in 52 patients after 12 months of treatment with either 0.2mg/kg agalsidase alppha (10 males, 8 females) or beta (8 males, 5 females) or 1.0mg/kg agalsidase beta (10 males, 11 females). Antibodies were detected in 18/28 male patients after 6 months. None of the females developed antibodies. Following 12 months of 0.2mg/kg treatment, urinary GL-3 decreased in antibody negative (AB-) but increased in antibody positive (AB+) patients. Treatment with 1.0mg/kg gave a reduction in urinary GL-3 in both AB- and AB+ patients. Levels of plasma GL-3 and chitotriosidase decreased in all patient groups. Twelve months of 0.2mg/kg treatment did not change renal function or left ventricular mass. Further, no change in renal function was seen following 1.0mg/kg treatment and left ventricular mass decreased in both AB- and AB+ patients. In summary, alpha-Gal A antibodies frequently develop in male Fabry disease patients and interfere with urinary GL-3 excretion. Infusion of a dose of 1.0mg/kg results in a more robust decline in GL-3, less impact, if any of antibodies, stable renal function and reduction of LVMass.

Elevated globotriaosylsphingosine is a hallmark of Fabry disease.

Fabry disease is an X-linked lysosomal storage disease caused by deficiency of alpha-galactosidase A that affects males and shows disease expression in heterozygotes. The characteristic progressive renal insufficiency, cardiac involvement, and neuropathology usually are ascribed to globotriaosylceramide accumulation in the endothelium. However, no direct correlation exists between lipid storage and clinical manifestations, and treatment of patients with recombinant enzymes does not reverse several key signs despite clearance of lipid from the endothelium. We therefore investigated the possibility that globotriaosylceramide metabolites are a missing link in the pathogenesis. We report that deacylated globotriaosylceramide, globotriaosylsphingosine, and a minor additional metabolite are dramatically increased in plasma of classically affected male Fabry patients and plasma and tissues of Fabry mice. Plasma globotriaosylceramide levels are reduced by therapy. We show that globotriaosylsphingosine is an inhibitor of alpha-galactosidase A activity. Furthermore, exposure of smooth muscle cells, but not fibroblasts, to globotriaosylsphingosine at concentrations observed in plasma of patients promotes proliferation. The increased intima-media thickness in Fabry patients therefore may be related to the presence of this metabolite. Our findings suggest that measurement of circulating globotriaosylsphingosine will be useful to monitor Fabry disease and may contribute to a better understanding of the disorder.

Detection of mutant protein in complex biological samples: glucocerebrosidase mutations in Gaucher's disease.

We report a sensitive method to detect point mutations in proteins from complex samples. The method is based on surface-enhanced laser desorption/ionization time-of-flight (SELDI-ToF) MS but can be extended to other MS platforms. The target protein in this study is the lysosomal enzyme glucocerebrosidase (GC), the key enzyme in Gaucher's disease. Deficiency of GC activity results in accumulation of glucosylceramide in macrophages. The relationship between GC genotypes and Gaucher's patient phenotypes is not strict. The possibility to measure protein levels of GC in clinical samples may provide deeper insight into the phenomenology of Gaucher's disease. For this purpose, GC was isolated in a single enrichment step through interaction with an immobilized monoclonal antibody, 8E4. After on-chip digestion of the antibody-antigen complex with trypsin, a total of 25 GC peptides were identified (sequence coverage approximately 60%), including several peptides containing mutated amino acid residues. The described methodology allows mutational analysis on the protein level, directly measured on complex biological samples without the necessity of elaborate purification procedures.

Identification of the non-lysosomal glucosylceramidase as beta-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.