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Biallelic mutations in the glucocerebrosidase (GBA1) gene cause Gaucher disease, characterized by lysosomal accumulation of glucosylceramide and glucosylsphingosine in macrophages. This and other lysosomal diseases occur with high frequency in Ashkenazi Jews. It has been proposed that the underlying mutations confer a selective advantage, in particular conferring protection against tuberculosis. Here, using a zebrafish Gaucher disease model, we find that the mutationGBA1N370S, predominant among Ashkenazi Jews, increases resistance to tuberculosis through the microbicidal activity of glucosylsphingosine in macrophage lysosomes. Consistent with lysosomal accumulation occurring only in homozygotes, heterozygotes remain susceptible to tuberculosis. Thus, our findings reveal a mechanistic basis for protection against tuberculosis byGBA1N370S and provide biological plausibility for its selection if the relatively mild deleterious effects in homozygotes were offset by significant protection against tuberculosis, a rampant killer of the young in Europe through the Middle Ages into the 19thcentury.Significance StatementGaucher disease is a recessively inherited disorder in which the lipids glucosylceramide and glucosylsphingosine accumulate in lysosomes of macrophages. Macrophages are the first immune cells to engulf infecting bacteria and we find that glucosylsphingosine increases their ability to killMycobacterium tuberculosisthat causes tuberculosis. Gaucher disease due to a particular mutation is frequent in Ashkenazi Jews. Since from the middle ages they were often confined to areas of high tuberculosis prevalence, it has been proposed that the mutation prevailed because heterozygotes, who do not accumulate lipids nor manifest Gaucher disease, were protected. Our findings raise the possibility that selection operated on homozygotes manifesting mild forms of Gaucher disease who were protected against tuberculosis which would often have been fatal.

Glucocerebrosidase (GCase), encoded by the GBA gene, degrades the ubiquitous glycosphingolipid glucosylceramide. Inherited GCase deficiency causes Gaucher disease (GD). In addition, carriers of an abnormal GBA allele are at increased risk for Parkinson's disease. GCase undergoes extensive modification of its four N-glycans en route to and inside the lysosome that is reflected in changes in molecular weight as detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent activity-based probes (ABPs) that covalently label GCase in reaction-based manner in vivo and in vitro allow sensitive visualization of GCase molecules. Using these ABPs, we studied the life cycle of GCase in cultured fibroblasts and macrophage-like RAW264.7 cells. Specific attention was paid to the impact of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) supplementation to bicarbonate-buffered medium. Here, we report how HEPES-buffered medium markedly influences processing of GCase, its lysosomal degradation, and the total cellular enzyme level. HEPES-containing medium was also found to reduce maturation of other lysosomal enzymes (alpha-glucosidase and beta-glucuronidase) in cells. The presence of HEPES in bicarbonate containing medium increases GCase activity in GD-patient derived fibroblasts, illustrating how the supplementation of HEPES complicates the use of cultured cells for diagnosing GD.

Exo-beta-mannosidases are a broad class of stereochemically retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo-beta-mannosidases in complex biological samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogues selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compounds. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biological samples. Towards the development of ABPs for exo-beta-mannosidases, we present a concise synthesis of beta-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-beta-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudoanomeric centre to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity-based protein profiling using an N-alkyl aziridine derivative by specifically labelling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogues hold promise for detecting exo-beta-mannosidases in biological and biomedical research.

Gaucher disease (GD) is a lysosomal storage disorder caused by inherited deficiencies in beta-glucocerebrosidase (GBA). Current treatments require rapid disease diagnosis and a means of monitoring therapeutic efficacy, both of which may be supported by the use of GBA-targeting activity-based probes (ABPs). Here, we report the synthesis and structural analysis of a range of cyclophellitol epoxide and aziridine inhibitors and ABPs for GBA. We demonstrate their covalent mechanism-based mode of action and uncover binding of the new N-functionalised aziridines to the ligand binding cleft. These inhibitors became scaffolds for the development of ABPs; the O6-fluorescent tags of which bind in an allosteric site at the dimer interface. Considering GBA's preference for O6- and N-functionalised reagents, a bi-functional aziridine ABP was synthesized as a potentially more powerful imaging agent. Whilst this ABP binds to two unique active site clefts of GBA, no further benefit in potency was achieved over our first generation ABPs. Nevertheless, such ABPs should serve useful in the study of GBA in relation to GD and inform the design of future probes.

Glucocerebrosidase (GBA), a lysosomal retaining β‐d‐glucosidase, has recently been shown to hydrolyze β‐d‐xylosides and to transxylosylate cholesterol. Genetic defects in GBA cause the lysosomal storage disorder Gaucher disease (GD), and also constitute a risk factor for developing Parkinson's disease. GBA and other retaining glycosidases can be selectively visualized by activity‐based protein profiling (ABPP) using fluorescent probes composed of a cyclophellitol scaffold having a configuration tailored to the targeted glycosidase family. GBA processes β‐d‐xylosides in addition to β‐d‐glucosides, this in contrast to the other two mammalian cellular retaining β‐d‐glucosidases, GBA2 and GBA3. Here we show that the xylopyranose preference also holds up for covalent inhibitors: xylose‐configured cyclophellitol and cyclophellitol aziridines selectively react with GBA over GBA2 and GBA3 in vitro and in vivo, and that the xylose‐configured cyclophellitol is more potent and more selective for GBA than the classical GBA inhibitor, conduritol B‐epoxide (CBE). Both xylose‐configured cyclophellitol and cyclophellitol aziridine cause accumulation of glucosylsphingosine in zebrafish embryo, a characteristic hallmark of GD, and we conclude that these compounds are well suited for creating such chemically induced GD models., New selective GBA inhibitor: We show that the β‐d‐xylose configured epoxide and aziridine are potent selective inhibitors towards GBA in vitro and in vivo, and their selectivity and inhibitory effect towards GBA are superior to the widely used GBA inhibitor Conduritol B‐epoxide (CBE).

Glycoside hydrolases (GHs) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GHs can perform transglycosylation under specific conditions. In nature, oligosaccharide synthesis is performed by glycosyltransferases (GTs); however, the industrial use of GTs is limited by their instability in solution. A key difference between GTs and GHs is the flexibility of their binding site architecture. We have used the xylanase from Bacillus circulans (BCX) to study the interplay between active‐site flexibility and transglycosylation. Residues of the BCX “thumb” were substituted to increase the flexibility of the enzyme binding site. Replacement of the highly conserved residue P116 with glycine shifted the balance of the BCX enzymatic reaction toward transglycosylation. The effects of this point mutation on the structure and dynamics of BCX were investigated by NMR spectroscopy. The P116G mutation induces subtle changes in the configuration of the thumb and enhances the millisecond dynamics of the active site. Based on our findings, we propose the remodelling of the GH enzymes glycon site flexibility as a strategy to improve the transglycosylation efficiency of these biotechnologically important catalysts., Shifting the balance: Glycoside hydrolases are attractive tools for the enzymatic synthesis of carbohydrates. We used the xylanase from B. circulans (BCX) to study the interplay between active‐site flexibility and the transglycosylation reaction. A point mutation that enhances the flexibility and dynamics of BCX active site shifts the balance of the enzymatic reaction toward transglycosylation.

Parkinson's disease (PD) is a progressive neurodegenerative disease where dopaminergic neurons in the substantia nigra are lost, resulting in a decrease in striatal dopamine and, consequently, motor control. Dopaminergic degeneration is associated with the appearance of Lewy bodies, which contain membrane structures and proteins, including α-synuclein (α-Syn), in surviving neurons. PD displays a multifactorial pathology and develops from interactions between multiple elements, such as age, environmental conditions, and genetics. Mutations in the GBA1 gene represent one of the major genetic risk factors for PD. This gene encodes an essential lysosomal enzyme called β-glucocerebrosidase (GCase), which is responsible for degrading the glycolipid glucocerebroside into glucose and ceramide. GCase can generate glucosylated cholesterol via transglucosylation and can also degrade the sterol glucoside. Although the molecular mechanisms that predispose an individual to neurodegeneration remain unknown, the role of cholesterol in PD pathology deserves consideration. Disturbed cellular cholesterol metabolism, as reflected by accumulation of lysosomal cholesterol in GBA1-associated PD cellular models, could contribute to changes in lipid rafts, which are necessary for synaptic localization and vesicle cycling and modulation of synaptic integrity. α-Syn has been implicated in the regulation of neuronal cholesterol, and cholesterol facilitates interactions between α-Syn oligomers. In this review, we integrate the results of previous studies and describe the cholesterol landscape in cellular homeostasis and neuronal function. We discuss its implication in α-Syn and Lewy body pathophysiological mechanisms underlying PD, focusing on the role of GCase and cholesterol. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society., This work was supported by grants from the Span-ish Ministries of Innovation, Science and Universities and Health, SocialServices and Equality and ISCIII, CIBERNED: PCIN2015-098,PID2019-111693RB-I00, CB06/05/0055, and PI2019/09-3; RamónAreces Foundation (172275); European Union’s Horizon 2020 researchand innovation program, AND-PD, grant agreement no. 848002 to R.M.;and NWO (grant no. BBOL-2007247202 to J.M.F.G.A.)

Funder: Maratona da Saúde, Funder: Royal Society Wolfson, Funder: Wellcome; Id: http://dx.doi.org/10.13039/100010269, Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.

Macrophages are key multi-talented cells of the innate immune system and are equipped with receptors involved in damage and pathogen recognition with connected immune response guiding signaling systems. In addition, macrophages have various systems that are involved in the uptake of extracellular and intracellular cargo. The lysosomes in macrophages play a central role in the digestion of all sorts of macromolecules and the entry of nutrients to the cytosol, and, thus, the regulation of endocytic processes and autophagy. Simplistically viewed, two macrophage phenotype extremes exist. On one end of the spectrum, the classically activated pro-inflammatory M1 cells are present, and, on the other end, alternatively activated anti-inflammatory M2 cells. A unique macrophage population arises when lipid accumulation occurs, either caused by flaws in the catabolic machinery, which is observed in lysosomal storage disorders, or as a result of an acquired condition, which is found in multiple sclerosis, obesity, and cardiovascular disease. The accompanying overload causes a unique metabolic activation phenotype, which is discussed here, and, consequently, a unifying phenotype is proposed.

Non-invasive and real-time recording of processes in living cells has been limited to detection of small cellular components such as soluble proteins and metabolites. Here we report a multiphase NMR approach using magic-angle spinning NMR to synchronously follow microbial processes of fermentation, lipid metabolism and structural dynamic changes in live microalgae cells. Chlamydomonas reinhardtii green algae were highly concentrated, introducing dark fermentation and anoxia conditions. Single-pulse NMR experiments were applied to obtain temperature-dependent kinetic profiles of the formed fermentation products. Through dynamics-based spectral editing NMR, simultaneous conversion of galactolipids into TAG and free fatty acids was observed and rapid loss of rigid lipid structures. This suggests that lipolysis under dark and anoxia conditions finally results in the breakdown of cell and organelle membranes, which could be beneficial for recovery of intracellular microbial useful products.

The single‐domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, which is one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, which is attributed to an enzyme‐induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction., The enzyme xylanase from Bacillus circulans (BCX) is involved in the breakdown of xylan, which is one of the most abundant materials in wood. NMR experiments demonstrate that in solution the enzyme is rigid and does not change its structure during turnover. Binding of the substrate occurs in many orientations, including a distorted form. A model is presented for the effect of substrate dynamics on enzyme function.

Purpose To assess the utility of globotriaosylsphingosine (lyso-Gb(3)) for clinical monitoring of treatment response in patients with Fabry disease receiving migalastat. Methods A post hoc analysis evaluated data from 97 treatment-naive and enzyme replacement therapy (ERT)-experienced patients with migalastat-amenableGLAvariants from FACETS (NCT00925301) and ATTRACT (NCT01218659) and subsequent open-label extension studies. The relationship between plasma lyso-Gb(3)and measures of Fabry disease progression (left ventricular mass index [LVMi], estimated glomerular filtration rate [eGFR], and pain) and the relationship between lyso-Gb(3)and incidence of Fabry-associated clinical events (FACEs) were assessed in both groups. The relationship between changes in lyso-Gb(3)and kidney interstitial capillary (KIC) globotriaosylceramide (Gb(3)) inclusions was assessed in treatment-naive patients. Results No significant correlations were identified between changes in lyso-Gb(3)and changes in LVMi, eGFR, or pain. Neither baseline lyso-Gb(3)levels nor the rate of change in lyso-Gb(3)levels during treatment predicted FACE occurrences in all patients or those receiving migalastat for >= 24 months. Changes in lyso-Gb(3)correlated with changes in KIC Gb(3)inclusions in treatment-naive patients. Conclusions Although used as a pharmacodynamic biomarker in research and clinical studies, plasma lyso-Gb(3)may not be a suitable biomarker for monitoring treatment response in migalastat-treated patients.

Inherited cutis laxa, or inelastic, sagging skin is a genetic condition of premature and generalised connective tissue ageing, affecting various elastic components of the extracellular matrix. Several cutis laxa syndromes are inborn errors of metabolism and lead to severe neurological symptoms. In a patient with cutis laxa, a choreoathetoid movement disorder, dysmorphic features and intellectual disability we performed exome sequencing to elucidate the underlying genetic defect. We identified the amino acid substitution R275W in phosphatidylinositol 4‐kinase type IIα, caused by a homozygous missense mutation in the PI4K2A gene. We used lipidomics, complexome profiling and functional studies to measure phosphatidylinositol 4‐phosphate synthesis in the patient and evaluated PI4K2A deficient mice to define a novel metabolic disorder. The R275W residue, located on the surface of the protein, is involved in forming electrostatic interactions with the membrane. The catalytic activity of PI4K2A in patient fibroblasts was severely reduced and lipid mass spectrometry showed that particular acyl‐chain pools of PI4P and PI(4,5)P2 were decreased. Phosphoinositide lipids play a major role in intracellular signalling and trafficking and regulate the balance between proliferation and apoptosis. Phosphatidylinositol 4‐kinases such as PI4K2A mediate the first step in the main metabolic pathway that generates PI4P, PI(4,5)P2 and PI(3,4,5)P3. Although neurologic involvement is common, cutis laxa has not been reported previously in metabolic defects affecting signalling. Here we describe a patient with a complex neurological phenotype, premature ageing and a mutation in PI4K2A, illustrating the importance of this enzyme in the generation of inositol lipids with particular acylation characteristics.

As the scope of modern genomics technologies increases, so does the need for informative chemical tools to study functional biology. Activity-based probes (ABPs) provide a powerful suite of reagents to probe the biochemistry of living organisms. These probes, featuring a specificity motif, a reactive chemical group and a reporter tag, are opening-up large swathes of protein chemistry to investigation in vitro, as well as in cellular extracts, cells and living organisms in vivo. Glycoside hydrolases, by virtue of their prominent biological and applied roles, provide a broad canvas on which ABPs may illustrate their functions. Here we provide an overview of glycosidase ABP mechanisms, and review recent ABP work in the glycoside hydrolase field, encompassing their use in medical diagnosis, their application for generating chemical genetic disease models, their fine-tuning through conformational and reactivity insight, their use for high-throughput inhibitor discovery, and their deployment for enzyme discovery and dynamic characterization.

Glycosphingolipids are important building blocks of the outer leaflet of the cell membrane. They are continuously recycled, involving fragmentation inside lysosomes by glycosidases. Inherited defects in degradation cause lysosomal glycosphingolipid storage disorders. The relatively common glycosphingolipidosis Gaucher disease is highlighted here to discuss new insights in the molecular basis and pathophysiology of glycosphingolipidoses reached by fundamental research increasingly using chemical biology tools. We discuss improvements in the detection of glycosphingolipid metabolites by mass spectrometry and review new developments in laboratory diagnosis and disease monitoring as well as therapeutic interventions.

Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disease (LSD) in which sphingomyelin accumulates due to deficient acid sphingomyelinase. In the chronic visceral subtype, organ manifestations are generally limited to the spleen, liver, and lungs. We report a male patient with the chronic visceral subtype who developed proteinuria and renal insufficiency at the age of 49. In renal tissue, foam cells were observed in the glomeruli as well as sphingomyelin accumulation within podocytes, mesangial cells, endothelial cells, and tubular epithelial cells. Although macrophages are the primary storage cells in both ASMD and Gaucher disease, comparison to the histopathological findings in Gaucher and Fabry disease revealed a diffuse storage pattern in multiple renal cell types, closer resembling the pattern found in Fabry disease.

Apolipoprotein ε4 (APOE)4 is a strong risk factor for the development of Alzheimer’s disease (AD) and aberrant sphingolipid levels have been implicated in AD. We tested the hypothesis that the APOE4 genotype affects brain sphingolipid levels in AD. Seven ceramides and sphingosine-1-phosphate (S1P) were quantified by LC-MSMS in hippocampus, cortex, cerebellum, and plasma of 5 months old human APOE3 and APOE4-targeted replacement mice with or without the familial AD (FAD) background of both sexes (145 animals). APOE4 mice had higher Cer(d18:1/24:0) levels in the cortex (1.7-fold, p = 0.002) than APOE3 mice. Mice with AD background showed higher levels of Cer(d18:1/24:1) in the cortex than mice without (1.4-fold, p = 0.003). S1P levels were higher in all three brain regions of older mice than of young mice (1.7-1.8-fold, all p ≤ 0.001). In female mice, S1P levels in hippocampus (r = −0.54 [−0.70, −0.35], p < 0.001) and in cortex correlated with those in plasma (r = −0.53 [−0.71, −0.32], p < 0.001). Ceramide levels were lower in the hippocampus (3.7–10.7-fold, all p < 0.001), but higher in the cortex (2.3–12.8-fold, p < 0.001) of female than male mice. In cerebellum and plasma, sex effects on individual ceramides depended on acyl chain length (9.5-fold lower to 11.5-fold higher, p ≤ 0.001). In conclusion, sex is a stronger determinant of brain ceramide levels in mice than APOE genotype, AD background, or age. Whether these differences impact AD neuropathology in men and women remains to be investigated.

Relative humidity (RH) levels vary continuously in vivo, although during in vitro generation of three-dimensional human skin equivalents (HSEs) these remain high (90–95%) to prevent evaporation of the cell-culture medium. However, skin functionality is directly influenced by environmental RH. As the barrier formation in HSEs is different, there is a need to better understand the role of cell-culture conditions during the generation of HSEs. In this study, we aim to investigate the effects of RH on epidermal morphogenesis and lipid barrier formation in HSEs. Therefore, two types of HSEs were developed at 90% or at 60% RH. Assessments were performed to determine epidermal morphogenesis by immunohistochemical analyses, ceramide composition by lipidomic analysis, and lipid organization by Fourier transform infrared spectroscopy and small-angle X-ray diffraction. We show that reduction of RH mainly affected the uppermost viable epidermal layers in the HSEs, including an enlargement of the granular cells and induction of epidermal cell activation. Neither the composition nor the organization of the lipids in the intercorneocyte space were substantially altered at reduced RH. In addition, lipid processing from glucosylceramides to ceramides was not affected by reduced RH in HSEs as shown by enzyme expression, enzyme activity, and substrate-to-product ratio. Our results demonstrate that RH directly influences epidermal morphogenesis, albeit the in vitro lipid barrier formation is comparable at 90% and 60% RH. Electronic supplementary material The online version of this article (10.1007/s00403-019-01948-3) contains supplementary material, which is available to authorized users.

The nonlysosomal glucosylceramidase β2 (GBA2) catalyzes the hydrolysis of glucosylceramide to glucose and ceramide. Mutations in the human GBA2 gene have been associated with hereditary spastic paraplegia (HSP), autosomal-recessive cerebellar ataxia (ARCA), and the Marinesco-Sjögren–like syndrome. However, the underlying molecular mechanisms are ill-defined. Here, using biochemistry, immunohistochemistry, structural modeling, and mouse genetics, we demonstrate that all but one of the spastic gait locus #46 (SPG46)-connected mutations cause a loss of GBA2 activity. We demonstrate that GBA2 proteins form oligomeric complexes and that protein–protein interactions are perturbed by some of these mutations. To study the pathogenesis of GBA2-related HSP and ARCA in vivo, we investigated GBA2-KO mice as a mammalian model system. However, these mice exhibited a high phenotypic variance and did not fully resemble the human phenotype, suggesting that mouse and human GBA2 differ in function. Whereas some GBA2-KO mice displayed a strong locomotor defect, others displayed only mild alterations of the gait pattern and no signs of cerebellar defects. On a cellular level, inhibition of GBA2 activity in isolated cerebellar neurons dramatically affected F-actin dynamics and reduced neurite outgrowth, which has been associated with the development of neurological disorders. Our results shed light on the molecular mechanism underlying the pathogenesis of GBA2-related HSP and ARCA and reveal species-specific differences in GBA2 function in vivo.

Glucocerebrosidase (GBA) is a lysosomal β‐glucosidase‐degrading glucosylceramide. Its deficiency causes Gaucher disease (GD), a common lysosomal storage disorder. Carrying a genetic abnormality in GBA constitutes at present the largest genetic risk factor for Parkinson's disease (PD). Conduritol B epoxide (CBE), a mechanism‐based irreversible inhibitor of GBA, is used to generate cell and animal models for investigations on GD and PD. However, CBE may have additional glycosidase targets besides GBA. Here, we present the first in vivo target engagement study for CBE, employing a suite of activity‐based probes to visualize catalytic pocket occupancy of candidate off‐target glycosidases. Only at significantly higher CBE concentrations, nonlysosomal glucosylceramidase (GBA2) and lysosomal α‐glucosidase were identified as major off‐targets in cells and zebrafish larvae. A tight, but acceptable window for selective inhibition of GBA in the brain of mice was observed. On the other hand, cyclophellitol, a closer glucose mimic, was found to inactivate with equal affinity GBA and GBA2 and therefore is not suitable to generate genuine GD‐like models. Enzymes Glucocerebrosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/45.html), nonlysosomal β‐glucocerebrosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/45.html); cytosolic β‐glucosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/21.html); α‐glucosidases (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/20.html); β‐glucuronidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/31.html)., In vivo target engagement of mechanism‐based glucocerebrosidase (GBA) inhibitors—conduritol B epoxide (CBE) and cyclophellitol (CP)—were examined in cultured cells, zebrafish larvae and mice by competitive activity‐based protein profiling (ABPP). This method utilizes suicide fluorescent enzyme reporter molecules to assess active site occupancy of target glycosidases by inhibitors. The in vivo targets of CBE and CP and their selectivity towards GBA were revealed.

Gaucher disease is caused by inherited deficiency in glucocerebrosidase (GBA, a retaining β-glucosidase), and deficiency in GBA constitutes the largest known genetic risk factor for Parkinson's disease. In the past, animal models of Gaucher disease have been generated by treatment with the mechanism-based GBA inhibitors, conduritol B epoxide (CBE), and cyclophellitol. Both compounds, however, also target other retaining glycosidases, rendering generation and interpretation of such chemical knockout models complicated. Here we demonstrate that cyclophellitol derivatives carrying a bulky hydrophobic substituent at C8 are potent and selective GBA inhibitors and that an unambiguous Gaucher animal model can be readily generated by treatment of zebrafish with these.

Cyclophellitol aziridine and its configurational and functional isomers are powerful covalent inhibitors of retaining glycosidases, and find application in fundamental studies on glycosidases, amongst others in relation to inherited lysosomal storage disorders caused by glycosidase malfunctioning. Few direct and stereoselective aziridination methodologies are known for the synthesis of cyclophellitol aziridines. Herein, we present our studies on the scope of direct 3-amino-2-(trifluoromethyl)quinazolin-4(3H)-one-mediated aziridination on a variety of configurational and functional cyclohexenol isosters. We demonstrate that the aziridination can be directed by an allylic or homoallylic hydroxyl through H-bonding and that steric hindrance plays a key role in the diastereoselectivity of the reaction.

Iminosugars are an important class of natural products and have been subject to extensive studies in organic synthesis, bioorganic chemistry and medicinal chemistry, yet only a limited number of these studies are on glycosylated iminosugars. Here, a general route of synthesis is presented towards glycosylated 1‐deoxynojirimycin derivatives based on the oxidation–reductive amination protocol that in the past has also been shown to be a versatile route towards 1‐deoxynojirimycin. The strategy can be applied on commercial disaccharides, as shown in four examples, as well as on disaccharides that are not commercially available and are synthesized for this purpose, as shown by a fifth example.

We thank The Netherlands Organization for Scientific Research (NWO-CW, ChemThem grant to J.M.F.G.A. and H.S.O.), the European Research Council (ERC-2011-AdG290836 “Chembiosphing”, to H.S.O.) and Sanofi Genzyme (research grant to J.M.F.G.A. and H.S.O. and postdoctoral contract to M.A.). We acknowledge ChemAxon for kindly providing the Instant JChem software to manage our compound library. T.M.G., V.O. and S.A.M. are supported by a Wellcome Trust Research Career Development Fellowship and Institutional Strategic Support Funding. Cyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity‐based glycosidase probes (ABPs). Direct 3‐amino‐2‐(trifluoromethyl)quinazolin‐4(3H)‐one‐mediated aziridination of ʟ‐ido‐configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α‐ʟ‐iduronidase, deficiency of which underlies the lysosomal storage disorder mucopolysaccharidosis type I (MPS I). The iduronidase ABPs react covalently and irreversibly in an activity‐based manner with human recombinant α‐ʟ‐iduronidase (rIDUA, Aldurazyme®). The structures of IDUA when complexed with the inhibitors in a non‐covalent transition state mimicking form and a covalent enzyme‐bound form provide insights into its conformational itinerary. Inhibitors 1–3 adopt a half‐chair conformation in solution (4H3 and 3H4), as predicted by DFT calculations, which is different from the conformation of the Michaelis complex observed by crystallographic studies. Consequently, 1–3 may need to overcome an energy barrier in order to switch from the 4H3 conformation to the transition state (2, 5B) binding conformation before reacting and adopting a covalent 5S1 conformation. rIDUA can be labeled with fluorescent Cy5 ABP 2 , which allows monitoring of the delivery of therapeutic recombinant enzyme to lysosomes, as is intended in enzyme replacement therapy for the treatment of MPS I patients. Publisher PDF

Obesity and dyslipidaemia are features of the metabolic syndrome and risk factors for chronic kidney disease. The cellular mechanisms connecting metabolic syndrome with chronic kidney disease onset and progression remain largely unclear. We show that proximal tubular epithelium is a target site for lipid deposition upon overnutrition with a cholesterol-rich Western-type diet. Affected proximal tubule epithelial cells displayed giant vacuoles of lysosomal or autophagosomal origin, harbouring oxidised lipoproteins and concentric membrane layer structures (multilamellar bodies), reminiscent of lysosomal storage diseases. Additionally, lipidomic analysis revealed renal deposition of cholesterol and phospholipids, including lysosomal phospholipids. Proteomic profiles of renal multilamellar bodies were distinct from those of epidermis or lung multilamellar bodies and of cytoplasmic lipid droplets. Tubular multilamellar bodies were observed in kidney biopsies of obese hypercholesterolaemic patients, and the concentration of the phospholipidosis marker di-docosahexaenoyl (22:6)-bis(monoacylglycerol) phosphate was doubled in urine from individuals with metabolic syndrome and chronic kidney disease. The enrichment of proximal tubule epithelial cells with phospholipids and multilamellar bodies was accompanied by enhanced inflammation, fibrosis, tubular damage markers, and higher urinary electrolyte content. Concomitantly to the intralysosomal lipid storage, a renal transcriptional response was initiated to enhance lysosomal degradation and lipid synthesis. In cultured proximal tubule epithelial cells, inhibition of cholesterol efflux transport or oxysterol treatment induced effects very similar to the in vivo situation, such as multilamellar body and phospholipid amassing, and induction of damage, inflammatory, fibrotic, and lipogenic molecules. The onset of phospholipidosis in proximal tubule epithelial cells is a novel pathological trait in metabolic syndrome-related chronic kidney disease, and emphasises the importance of healthy lysosomes and nutrition for kidney well-being. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Funding Information: This work was supported by the NWO-Building Blocks of Life: GlcCer grant to J.M.F.G.A: BBOL-2007247202. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. The lysosomal storage disease Niemann–Pick type C (NPC) is caused by impaired cholesterol efflux from lysosomes, which is accompanied by secondary lysosomal accumulation of sph-ingomyelin and glucosylceramide (GlcCer). Similar to Gaucher disease (GD), patients deficient in glucocerebrosidase (GCase) degrading GlcCer, NPC patients show an elevated glucosylsphingosine and glucosylated cholesterol. In livers of mice lacking the lysosomal cholesterol efflux transporter NPC1, we investigated the expression of established biomarkers of lipid-laden macrophages of GD patients, their GCase status, and content on the cytosol facing glucosylceramidase GBA2 and lysoso-mal integral membrane protein type B (LIMP2), a transporter of newly formed GCase to lysosomes. Livers of 80-week-old Npc1−/− mice showed a partially reduced GCase protein and enzymatic activity. In contrast, GBA2 levels tended to be reciprocally increased with the GCase deficiency. In Npc1−/− liver, increased expression of lysosomal enzymes (cathepsin D, acid ceramidase) was observed as well as increased markers of lipid-stressed macrophages (GPNMB and galectin-3). Im-munohistochemistry showed that the latter markers are expressed by lipid laden Kupffer cells. Earlier reported increase of LIMP2 in Npc1−/− liver was confirmed. Unexpectedly, immunohistochemistry showed that LIMP2 is particularly overexpressed in the hepatocytes of the Npc1−/− liver. LIMP2 in these hepatocytes seems not to only localize to (endo)lysosomes. The recent recognition that LIMP2 harbors a cholesterol channel prompts the speculation that LIMP2 in Npc1−/− hepatocytes might mediate export of cholesterol into the bile and thus protects the hepatocytes. publishersversion published

Human GBA1 encodes lysosomal acid β-glucocerebrosidase (GCase), which hydrolyzes cleavage of the beta-glucosidic linkage of glucosylceramide (GlcCer). Mutations in this gene lead to reduced GCase activity, accumulation of glucosylceramide and glucosylsphingosine, and development of Gaucher disease (GD). Drosophila melanogaster has two GBA1 orthologs. Thus far, GBA1b was documented as a bone fide GCase-encoding gene, while the role of GBA1a encoded protein remained unclear. In the present study, we characterized a mutant variant of the fly GBA1a, which underwent ERAD and mildly activated the UPR machinery. RNA-seq analyses of homozygous mutant flies revealed upregulation of inflammation-associated as well as of cell-cycle related genes and reduction in programmed cell death (PCD)-associated genes, which was confirmed by qRT-PCR. We also observed compromised cell death in the midgut of homozygous larvae and a reduction in pupation. Our results strongly indicated that GBA1a-encoded protein plays a role in midgut maturation during larvae development.

Amylases are key enzymes in the processing of starch in many kingdoms of life. They are important catalysts in industrial biotechnology where they are applied in, among others, food processing and the production of detergents. In man amylases are the first enzymes in the digestion of starch to glucose and arguably also the preferred target in therapeutic strategies aimed at the treatment of type 2 diabetes patients through down-tuning glucose assimilation. Efficient and sensitive assays that report selectively on retaining amylase activities irrespective of the nature and complexity of the biomaterial studied are of great value both in finding new and effective human amylase inhibitors and in the discovery of new microbial amylases with potentially advantageous features for biotechnological application. Activity-based protein profiling (ABPP) of retaining glycosidases is inherently suited for the development of such an assay format. We here report on the design and synthesis of 1,6-epi-cyclophellitol-based pseudodisaccharides equipped with a suite of reporter entities and their use in ABPP of retaining amylases from human saliva, murine tissue as well as secretomes from fungi grown on starch. The activity and efficiency of the inhibitors and probes are substantiated by extensive biochemical analysis, and the selectivity for amylases over related retaining endoglycosidases is validated by structural studies.

Deficiency of glucocerebrosidase (GBA), a lysosomal beta-glucosidase, causes Gaucher disease. The enzyme hydrolyzes beta-glucosidic substrates and transglucosylates cholesterol to cholesterol-p-glucoside. Here we show that recombinant human GBA also cleaves beta-xylosides and transxylosylates cholesterol. The xylosyl-cholesterol formed acts as an acceptor for the subsequent formation of di-xylosyl-cholesterol. Common mutant forms of GBA from patients with Gaucher disease with reduced beta-glucosidase activity were similarly impaired in beta-xylosidase, transglucosidase, and transxylosidase activities, except for a slightly reduced xylosidase/glucosidase activity ratio of N370S GBA and a slightly reduced transglucosylation/glucosidase activity ratio of D409H GBA. XylChol was found to be reduced in spleen from patients with Gaucher disease. The origin of newly identified XylChol in mouse and human tissues was investigated. Cultured human cells exposed to exogenous beta-xylosides generated XylChol in a manner dependent on active lysosomal GBA but not the cytosol-facing beta-glucosidase GBA2. We later sought an endogenous beta-xyloside acting as donor in transxylosylation reactions, identifying xylosylated ceramide (XylCer) in cells and tissues that serve as donor in the formation of XylChol. UDP-glucosylceramide synthase (GCS) was unable to synthesize XylChol but could catalyze the formation of XylCer. Thus, food-derived beta-D-xyloside and XylCer are potential donors for the GBA-mediated formation of XylChol in cells. The enzyme GCS produces XylCer at a low rate. Our findings point to further catalytic versatility of GBA and prompt a systematic exploration of the distribution and role of xylosylated lipids.

To assess the utility of globotriaosylsphingosine (lyso-GbA post hoc analysis evaluated data from 97 treatment-naive and enzyme replacement therapy (ERT)-experienced patients with migalastat-amenable GLA variants from FACETS (NCT00925301) and ATTRACT (NCT01218659) and subsequent open-label extension studies. The relationship between plasma lyso-GbNo significant correlations were identified between changes in lyso-GbAlthough used as a pharmacodynamic biomarker in research and clinical studies, plasma lyso-Gb

Lyso-glycosphingolipids are generated in excess in glycosphingolipid storage disorders. In the course of these pathologies glycosylated sphingolipid species accumulate within lysosomes due to flaws in the respective lipid degrading machinery. Deacylation of accumulating glycosphingolipids drives the formation of lyso-glycosphingolipids. In lysosomal storage diseases such as Gaucher Disease, Fabry Disease, Krabbe disease, GM1 -and GM2 gangliosidosis, Niemann Pick type C and Metachromatic leukodystrophy massive intra-lysosomal glycosphingolipid accumulation occurs. The lysosomal enzyme acid ceramidase generates the deacylated lyso-glycosphingolipid species. This review discusses how the various lyso-glycosphingolipids are synthesized, how they may contribute to abnormal immunity in glycosphingolipid storing lysosomal diseases and what therapeutic opportunities exist.

Gaucher disease type 1 (GD1) is an inherited lysosomal disorder with multisystemic effects in patients. Hallmark symptoms include hepatosplenomegaly, cytopenias, and bone disease with varying degrees of severity. Mutations in a single gene, glucosidase beta acid 1 (GBA1), are the underlying cause for the disorder, resulting in insufficient activity of the enzyme glucocerebrosidase, which in turn leads to a progressive accumulation of the lipid component glucocerebroside. In this study, we treat mice with signs consistent with GD1, with hematopoietic stem/progenitor cells transduced with a lentiviral vector containing an RNA transcript that, after reverse transcription, results in codon-optimized cDNA that, upon its integration into the genome encodes for functional human glucocerebrosidase. Five months after gene transfer, a highly significant reduction in glucocerebroside accumulation with subsequent reversal of hepatosplenomegaly, restoration of blood parameters, and a tendency of increased bone mass and density was evident in vector-treated mice compared to non-treated controls. Furthermore, histopathology revealed a prominent reduction of Gaucher cell infiltration after gene therapy. The vector displayed an oligoclonal distribution pattern but with no sign of vector-induced clonal dominance and a typical lentiviral vector integration profile. Cumulatively, our findings support the initiation of the first clinical trial for GD1 using the lentiviral vector described here., Graphical Abstract, This study evaluates a clinically applicable lentiviral vector for treatment of Gaucher disease type 1. Hematopoietic stem cells transduced with the vector and transplanted into a mouse model successfully halted or reversed pathology. These data were used as proof-of-concept for regulatory filing enabling the commencement of an international phase 1/2 clinical trial.

Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GIcNAc-Man(5)GlcNAc(2) to produce GlcNAcMan(3) GlcNAc(2), the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce alpha-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of alpha-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.

The existence of glucosylated cholesterol (GlcChol) in tissue has recently been recognized. GlcChol is generated from glucosylceramide (GlcCer) and cholesterol through transglucosylation by two retaining β-glucosidases, GBA and GBA2. Given the abundance of GBA, GlcCer and cholesterol in the skin's stratum corneum (SC), we studied the occurrence of GlcChol. A significant amount of GlcChol was detected in SC (6 pmol/mg weight). The ratio GlcChol/GlcCer is higher in SC than epidermis, 0.083 and 0.011, respectively. Examination of GlcChol in patients with Netherton syndrome revealed comparable levels (11 pmol/mg). Concluding, GlcChol was identified as a novel component in SC and is likely locally metabolized by GBA. The physiological function of GlcChol in the SC warrants future investigation.

Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcMan

Enzyme replacement therapy (ERT) has been shown to stabilize certain aspects of Fabry disease (FD). However, in some patients on ERT, high antibody titres have been documented, with limited clinical improvement in systemic manifestations and often with significant adverse drug reactions. We present two related adolescent males with a 4.5 kb GLA deletion, not amenable to chaperone therapy, leading to profound reduction in alpha-galactosidase A (alpha-gal A) enzyme activity. Over a 3-year period of ERT, increasing IgG antibody titres against alpha-gal A were noted. After starting ERT serial urine globotriaosylceramide (Gb(3)) measurements showed an upward trend from 333 to 2260 mu g/mmol creatinine for patient 1 and 1165 to 2260 mu g/mmol creatinine for patient 2. Markedly increased levels of urine and plasma globotriaosylsphingosine (Lyso-Gb(3)) analogues were also found. The patients experienced recurrent infusion-associated reactions necessitating premedication and prolonged infusion times. Over the 3-year period of ERT, the patients experienced continued malaise, gastrointestinal symptoms and neuropathic pain. In addition, they had increasing anxiety related to their disease and apparent lack of response to ERT which led to a decision to ultimately stop ERT. No other approved treatment options are currently available for these patients. It is possible that the rapid development of the high antidrug neutralizing antibody (ADA) titres is related to the large GLA deletion leading to virtually absent enzyme activity. It remains unclear if their symptomatology during the period of receiving ERT is related to lack of its efficacy, the rising ADA titres, or both. These two patients highlight the need for further research into the management of antidrug antibodies and additional therapeutic approaches for FD.Synopsis: The development of very high antidrug antibody titres in response to ERT in two related adolescent males with FD highlight the need for other therapeutic options for patients in whom ERT or other currently approved therapies does not meet their treatment needs.