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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.

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).

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.

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.

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.

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.

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

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

The cellular recycling of glycosphingolipids (GSLs) is mediated by specific lysosomal glycosidases. Inherited deficiencies in these enzymes cause lysosomal storage disorders. Some of the common disorders are Gaucher disease (GD) and Fabry disease (FD) resulting from the defects in lysosomal glucocerebrosidase (GBA) degrading glucosylceramide and α‐galactosidase A (GLA) degrading globotriaosylceramide. Here, GSL accumulation in tissues slows down with age despite ongoing lysosomal turnover of endogenous and endocytosed GSLs. Biochemical adaptations might explain this phenomenon. One crucial adaptation is the deacylation of accumulating GSLs in lysosomes by acid ceramidase. The soluble bases glucosylsphingosine in GD and globotriaosylsphingosine in FD are capable of leaving lysosomes and cells. In the case of GD, a further adaptation involves the cytosol‐faced enzyme GBA2. This enzyme allows extra‐lysosomal degradation of GlcCer while possibly generating glucosylated cholesterol. The beneficial and harmful effects of these adaptations are discussed. Key concepts: Glycosphingolipids (GSLs) are membrane constituents composed of a ceramide with one or more sugars. The simplest GSL is glucosylceramide (GlcCer). Ongoing recycling of GSLs in cells includes lysosomal degradation by the sequential action of glycosidases and acid ceramidase. Deficiency of lysosomal glycosidase leads to lysosomal storage diseases caused by accumulation of the corresponding substrate in lysosomes. The most common glycosphingolipidoses are Gaucher disease (GD) and Fabry disease (FD). GD is an autosomal recessive disorder caused by deficient activity of the lysosomal enzyme acid β‐glucosidase (glucocerebrosidase; GBA) resulting in lysosomal accumulation of GlcCer. FD is an X‐linked disorder caused by deficient activity of the lysosomal enzyme α‐galactosidase A (GLA) resulting in lysosomal accumulation of globotriaosylceramide (Gb3). Accumulation of storage lipids during GBA and GLA tends to slow down with age, likely partly due to poorly appreciated biochemical adaptations. Active conversion of accumulating GlcCer in lysosomes of GBA‐deficient cells is mediated by acid ceramidase, resulting in the formation of water‐soluble glucosylsphingosine (GlcSph). Likewise, globotriaosylsphingosine (lysoGb3) is formed from accumulating in lysosomes of GLA‐deficient cells. Elevated plasma GlcSph and lysoGb3 levels can be sensitively measured LC–MS and may assist in diagnosing and monitoring of the disease and response to treatment in GD and FD patients, respectively. Increased GlcSph level in GD patients acts as an autoantigen, causing ongoing B‐cell proliferation, leading to multiple myeloma. Increased lysoGb3 level in FD patients is thought to cause damage to nociceptive neurons and podocytes, thus contributing to pain and renal failure. In GD, the cytosol‐faced enzyme β‐glucosidase GBA2 allows degradation of GlcCer outside lysosomes. Through transglycosylation, GBA2 may generate glucosylcholesterol and ceramide from GlcCer and cholesterol. The toxic effects of secondary metabolites such as glycosphingoid bases (GlcSph in GD and lysoGb3 in FD) and glucosylated metabolites (GlcChol in GD) warrant further investigations. info:eu-repo/semantics/publishedVersion

β-glucosidases (GBA1 [glucocerebrosidase], GBA2, and GBA3) are ubiquitous, essential enzymes. Lysosomal GBA1 and cytosol-facing GBA2 degrade glucosylceramide (GlcCer); GBA1 deficiency causes Gaucher disease (GD), a lysosomal storage disorder characterized by lysosomal accumulation of GlcCer, which is partly converted to glucosylsphingosine (GlcSph). GBA1 and GBA2 also may transfer glucose from GlcCer to cholesterol, yielding glucosylated cholesterol (GlcChol). Here, we aimed to clarify the role of zebrafish Gba2 in glycosphingolipid metabolism during Gba1 deficiency in zebrafish (Danio rerio), which are able to survive total Gba1 deficiency. We developed Gba1 and Gba2 zebrafish knockouts (gba1-/- and gba2-/-, respectively) using CRISPR/Cas9, modulated glucosidases genetically and pharmacologically, studied GlcCer metabolism in individual larvae, and explored the feasibility of pharmacologic or genetic interventions. Activity-based probes and quantification of relevant glycolipid metabolites confirmed enzyme deficiency. GlcSph increased in gba1-/- larvae (0.09 pmol/fish) but did not increase more in gba1-/-:gba2-/- larvae. GlcCer was comparable in gba1-/- and wild-type (WT) larvae but increased in gba2-/- and gba1-/-:gba2-/- larvae. Independent of Gba1 status, GlcChol was low in all gba2-/- larvae (0.05 vs. 0.18. pmol/fish in WT). Pharmacologic inactivation of zebrafish Gba1 comparably increased GlcSph. Inhibition of glucosylceramide synthase in Gba1-deficient larvae reduced GlcCer and GlcSph, and concomitant inhibition of glucosylceramide synthase and Gba2 with iminosugars also reduced excessive GlcChol. Finally, overexpression of human GBA1 and injection of recombinant GBA1 both decreased GlcSph. We determined that zebrafish larvae offer an attractive model to study glucosidase actions in glycosphingolipid metabolism in vivo, and we identified distinguishing characteristics of zebrafish Gba2 deficiency.

Background Cervical spondylotic myelopathy (CSM) is a degenerative cervical disease in which the spinal cord is compressed. Patients with CSM experience balance disturbance because of impaired proprioception. The weighting of the sensory inputs for postural control in patients with CSM is unclear. Therefore, this study investigated the weighting of sensory systems in patients with CSM. Method Twenty-four individuals with CSM (CSM group) and 24 age-matched healthy adults (healthy control group) were analyzed in this observational study. The functional outcomes (modified Japanese Orthopaedic Association Scale [mJOA], Japanese Orthopaedic Association Cervical Myelopathy Questionnaire [JOACMEQ], Nurick scale) and static balance (eyes-open and eyes-closed conditions) were assessed for individuals with CSM before surgery, 3 and 6 months after surgery. Time-domain and time–frequency-domain variables of the center of pressure (COP) were analyzed to examine the weighting of the sensory systems. Results In the CSM group, lower extremity function of mJOA and Nurick scale significantly improved 3 and 6 months after surgery. Before surgery, the COP mean velocity and total energy were significantly higher in the CSM group than in the control group for both vision conditions. Compared with the control group, the CSM group exhibited lower energy content in the moderate-frequency band (i.e., proprioception) and higher energy content in the low-frequency band (i.e., cerebellar, vestibular, and visual systems) under the eyes-open condition. The COP mean velocity of the CSM group significantly decreased 3 months after surgery. The energy content in the low-frequency band (i.e., visual and vestibular systems) of the CSM group was closed to that of the control group 6 months after surgery under the eyes-open condition. Conclusion Before surgery, the patients with CSM may have had compensatory sensory weighting for postural control, with decreased weighting on proprioception and increased weighting on the other three sensory inputs. After surgery, the postural control of the patients with CSM improved, with decreased compensation for the proprioceptive system from the visual and vestibular inputs. However, the improvement remained insufficient because the patients with CSM still had lower weighting on proprioception than the healthy adults did. Therefore, patients with CSM may require balance training and posture education after surgery. Trial registration Trial Registration number: NCT03396055 Name of the registry: ClinicalTrials.gov Date of registration: January 10, 2018 - Retrospectively registered Date of enrolment of the first participant to the trial: October 19, 2015

The development of small molecule activity-based probes (ABPs) is an evolving and powerful area of chemistry. There is a major need for synthetically accessible and specific ABPs to advance our understanding of enzymes in health and disease. α-Glucosidases are involved in diverse physiological processes including carbohydrate assimilation in the gastrointestinal tract, glycoprotein processing in the endoplasmic reticulum (ER), and intralysosomal glycogen catabolism. Inherited deficiency of the lysosomal acid α-glucosidase (GAA) causes the lysosomal glycogen storage disorder, Pompe disease. Here, we design a synthetic route for fluorescent and biotin-modified ABPs for in vitro and in situ monitoring of α-glucosidases. We show, through mass spectrometry, gel electrophoresis, and X-ray crystallography, that α-glucopyranose configured cyclophellitol aziridines label distinct retaining α-glucosidases including GAA and ER α-glucosidase II, and that this labeling can be tuned by pH. We illustrate a direct diagnostic application in Pompe disease patient cells, and discuss how the probes may be further exploited for diverse applications., α-Glucosidases control food digestion, protein folding, and glycogen turnover. Designer probes report on tissue- and pH-specific α-glucosidase activity and reveal loss of α-glucosidase activity in Pompe disease patient material.

Glycosidases mediate the fragmentation of glycoconjugates in the body, including the vital recycling of endogenous molecules. Several inherited diseases in man concern deficiencies in lysosomal glycosidases degrading glycosphingolipids. Prominent is Gaucher disease caused by an impaired lysosomal β-glucosidase (glucocerebrosidase, GBA) and resulting in pathological lysosomal storage of glucosylceramide (glucocerebroside) in tissue macrophages. GBA is a retaining glucosidase with a characteristic glycosyl-enzyme intermediate formed during catalysis. Using the natural suicide inhibitor cyclophellitol as a lead, we developed mechanism-based irreversible inhibitors of GBA equipped with a fluorescent reporter. These reagents covalently link to the catalytic nucleophile residue of GBA and permit specific and sensitive visualization of active enzyme molecules. The amphiphilic activity-based probes (ABPs) allow in situ detection of active GBA in cells and organisms. Furthermore, they may be used to biochemically confirm the diagnosis of Gaucher disease and they might assist in screening for small compounds interacting with the catalytic pocket. While the focus of this chapter is ABPs for β-glucosidases and Gaucher disease, the described concept has meanwhile been extended to other retaining glycosidases and related disease conditions as well.

Glycosidases mediate the fragmentation of glycoconjugates in the body, including the vital recycling of endogenous molecules. Several inherited diseases in man concern deficiencies in lysosomal glycosidases degrading glycosphingolipids. Prominent is Gaucher disease caused by an impaired lysosomal β-glucosidase (glucocerebrosidase, GBA) and resulting in pathological lysosomal storage of glucosylceramide (glucocerebroside) in tissue macrophages. GBA is a retaining glucosidase with a characteristic glycosyl–enzyme intermediate formed during catalysis. Using the natural suicide inhibitor cyclophellitol as a lead, we developed mechanism-based irreversible inhibitors of GBA equipped with a fluorescent reporter. These reagents covalently link to the catalytic nucleophile residue of GBA and permit specific and sensitive visualization of active enzyme molecules. The amphiphilic activity-based probes (ABPs) allow in situ detection of active GBA in cells and organisms. Furthermore, they may be used to biochemically confirm the diagnosis of Gaucher disease and they might assist in screening for small compounds interacting with the catalytic pocket. While the focus of this chapter is ABPs for β-glucosidases and Gaucher disease, the described concept has meanwhile been extended to other retaining glycosidases and related disease conditions as well.

Activity-based protein profiling has emerged as a powerful tool for visualizing glycosidases in complex biological samples. Several configurational cyclophellitol isomers have been shown to display high selectivity as probes for glycosidases processing substrates featuring the same configuration. Here, a set of deoxygenated cyclophellitols are presented which enable inter-class profiling of [small beta]-glucosidases and [small beta]-galactosidases.

The title linker is stable under various biological conditions and can be cleaved chemoselectively with hydrazine. Its use is demonstrated in the activity-based enrichment and identification of proteasome active subunits from cell extracts.

[This corrects the article DOI: 10.1021/acscentsci.6b00057.].