Publications by authors named "Johannes M F G Aerts"

170 Publications

GCase and LIMP2 Abnormalities in the Liver of Niemann Pick Type C Mice.

Int J Mol Sci 2021 Mar 3;22(5). Epub 2021 Mar 3.

Department Medical Biochemistry, Leiden University, 2333 CC Leiden, The Netherlands.

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 sphingomyelin 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 lysosomal integral membrane protein type B (LIMP2), a transporter of newly formed GCase to lysosomes. Livers of 80-week-old mice showed a partially reduced GCase protein and enzymatic activity. In contrast, GBA2 levels tended to be reciprocally increased with the GCase deficiency. In 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). Immunohistochemistry showed that the latter markers are expressed by lipid laden Kupffer cells. Earlier reported increase of LIMP2 in liver was confirmed. Unexpectedly, immunohistochemistry showed that LIMP2 is particularly overexpressed in the hepatocytes of the 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 hepatocytes might mediate export of cholesterol into the bile and thus protects the hepatocytes.
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http://dx.doi.org/10.3390/ijms22052532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959463PMC
March 2021

Fabry Disease: Molecular Basis, Pathophysiology, Diagnostics and Potential Therapeutic Directions.

Biomolecules 2021 Feb 12;11(2). Epub 2021 Feb 12.

Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.

Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by the deficiency of α-galactosidase A (α-GalA) and the consequent accumulation of toxic metabolites such as globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3). Early diagnosis and appropriate timely treatment of FD patients are crucial to prevent tissue damage and organ failure which no treatment can reverse. LSDs might profit from four main therapeutic strategies, but hitherto there is no cure. Among the therapeutic possibilities are intravenous administered enzyme replacement therapy (ERT), oral pharmacological chaperone therapy (PCT) or enzyme stabilizers, substrate reduction therapy (SRT) and the more recent gene/RNA therapy. Unfortunately, FD patients can only benefit from ERT and, since 2016, PCT, both always combined with supportive adjunctive and preventive therapies to clinically manage FD-related chronic renal, cardiac and neurological complications. Gene therapy for FD is currently studied and further strategies such as substrate reduction therapy (SRT) and novel PCTs are under investigation. In this review, we discuss the molecular basis of FD, the pathophysiology and diagnostic procedures, together with the current treatments and potential therapeutic avenues that FD patients could benefit from in the future.
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http://dx.doi.org/10.3390/biom11020271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918333PMC
February 2021

Tuning the Transglycosylation Reaction of a GH11 Xylanase by a Delicate Enhancement of its Thumb Flexibility.

Chembiochem 2021 Feb 3. Epub 2021 Feb 3.

Department of Macromolecular Biochemistry, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.

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.
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http://dx.doi.org/10.1002/cbic.202000856DOI Listing
February 2021

Correction of pathology in mice displaying Gaucher disease type 1 by a clinically-applicable lentiviral vector.

Mol Ther Methods Clin Dev 2021 Mar 3;20:312-323. Epub 2020 Dec 3.

Department of Molecular Medicine and Gene Therapy, Lund University, Lund, Sweden.

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 (), 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.
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http://dx.doi.org/10.1016/j.omtm.2020.11.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806948PMC
March 2021

Activity-Based Protein Profiling of Retaining α-Amylases in Complex Biological Samples.

J Am Chem Soc 2021 Feb 26;143(5):2423-2432. Epub 2021 Jan 26.

Department of Chemistry, York Structural Biology Laboratory, University of York, Heslington, York YO10 5DD, United Kingdom.

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--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.
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http://dx.doi.org/10.1021/jacs.0c13059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883350PMC
February 2021

The Role of Cholesterol in α-Synuclein and Lewy Body Pathology in GBA1 Parkinson's Disease.

Mov Disord 2020 Nov 21. Epub 2020 Nov 21.

Instituto Cajal, CSIC, Madrid, Spain.

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.
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http://dx.doi.org/10.1002/mds.28396DOI Listing
November 2020

Glucosylated cholesterol in skin: Synthetic role of extracellular glucocerebrosidase.

Clin Chim Acta 2020 Nov 16;510:707-710. Epub 2020 Sep 16.

Medical Biochemistry Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cca.2020.09.017DOI Listing
November 2020

Lyso-glycosphingolipids: presence and consequences.

Essays Biochem 2020 09;64(3):565-578

Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA, Leiden, The Netherlands.

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.
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http://dx.doi.org/10.1042/EBC20190090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7517347PMC
September 2020

Therapeutic challenges in two adolescent male patients with Fabry disease and high antibody titres.

Mol Genet Metab Rep 2020 Sep 24;24:100618. Epub 2020 Jun 24.

Department of Pediatrics and Child Health, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada.

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 deletion, not amenable to chaperone therapy, leading to profound reduction in α-galactosidase A (α-gal A) enzyme activity. Over a 3-year period of ERT, increasing IgG antibody titres against α-gal A were noted. After starting ERT serial urine globotriaosylceramide (Gb) measurements showed an upward trend from 333 to 2260 μg/mmol creatinine for patient 1 and 1165 to 2260 μg/mmol creatinine for patient 2. Markedly increased levels of urine and plasma globotriaosylsphingosine (Lyso-Gb 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 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.
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http://dx.doi.org/10.1016/j.ymgmr.2020.100618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322173PMC
September 2020

Manno--cyclophellitols Enable Activity-Based Protein Profiling of Human α-Mannosidases and Discovery of New Golgi Mannosidase II Inhibitors.

J Am Chem Soc 2020 07 16;142(30):13021-13029. Epub 2020 Jul 16.

Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, United Kingdom.

Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcManGlcNAc to produce GlcNAcManGlcNAc, the precursor for all complex -glycans, including the branched -glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-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--cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno--cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-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.
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http://dx.doi.org/10.1021/jacs.0c03880DOI Listing
July 2020

Dynamics of Ligand Binding to a Rigid Glycosidase*.

Angew Chem Int Ed Engl 2020 11 3;59(46):20508-20514. Epub 2020 Sep 3.

Department of Macromolecular Biochemistry, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.

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.
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http://dx.doi.org/10.1002/anie.202003236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693232PMC
November 2020

Novel defect in phosphatidylinositol 4-kinase type 2-alpha (PI4K2A) at the membrane-enzyme interface is associated with metabolic cutis laxa.

J Inherit Metab Dis 2020 11 26;43(6):1382-1391. Epub 2020 Jun 26.

Translational Metabolic Laboratory, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.

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)P 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)P and PI(3,4,5)P . 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.
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http://dx.doi.org/10.1002/jimd.12255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687218PMC
November 2020

Plant Glycosides and Glycosidases: A Treasure-Trove for Therapeutics.

Front Plant Sci 2020 7;11:357. Epub 2020 Apr 7.

Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, Netherlands.

Plants contain numerous glycoconjugates that are metabolized by specific glucosyltransferases and hydrolyzed by specific glycosidases, some also catalyzing synthetic transglycosylation reactions. The documented value of plant-derived glycoconjugates to beneficially modulate metabolism is first addressed. Next, focus is given to glycosidases, the central theme of the review. The therapeutic value of plant glycosidases is discussed as well as the present production in plant platforms of therapeutic human glycosidases used in enzyme replacement therapies. The increasing knowledge on glycosidases, including structure and catalytic mechanism, is described. The novel insights have allowed the design of functionalized highly specific suicide inhibitors of glycosidases. These so-called activity-based probes allow unprecedented visualization of glycosidases cross-species. Here, special attention is paid on the use of such probes in plant science that promote the discovery of novel enzymes and the identification of potential therapeutic inhibitors and chaperones.
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http://dx.doi.org/10.3389/fpls.2020.00357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154165PMC
April 2020

Skin barrier lipid enzyme activity in Netherton patients is associated with protease activity and ceramide abnormalities.

J Lipid Res 2020 06 7;61(6):859-869. Epub 2020 Apr 7.

Leiden Academic Centre for Drug Research, Leiden University, Leiden, The Netherlands. Electronic address:

Individuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we investigated how serine protease activity in NTS correlates with changes in the stratum corneum (SC) ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, β-glucocerebrosidase (GBA) and acid-sphingomyelinase (ASM). We compared in situ expression levels and activities of GBA and ASM between NTS patients and controls and correlated the expression and activities with ) SC ceramide profiles, ) in situ serine protease activity, and ) clinical presentation of patients. Using activity-based probe labeling, we visualized and localized active epidermal GBA, and a newly developed in situ zymography method enabled us to visualize and localize active ASM. Reduction in active GBA in NTS patients coincided with increased ASM activity, particularly in areas with increased serine protease activity. NTS patients with scaly erythroderma exhibited more pronounced anomalies in GBA and ASM activities than patients with ichthyosis linearis circumflexa. They also displayed a stronger increase in SC ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with ) altered SC ceramide composition in NTS patients, ) local serine protease activity, and ) the clinical manifestation of NTS.
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http://dx.doi.org/10.1194/jlr.RA120000639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269766PMC
June 2020

Glucocerebrosidase: Functions in and Beyond the Lysosome.

J Clin Med 2020 Mar 9;9(3). Epub 2020 Mar 9.

Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Faculty of Science, 2333 CC Leiden, The Netherlands.

Glucocerebrosidase (GCase) is a retaining β-glucosidase with acid pH optimum metabolizing the glycosphingolipid glucosylceramide (GlcCer) to ceramide and glucose. Inherited deficiency of GCase causes the lysosomal storage disorder named Gaucher disease (GD). In GCase-deficient GD patients the accumulation of GlcCer in lysosomes of tissue macrophages is prominent. Based on the above, the key function of GCase as lysosomal hydrolase is well recognized, however it has become apparent that GCase fulfills in the human body at least one other key function beyond lysosomes. Crucially, GCase generates ceramides from GlcCer molecules in the outer part of the skin, a process essential for optimal skin barrier property and survival. This review covers the functions of GCase in and beyond lysosomes and also pays attention to the increasing insight in hitherto unexpected catalytic versatility of the enzyme.
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http://dx.doi.org/10.3390/jcm9030736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141376PMC
March 2020

Glucocerebrosidases catalyze a transgalactosylation reaction that yields a newly-identified brain sterol metabolite, galactosylated cholesterol.

J Biol Chem 2020 04 6;295(16):5257-5277. Epub 2020 Mar 6.

RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan; Cellular Informatics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan. Electronic address:

β-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form β-cholesterylglucoside (β-GlcChol) β-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate β-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (β-GalChol), in addition to β-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish () revealed that GBA1 and GBA2 are responsible for β-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for β-GalChol formation. Liquid chromatography-tandem MS revealed that β-GlcChol and β-GalChol are present throughout development from embryo to adult in the mouse brain. We found that β-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of β-GalChol biosynthesis appeared to be during myelination. We also found that β-GlcChol and β-GalChol are secreted from neurons and glial cells in association with exosomes. enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form β-GalChol. This is the first report of the existence of β-GalChol in vertebrates and how β-GlcChol and β-GalChol are formed in the brain.
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http://dx.doi.org/10.1074/jbc.RA119.012502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170530PMC
April 2020

Skin of atopic dermatitis patients shows disturbed β-glucocerebrosidase and acid sphingomyelinase activity that relates to changes in stratum corneum lipid composition.

Biochim Biophys Acta Mol Cell Biol Lipids 2020 06 21;1865(6):158673. Epub 2020 Feb 21.

Leiden Academic Centre for Drug Research, Leiden, the Netherlands. Electronic address:

Patients with Atopic Dermatitis (AD) suffer from inflamed skin and skin barrier defects. Proper formation of the outermost part of the skin, the stratum corneum (SC), is crucial for the skin barrier function. In this study we analyzed the localization and activity of lipid enzymes β-glucocerebrosidase (GBA) and acid sphingomyelinase (ASM) in the skin of AD patients and controls. Localization of both the expression and activity of GBA and ASM in the epidermis of AD patients was altered, particularly at lesional skin sites. These changes aligned with the altered SC lipid composition. More specifically, abnormal localization of GBA and ASM related to an increase in specific ceramide subclasses [AS] and [NS]. Moreover we related the localization of the enzymes to the amounts of SC ceramide subclasses and free fatty acids (FFAs). We report a correlation between altered localization of active GBA and ASM and a disturbed SC lipid composition. Localization of antimicrobial peptide beta-defensin-3 (HBD-3) and AD biomarker Thymus and Activation Regulated Chemokine (TARC) also appeared to be diverging in AD skin compared to control. This research highlights the relation between correct localization of expressed and active lipid enzymes and a normal SC lipid composition for a proper skin barrier.
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http://dx.doi.org/10.1016/j.bbalip.2020.158673DOI Listing
June 2020

Biochemical and imaging parameters in acid sphingomyelinase deficiency: Potential utility as biomarkers.

Mol Genet Metab 2020 05 12;130(1):16-26. Epub 2020 Feb 12.

Amsterdam UMC, University of Amsterdam, Department of Endocrinology and Metabolism, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Electronic address:

Acid Sphingomyelinase Deficiency (ASMD), or Niemann-Pick type A/B disease, is a rare lipid storage disorder leading to accumulation of sphingomyelin and its precursors primarily in macrophages. The disease has a broad phenotypic spectrum ranging from a fatal infantile form with severe neurological involvement (the infantile neurovisceral type) to a primarily visceral form with different degrees of pulmonary, liver, spleen and skeletal involvement (the chronic visceral type). With the upcoming possibility of treatment with enzyme replacement therapy, the need for biomarkers that predict or reflect disease progression has increased. Biomarkers should be validated for their use as surrogate markers of clinically relevant endpoints. In this review, clinically important endpoints as well as biochemical and imaging markers of ASMD are discussed and potential new biomarkers are identified. We suggest as the most promising biomarkers that may function as surrogate endpoints in the future: diffusion capacity measured by spirometry, spleen volume, platelet count, low-density lipoprotein cholesterol, liver fibrosis measured with a fibroscan, lysosphingomyelin and walked distance in six minutes. Currently, no biomarkers have been validated. Several plasma markers of lipid-laden cells, fibrosis or inflammation are of high potential as biomarkers and deserve further study. Based upon current guidelines for biomarkers, recommendations for the validation process are provided.
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http://dx.doi.org/10.1016/j.ymgme.2020.02.002DOI Listing
May 2020

Glycosphingolipids and Infection. Potential New Therapeutic Avenues.

Front Cell Dev Biol 2019 6;7:324. Epub 2019 Dec 6.

Leiden Institute of Chemistry, Leiden University, Leiden, Netherlands.

Glycosphingolipids (GSLs), the main topic of this review, are a subclass of sphingolipids. With their glycans exposed to the extracellular space, glycosphingolipids are ubiquitous components of the plasma membrane of cells. GSLs are implicated in a variety of biological processes including specific infections. Several pathogens use GSLs at the surface of host cells as binding receptors. In addition, lipid-rafts in the plasma membrane of host cells may act as platform for signaling the presence of pathogens. Relatively common in man are inherited deficiencies in lysosomal glycosidases involved in the turnover of GSLs. The associated storage disorders (glycosphingolipidoses) show lysosomal accumulation of substrate(s) of the deficient enzyme. In recent years compounds have been identified that allow modulation of GSLs levels in cells. Some of these agents are well tolerated and already used to treat lysosomal glycosphingolipidoses. This review summarizes present knowledge on the role of GSLs in infection and subsequent immune response. It concludes with the thought to apply glycosphingolipid-lowering agents to prevent and/or combat infections.
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http://dx.doi.org/10.3389/fcell.2019.00324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908816PMC
December 2019

Direct Stereoselective Aziridination of Cyclohexenols with 3-Amino-2-(trifluoromethyl)quinazolin-4(3)-one in the Synthesis of Cyclitol Aziridine Glycosidase Inhibitors.

European J Org Chem 2019 Feb 11;2019(6):1397-1404. Epub 2019 Jan 11.

Department of Bio-organic Synthesis Leiden Institute of Chemistry Leiden University Einsteinweg 55 2333 CC Leiden The Netherlands.

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(3)-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.
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http://dx.doi.org/10.1002/ejoc.201801703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876648PMC
February 2019

Glycosphingolipids and lysosomal storage disorders as illustrated by gaucher disease.

Curr Opin Chem Biol 2019 12 26;53:204-215. Epub 2019 Nov 26.

Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA, Leiden, the Netherlands.

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.
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http://dx.doi.org/10.1016/j.cbpa.2019.10.006DOI Listing
December 2019

The Iminosugar AMP-DNM Improves Satiety and Activates Brown Adipose Tissue Through GLP1.

Diabetes 2019 12 2;68(12):2223-2234. Epub 2019 Oct 2.

Laboratory of Endocrinology, Department of Endocrinology and Metabolism, Amsterdam Gastroenterology & Metabolism, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands.

Obesity is taking on worldwide epidemic proportions, yet effective pharmacological agents with long-term efficacy remain unavailable. Previously, we designed the iminosugar N-adamantine-methyloxypentyl-deoxynojirimycin (AMP-DNM), which potently improves glucose homeostasis by lowering excessive glycosphingolipids. Here we show that AMP-DNM promotes satiety and activates brown adipose tissue (BAT) in obese rodents. Moreover, we demonstrate that the mechanism mediating these favorable actions depends on oral, but not central, administration of AMP-DNM, which ultimately stimulates systemic glucagon-like peptide 1 (GLP1) secretion. We evidence an essential role of brain GLP1 receptors (GLP1r), as AMP-DNM fails to promote satiety and activate BAT in mice lacking the brain GLP1r as well as in mice treated intracerebroventricularly with GLP1r antagonist exendin-9. In conclusion, AMP-DNM markedly ameliorates metabolic abnormalities in obese rodents by restoring satiety and activating BAT through central GLP1r, while improving glucose homeostasis by mechanisms independent of central GLP1r.
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http://dx.doi.org/10.2337/db19-0049DOI Listing
December 2019

Role of β-glucosidase 2 in aberrant glycosphingolipid metabolism: model of glucocerebrosidase deficiency in zebrafish.

J Lipid Res 2019 11 27;60(11):1851-1867. Epub 2019 Sep 27.

Department of Medical Biochemistry Leiden Institute of Chemistry, Leiden, The Netherlands

β-glucosidases [GBA1 (glucocerebrosidase) and GBA2] are ubiquitous essential enzymes. Lysosomal GBA1 and cytosol-facing GBA2 degrade glucosylceramide (GlcCer); GBA1 deficiency causes Gaucher disease, 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 (), which are able to survive total Gba1 deficiency. We developed Gba1 (), Gba2 (), and double () zebrafish knockouts using CRISPR/Cas9 and explored the effects of both genetic and pharmacological interventions on GlcCer metabolism in individual larvae. Activity-based probes and quantification of relevant glycolipid metabolites confirmed enzyme deficiency. GlcSph increased in larvae (0.09 pmol/fish) but did not increase more in larvae. GlcCer was comparable in and WT larvae but increased in and larvae. Independent of Gba1 status, GlcChol was low in all larvae (0.05 vs. 0.18 pmol/fish in WT). Pharmacologic inactivation of zebrafish Gba1 comparably increased GlcSph. Inhibition of GlcCer synthase (GCS) in Gba1-deficient larvae reduced GlcCer and GlcSph, and concomitant inhibition of GCS 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.
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http://dx.doi.org/10.1194/jlr.RA119000154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824494PMC
November 2019

Unravelling effects of relative humidity on lipid barrier formation in human skin equivalents.

Arch Dermatol Res 2019 Nov 18;311(9):679-689. Epub 2019 Jul 18.

Department of Dermatology, Leiden University Medical Center, Albinusdreef 2, Room C3-76, 2333 ZA, Leiden, The Netherlands.

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.
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http://dx.doi.org/10.1007/s00403-019-01948-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6787114PMC
November 2019

Adipocytes harbor a glucosylceramide biosynthesis pathway involved in iNKT cell activation.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 08 30;1864(8):1157-1167. Epub 2019 Apr 30.

Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, the Netherlands. Electronic address:

Background: Natural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis.

Results: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell activity.

Conclusion: Glucosylceramide biosynthesis pathway is important for endogenous lipid antigen activation of iNKT cells in adipocytes.

Significance: Unraveling adipocyte-iNKT cell communication may help to fight obesity-induced AT dysfunction. Overproduction and/or accumulation of ceramide and ceramide metabolites, including glucosylceramides, can lead to insulin resistance. However, glucosylceramides also fulfill important physiological functions. They are presented by antigen presenting cells (APC) as endogenous lipid antigens via CD1d to activate a unique lymphocyte subspecies, the CD1d-restricted invariant (i) natural killer T (NKT) cells. Recently, adipocytes have emerged as lipid APC that can activate adipose tissue-resident iNKT cells and thereby contribute to whole body energy homeostasis. Here we investigate the role of the glucosylceramide biosynthesis pathway in the activation of iNKT cells by adipocytes. UDP-glucose ceramide glucosyltransferase (Ugcg), the first rate limiting step in the glucosylceramide biosynthesis pathway, was inhibited via chemical compounds and shRNA knockdown in vivo and in vitro. β-1,4-Galactosyltransferase (B4Galt) 5 and 6, enzymes that convert glucosylceramides into potentially inactive lactosylceramides, were subjected to shRNA knock down. Subsequently, (pre)adipocyte cell lines were tested in co-culture experiments with iNKT cells (IFNγ and IL4 secretion). Inhibition of Ugcg activity shows that it regulates presentation of a considerable fraction of lipid self-antigens in adipocytes. Furthermore, reduced expression levels of either B4Galt5 or -6, indicate that B4Galt5 is dominant in the production of cellular lactosylceramides, but that inhibition of either enzyme results in increased iNKT cell activation. Additionally, in vivo inhibition of Ugcg by the aminosugar AMP-DNM results in decreased iNKT cell effector function in adipose tissue. Inhibition of endogenous glucosylceramide production results in decreased iNKT cells activity and cytokine production, underscoring the role of this biosynthetic pathway in lipid self-antigen presentation by adipocytes.
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http://dx.doi.org/10.1016/j.bbalip.2019.04.016DOI Listing
August 2019

Localization of active endogenous and exogenous β-glucocerebrosidase by correlative light-electron microscopy in human fibroblasts.

Traffic 2019 05;20(5):346-356

Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands.

β-Glucocerebrosidase (GBA) is the enzyme that degrades glucosylceramide in lysosomes. Defects in GBA that result in overall loss of enzymatic activity give rise to the lysosomal storage disorder Gaucher disease, which is characterized by the accumulation of glucosylceramide in tissue macrophages. Gaucher disease is currently treated by infusion of mannose receptor-targeted recombinant GBA. The recombinant GBA is thought to reach the lysosomes of macrophages, based on the impressive clinical response that is observed in Gaucher patients (type 1) receiving this enzyme replacement therapy. In this study, we used cyclophellitol-derived activity-based probes (ABPs) with a fluorescent reporter that irreversibly bind to the catalytic pocket of GBA, to visualize the active enzymes in a correlative microscopy approach. The uptake of pre-labeled recombinant enzyme was monitored by fluorescence and electron microscopy in human fibroblasts that stably expressed the mannose receptor. The endogenous active enzyme was simultaneously visualized by in situ labeling with the ABP containing an orthogonal fluorophore. This method revealed the efficient delivery of recombinant GBA to lysosomal target compartments that contained endogenous active enzyme.
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http://dx.doi.org/10.1111/tra.12641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519279PMC
May 2019

Cytosolic glucosylceramide regulates endolysosomal function in Niemann-Pick type C disease.

Neurobiol Dis 2019 07 12;127:242-252. Epub 2019 Mar 12.

School of Pharmacy, De Montfort University, The Gateway, Leicester LE1 9BH, UK. Electronic address:

Niemann-Pick type C disease (NPCD) is a neurodegenerative disease associated with increases in cellular cholesterol and glycolipids and most commonly caused by defective NPC1, a late endosomal protein. Using ratiometric probes we find that NPCD cells show increased endolysosomal pH. In addition U18666A, an inhibitor of NPC1, was found to increase endolysosomal pH, and the number, size and heterogeneity of endolysosomal vesicles. NPCD fibroblasts and cells treated with U18666A also show disrupted targeting of fluorescent lipid BODIPY-LacCer to high pH vesicles. Inhibiting non-lysosomal glucocerebrosidase (GBA2) reversed increases in endolysosomal pH and restored disrupted BODIPY-LacCer trafficking in NPCD fibroblasts. GBA2 KO cells also show decreased endolysosomal pH. NPCD fibroblasts also show increased expression of a key subunit of the lysosomal proton pump vATPase on GBA2 inhibition. The results are consistent with a model where both endolysosomal pH and Golgi targeting of BODIPY-LacCer are dependent on adequate levels of cytosolic-facing GlcCer, which are reduced in NPC disease.
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http://dx.doi.org/10.1016/j.nbd.2019.03.005DOI Listing
July 2019

Functionalized Cyclophellitols Are Selective Glucocerebrosidase Inhibitors and Induce a Bona Fide Neuropathic Gaucher Model in Zebrafish.

J Am Chem Soc 2019 03 4;141(10):4214-4218. Epub 2019 Mar 4.

Department of Chemistry, York Structural Biology Laboratory , University of York , Heslington, York YO10 5DD , United Kingdom.

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.
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http://dx.doi.org/10.1021/jacs.9b00056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418866PMC
March 2019

Species-specific differences in nonlysosomal glucosylceramidase GBA2 function underlie locomotor dysfunction arising from loss-of-function mutations.

J Biol Chem 2019 03 20;294(11):3853-3871. Epub 2019 Jan 20.

From the Institute of Innate Immunity, University Hospital, University of Bonn, 53127 Bonn, Germany,

The nonlysosomal glucosylceramidase β2 (GBA2) catalyzes the hydrolysis of glucosylceramide to glucose and ceramide. Mutations in the human 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 , 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 .
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http://dx.doi.org/10.1074/jbc.RA118.006311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422076PMC
March 2019

In vivo inactivation of glycosidases by conduritol B epoxide and cyclophellitol as revealed by activity-based protein profiling.

FEBS J 2019 02 2;286(3):584-600. Epub 2019 Feb 2.

Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, The Netherlands.

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 (EC 3.2.1.45), nonlysosomal β-glucocerebrosidase (EC 3.2.1.45); cytosolic β-glucosidase (EC 3.2.1.21); α-glucosidases (EC 3.2.1.20); β-glucuronidase (EC 3.2.1.31).
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http://dx.doi.org/10.1111/febs.14744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850446PMC
February 2019