A Sensitive Gel-based Method Combining Distinct Cyclophellitol-based Probes for the Identification of Acid/Base Residues in Human Retaining β-Glucosidases

Kallemeijn, Wouter W.; Witte, Martin D.; Voorn-Brouwer, Tineke; Walvoort, Marthe T. C.; Li, Kah‐Yee; Codée, Jeroen D. C.; Marel, Gijsbert A. van der; Boot, Rolf G.; Overkleeft, Herman S.; Aerts, Johannes M. F. G.

doi:10.1074/jbc.m114.593376

Cited by 21 publications

(20 citation statements)

References 46 publications

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“…The second class of ABPs consists of β-aziridine cyclophellitol with a linked fluorescent BODIDY group. Contrary to the cyclophellitol β-epoxide ABPs, the β-aziridine cyclophellitol-type structures label the entire class of retaining β-glucosidases, including GBA, GBA2, GBA3, and lactase/phoridzin hydrolase (LPH) [ 23 , 27 ]. This broad-specific ABP is here referred to as Anybody.…”

Section: Introductionmentioning

confidence: 99%

Visualization of Active Glucocerebrosidase in Rodent Brain with High Spatial Resolution following In Situ Labeling with Fluorescent Activity Based Probes

et al. 2015

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Gaucher disease is characterized by lysosomal accumulation of glucosylceramide due to deficient activity of lysosomal glucocerebrosidase (GBA). In cells, glucosylceramide is also degraded outside lysosomes by the enzyme glucosylceramidase 2 (GBA2) of which inherited deficiency is associated with ataxias. The interest in GBA and glucosylceramide metabolism in the brain has grown following the notion that mutations in the GBA gene impose a risk factor for motor disorders such as α-synucleinopathies. We earlier developed a β-glucopyranosyl-configured cyclophellitol-epoxide type activity based probe (ABP) allowing in vivo and in vitro visualization of active molecules of GBA with high spatial resolution. Labeling occurs through covalent linkage of the ABP to the catalytic nucleophile residue in the enzyme pocket. Here, we describe a method to visualize active GBA molecules in rat brain slices using in vivo labeling. Brain areas related to motor control, like the basal ganglia and motor related structures in the brainstem, show a high content of active GBA. We also developed a β-glucopyranosyl cyclophellitol-aziridine ABP allowing in situ labeling of GBA2. Labeled GBA2 in brain areas can be identified and quantified upon gel electrophoresis. The distribution of active GBA2 markedly differs from that of GBA, being highest in the cerebellar cortex. The histological findings with ABP labeling were confirmed by biochemical analysis of isolated brain areas. In conclusion, ABPs offer sensitive tools to visualize active GBA and to study the distribution of GBA2 in the brain and thus may find application to establish the role of these enzymes in neurodegenerative disease conditions such as α-synucleinopathies and cerebellar ataxia.

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Section: Introductionmentioning

confidence: 99%

Visualization of Active Glucocerebrosidase in Rodent Brain with High Spatial Resolution following In Situ Labeling with Fluorescent Activity Based Probes

et al. 2015

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“…The GH116 family comprises enzymes with diverse specificities and includes the glucosylceramidase GBA2 (55). The partial homology with -xylosidase/-glucosidase SSO1353 of Sulfolobus solfataricus assisted the identification of E527 as the nucleophile and D677 as the acid/base in GBA2 (55,56). At present, no 3D structure of GBA2 is available, but recently such a structure was reported for a GH116 -glucosidase of Thermoanaerobacterium xylanolyticum (TxGH116), alone and in complex with diverse ligands (57).…”

Section: Glycosylceramidases Of Family Gh116mentioning

confidence: 99%

“…These patients produce mutant GBA1 molecules that often are inefficiently folded in the ER and/or show reduced stability in lysosomes (17). IFG shows a higher binding affinity to GBA1 at neutral pH (as in the ER) compared with acidic pH (as in lysosomes) (56). IFG was envisioned to assist the folding and/or stability of (mutant) GBA1 and, because it crosses the blood-brain barrier, to have the potential to improve neuropathology in severely affected Gaucher disease patients.…”

Section: Therapeutic Noncovalent Inhibitorsmentioning

confidence: 99%

Distinguishing the differences in β-glycosylceramidase folds, dynamics, and actions informs therapeutic uses

Bdira

Artola

Overkleeft

et al. 2018

Journal of Lipid Research

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Glycoconjugates play essential roles in diverse biological processes and abnormalities and have been linked to multiple pathologies. The staggering structural diversity of glycoconjugates stems from the almost infinite possible combinations by which multiple monosaccharide building blocks may be linked with each other (1). For instance, 1,056 unique trisaccharides can be formed just from 3 different monosaccharides. Polysaccharides are very stable compounds: their spontaneous hydrolysis takes place at a rate of 10 15 s 1 , corresponding to a half-life of 4.7 million years (2). To allow for the efficient metabolism of glycoconjugates, enzymes have evolved as specialized catalysts. In most organisms, an estimated 1% to 3% of the genes encode carbohydrate-active enzymes (3). Among these are glycoside hydrolases (GHs) that can enhance the rate of hydrolysis of specific carbohydrate glycosidic bonds in glycoconjugates more than 10 17 times (2). These GH enzymes show marked specificity regarding number, position, and configuration of the hydroxyl groups in their substrate sugar. They are widely applied in biotechnology, for example, in biofuel production, paper pulp bleaching, and the food industry (4, 5). Likewise, specific GH inhibitors are extensively used as agrochemicals and therapeutic agents (6-8). Abnormalities in GHs underlie metabolic disorders in humans, for instance, inherited lysosomal storage disorders and lactose intolerance (9, 10). GH enzymes differ in substrate specificity, mode of enzymatic attack Abstract Glycosyl hydrolases (GHs) are carbohydrate-active enzymes that hydrolyze a specific -glycosidic bond in glycoconjugate substrates; -glucosidases degrade glucosylceramide, a ubiquitous glycosphingolipid. GHs are grouped into structurally similar families that themselves can be grouped into clans. GH1, GH5, and GH30 glycosidases belong to clan A hydrolases with a catalytic (/) 8 TIM barrel domain, whereas GH116 belongs to clan O with a catalytic (/) 6 domain. In humans, GH abnormalities underlie metabolic diseases. The lysosomal enzyme glucocerebrosidase (family GH30), deficient in Gaucher disease and implicated in Parkinson disease etiology, and the cytosol-facing membrane-bound glucosylceramidase (family GH116) remove the terminal glucose from the ceramide lipid moiety. Here, we compare enzyme differences in fold, action, dynamics, and catalytic domain stabilization by binding site occupancy. We also explore other glycosidases with reported glycosylceramidase activity, including human cytosolic -glucosidase, intestinal lactase-phlorizin hydrolase, and lysosomal galactosylceramidase. Last, we describe the successful translation of research to practice: recombinant glycosidases and glucosylceramide metabolism modulators are approved drug products (enzyme replacement therapies). Activity-based probes now facilitate the diagnosis of enzyme deficiency and screening for compounds that interact with the catalytic pocket of glycosidases. Future research may deepen the understanding of the function...

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“…Computational studies provided insight into the potent activity of analogues bearing the substituted guanidine moiety in the inhibition of lysosomal glucocerebrosidase (GBA).Creating potent and selective glycosidase inhibitors is an important goal in medicinal chemistry [1] due to their therapeutic potentiali nt he treatment of av ariety of carbohydrate-mediated diseases. [2][3][4][5][6][7][8][9][10][11][12] In this respect, iminosugars are privileged lead compounds because of their complementarity to glycosidase active sites and aspects of the relevant transition states in the hydrolysisp rocesses catalyzed by glycosidases. [13] Glycomimetics that comprise an endocyclic nitrogen, such as the naturally occurring 1-deoxynojirimycin( DNJ, 1,F igure 1) as well as 1,5dideoxy-1,5-imino-d-xylitol (DIX, 4)a nd their closely related un-naturalr elative isofagomine (IFG, 6), are of particulari nterest [14,15] and an umber of syntheses of these compounds have been reported.…”

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confidence: 99%

N‐Guanidino Derivatives of 1,5‐Dideoxy‐1,5‐imino‐d‐xylitol are Potent, Selective, and Stable Inhibitors of β‐Glucocerebrosidase

et al. 2017

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A series of lipidated guanidino and urea derivatives of 1,5-dideoxy-1,5-imino-d-xylitol were prepared from d-xylose using a concise synthetic protocol. Inhibition assays with a panel of glycosidases revealed that the guanidino analogues display potent inhibition against human recombinant β-glucocerebrosidase with IC values in the low nanomolar range. Related urea analogues of 1,5-dideoxy-1,5-imino-d-xylitol were also synthesized and evaluated in the same fashion and found to be selective for β-galactosidase from bovine liver. No inhibition of human recombinant β-glucocerebrosidase was observed for the urea analogues. Computational studies provided insight into the potent activity of analogues bearing the substituted guanidine moiety in the inhibition of lysosomal glucocerebrosidase (GBA).

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A Sensitive Gel-based Method Combining Distinct Cyclophellitol-based Probes for the Identification of Acid/Base Residues in Human Retaining β-Glucosidases

Cited by 21 publications

References 46 publications

Visualization of Active Glucocerebrosidase in Rodent Brain with High Spatial Resolution following In Situ Labeling with Fluorescent Activity Based Probes

Visualization of Active Glucocerebrosidase in Rodent Brain with High Spatial Resolution following In Situ Labeling with Fluorescent Activity Based Probes

Distinguishing the differences in β-glycosylceramidase folds, dynamics, and actions informs therapeutic uses

N‐Guanidino Derivatives of 1,5‐Dideoxy‐1,5‐imino‐d‐xylitol are Potent, Selective, and Stable Inhibitors of β‐Glucocerebrosidase

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