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

Abstract

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...

Glucocerebrosidase: Functions in and Beyond the Lysosome

“…This reaction is catalyzed by GCase, a 495 amino acid glycoprotein with four N-linked glycans [2,44]. GCase, based on its structural features, is classified in the glycoside hydrolase family GH30 (formerly in the related family GH5) [45]. The 3-D structure of GCase was resolved by crystallography [46,47].…”

“…GCase, like other GH5 and GH30 glycosidases, has an (α/β) 8 TIM barrel catalytic domain. In the case of GCase this is fused with a β-structure consisting of an immunoglobulin-like fold [45]. GCase is a retaining β-glucosidase hydrolyzing a glucosidic substrate with net retention of glucose stereochemistry ( Figure 1B).…”

CLN3 deficiency leads to neurological and metabolic perturbations during early development

“…Accordingly, BMP seems to play a key role in lipid metabolism and membrane composition by facilitating the adhesion of soluble, positively charged hydrolases and activator proteins such as sphingolipid activator proteins SapsA-D and the GM2 activator protein to intracellular membranes (Gallala and Sandhoff 2011). It is therefore possible that the BMP depletion in CLN3 deficient cells also negatively affects the function of glucocerebrosidases, which may then possibly explain the increased levels of glycosyl-ceramide and acyl steryl glycoside species that we observed in MUT1 larvae (Ben Bdira et al 2018). Moreover, BMPs have a cone-like shape that affects membrane curvature, playing an important role for endosomal vesicularisation and hence also in the maturation of late endosomes (Gallala and Sandhoff 2011).…”

Plant Glycosides and Glycosidases: A Treasure-Trove for Therapeutics

“…These enzymes play a variety of functions, including the lysosomal metabolism of glycolipids in animals, the catabolism of cell wall polysaccharides in plants and biomass conversion in microorganisms (Leah et al, 1995;Ketudat Cairns and Esen, 2010). More than 160 GH families have been listed in the Carbohydrate Active EnZymes (CAZy) database using a classification system based on amino acid sequence and secondary structure similarities (Henrissat, 1991;Ben Bdira et al, 2018). This classification system is regularly updated and new families are continuously discovered.…”