The molecular defect leading to Fabry disease: structure of human alpha-galactosidase

Fabry disease is an X-linked lysosomal storage disease afflicting 1 in 40,000 males with chronic pain, vascular degeneration, cardiac impairment, and other symptoms. Deficiency in the lysosomal enzyme alpha-galactosidase (alpha-GAL) causes an accumulation of its substrate, which ultimately leads to Fabry disease symptoms. Here, we present the structure of the human alpha-GAL glycoprotein determined by X-ray crystallography. The structure is a homodimer with each monomer containing a (beta/alpha)8 domain with the active site and an antiparallel beta domain. N-linked carbohydrate appears at six sites in the glycoprotein dimer, revealing the basis for lysosomal transport via the mannose-6-phosphate receptor. To understand how the enzyme cleaves galactose from glycoproteins and glycolipids, we also determined the structure of the complex of alpha-GAL with its catalytic product. The catalytic mechanism of the enzyme is revealed by the location of two aspartic acid residues (D170 and D231), which act as a nucleophile and an acid/base, respectively. As a point mutation in alpha-GAL can lead to Fabry disease, we have catalogued and plotted the locations of 245 missense and nonsense mutations in the three-dimensional structure. The structure of human alpha-GAL brings Fabry disease into the realm of molecular diseases, where insights into the structural basis of the disease phenotypes might help guide the clinical treatment of patients.     

Gaucher disease and Fabry disease: New markers and insights in pathophysiology for two distinct glycosphingolipidoses

Gaucher disease (GD) and Fabry disease (FD) are two relatively common inherited glycosphingolipidoses caused by deficiencies in the lysosomal glycosidases glucocerebrosidase and alpha-galactosidase A, respectively. For both diseases enzyme supplementation is presently used as therapy. Cells and tissues of GD and FD patients are uniformly deficient in enzyme activity, but the two diseases markedly differ in cell types showing lysosomal accumulation of the glycosphingolipid substrates glucosylceramide and globotriaosylceramide, respectively. The clinical manifestation of Gaucher disease and Fabry disease is consequently entirely different and the response to enzyme therapy is only impressive in the case of GD patients. This review compares both glycosphingolipid storage disorders with respect to similarities and differences. Presented is an update on insights regarding pathophysiological mechanisms as well as recently available biochemical markers and diagnostic tools for both disorders. Special attention is paid to sphingoid bases of the primary storage lipids in both diseases. The value of elevated glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease for diagnosis and monitoring of disease is discussed as well as the possible contribution of the sphingoid bases to (patho)physiology. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Characterization of gana-1, a Caenorhabditis elegans gene encoding a single ortholog of vertebrate α-galactosidase and α-N-acetylgalactosaminidase

Background  

Human α-galactosidase A (α-GAL) and α-N-acetylgalactosaminidase (α-NAGA) are presumed to share a common ancestor. Deficiencies of these enzymes cause two well-characterized human lysosomal storage disorders (LSD) – Fabry (α-GAL deficiency) and Schindler (α-NAGA deficiency) diseases. Caenorhabditis elegans was previously shown to be a relevant model organism for several late endosomal/lysosomal membrane proteins associated with LSDs. The aim of this study was to identify and characterize C. elegans orthologs to both human lysosomal luminal proteins α-GAL and α-NAGA.     

Sesonal changes in acid glycosidases from gander testes

The goal of this study was to assess the contributions of the most important acid glycosidases to the processes connected with testes involution (in the summer) and spermatogenesis during the reproductive season (the spring) in ganders. Statistically significant increases in the specific activity of N-acetyl-beta-D-hexosaminidase, alpha-D-galactosidase, beta-D-galactosidase, and alpha-L-fucosidase during the period of testes involution were detected. Alpha-D-galactosidase, beta-D-galactosidase, and alpha-D-glucosidase showed an increase in the relative contribution of those multiple forms which are characterized by less acidic values of the pI during the reproductive season. It is suggested that the observed increases in the specific activity of beta-HEX, alpha-GAL, beta-GAL and alpha-FUC may be connected with the catabolism of glycoconjugates, when the spermatogenic activity of the testes declines. The increases in the relative contribution of less acidic forms of alpha-GAL, beta-GAL, and alpha-GLU during the reproductive season may be linked to the rise in the number of spermatocytes, spermatids and spermatozoa during spermatogenesis.     

Potential role of glycosidase inhibitors in industrial biotechnological applications

The nutrient content of food and animal feed may be improved through new knowledge about enzymatic changes in complex carbohydrates. Enzymatic hydrolysis of complex carbohydrates containing alpha or beta glycosidic bonds is very important in nutrition and in several technological processes. These enzymes are called glycosidases (Enzyme Class 3.2.1) and include amylases, pectinases and xylanases. They are present in many foods such as cereals, but their microbial analogues are often produced and added in many food processes, for instance to improve the shelf-life of bakery products, clear beer, produce glucose, fructose or dextrins, hydrolyse lactose, modify food pectins, or improve processes. However, many plant foods also contain endogenous inhibitors, which reduce the activity of glycosidases, in particular, proteins, peptides, complexing agents and phenolic compounds. The plant proteinaceous inhibitors of glycosidases are in focus in this review whose objective is to report the effect and implications of these inhibitors in industrial processes and applications. These studies will contribute to the optimisation of industrial processes by using modified enzymes not influenced by the natural inhibitors. They will also allow careful selection of raw material and reaction conditions, and future development of new genetic varieties low in inhibitors. These are all new and very promising concepts for the food and feed sector.     

Potential physiological role of plant glycosidase inhibitors

Carbohydrate-active enzymes including glycosidases, transglycosidases, glycosyltransferases, polysaccharide lyases and carbohydrate esterases are responsible for the enzymatic processing of carbohydrates in plants. A number of carbohydrate-active enzymes are produced by microbial pathogens and insects responsible of severe crop losses. Plants have evolved proteinaceous inhibitors to modulate the activity of several of these enzymes. The continuing discovery of new inhibitors indicates that this research area is still unexplored and may lead to new exciting developments. To date, the role of the inhibitors is not completely understood. Here we review recent results obtained on the best characterised inhibitors, pointing to their possible biological role in vivo. Results recently obtained with plant transformation technology indicate that this class of inhibitors has potential biotechnological applications.     

Removal of carbohydrate from influenza A virus and its hemagglutinin and the effect on biological activities.

Treatment of influenza virus and its purified hemagglutining with glycosidases from Diplococcus pneumoniae, which included beta-galactosidase, beta-N-acetylglucosminidase, and endoglycosidase D, released amino and neutral sugars from the virus and these as well as large oligosaccharides from the purified hemagglutinin. The released glucosamine-containing oligosaccharides were of one discrete size. Large oligosaccharides not removed by the glycosidases were found on the virus as well as the hemagglutinin. Some oligosaccharides on the virus were inaccessible to the enzymes, since they could be removed only from the purified hemagglutinin. Approximately 50% of the hemagglutinin carbohydrates could be removed without effect on hemagglutinating activity. Similarly, removal of 20 to 25% of the carbohydrates from intact virus particles did not alter infectivity.     

Synthesis and glycosidase inhibitory profiles of functionalised morpholines and oxazepanes

In this work libraries of morpholines and oxazepanes have been prepared via the reductive amination reaction between dialdehydes, derived from carbohydrates, and a range of amines. In this way, functionalised morpholines and oxazepanes have been prepared that include N-alkylated derivatives, disaccharide analogues, and ester containing derivatives. The abilities of these functionalised morpholines and oxazepanes to inhibit a broad panel of glycosidase enzymes, that are associated with a range of diseases, have been probed and in this way new inhibitors of a range of glycosidases, but particularly β-d-galactosidase derived from Bovine kidney, have been discovered. N-Alkyl morpholines demonstrated the best inhibition profiles for this enzyme and derivatives (15a)-(15d) acted as non-competitive inhibitors with IC(50) values of 55.1-88.6 μM. Within this study, some preliminary structure-activity relationships are proposed, and it is demonstrated that N-substituted morpholines display better inhibitory profiles for the enzymes analysed than any of the N-substituted oxazepanes.     

The localization of genes for HPRT, G6PD, and alpha-GAL onto the X-chromosome of domestic pig (Sus scrofa domesticus)

Pig--mouse somatic cell hybrids were obtained from fusion of HPRT--mouse cells (RAG) and pig lymphocytes. The pig-mouse hybrids examined apparently retained on the average only 9 to 15 pig chromosomes. Seven of the hybrid clones were karyotyped to determine the pig chromosome constitution, and the same hybrid clones were tested electrophoretically for the expression of pig hypoxanthine-guanine phosphoribosyltransferase (HPRT), glucose-6-phosphate dehydrogenase (G6PD), and alpha-galactosidase (alpha-GAL) phenotypes. All five of the hybrid clones which had retained the pig X-chromosome exhibited concordant expression of pig HPRT, G6PD, and alpha-GAL enzymes. These data indicate that the genes HPRT, G6PD, and alpha-GAL are located on the X-chromosome of the domestic pig.     

An anti-A-like lectin of Rana catesbiana eggs showing unusual reactivity.

A lectin was isolated from Rana catesbiana eggs that agglutinated blood group A-erythrocytes but did not agglutinate blood group B- or 0-erythrocytes. The lectin was purified by Sephadex G-75 gel filtration and by acrylamide gel electrophoresis at pH 4.3 and was proved to be homogeneous on electrophoresis, and the molecular weight was determined as 210 000. The specificity of A-like activity seems to direct towards three monosaccharide units: GalNAcalpha1 leads to 3(or 4)-Galbeta1 leads to 4(or 3)GlcNAcbeta1 leads to R based on inhibition of A-like hemagglutination by various monosaccharides, oligosaccharides and glycolipids, and based on precipitin reaction with various glycolipids and glycoproteins with known structures. Uniquely, A-like agglutination was inhibited not only by alpha-N-acetylgalactosamine analogs but also by N-acetyllactosamine analogs. The lectin showed therefore, two correlated specificities: one directed towards alpha-N-acetylgalactosamine residue at the terminal, and the other towards the subterminal Galbeta1 leads to 4betaGlcNAc (N-acetyllactosaminyl) residue. The reactivity due to the N-acetyllactosamine structure which is also found in erythrocyte ganglioside and in H-active chain might be blocked by sialyl or alpha-L-fucosyl substitution at the terminal, as the reactivity appeared after elimination of these sugar residues. In the A structure the reactivity due to N-acetyllactosaminyl residue seems not to be blocked by the presence of alpha-N-acetylgalactosamine at the terminal as A-agglutination was strongly inhibited by N-acetyllactosamine and its analogs. Although the lectin showed a single band on electrophoresis under different conditions, there is a possibility that the lectin may be a mixture of two proteins with different specificities as mentioned above.     

Broad-range Glycosidase Activity Profiling

Plants produce hundreds of glycosidases. Despite their importance in cell wall (re)modeling, protein and lipid modification, and metabolite conversion, very little is known of this large class of glycolytic enzymes, partly because of their post-translational regulation and their elusive substrates. Here, we applied activity-based glycosidase profiling using cell-permeable small molecular probes that react covalently with the active site nucleophile of retaining glycosidases in an activity-dependent manner. Using mass spectrometry we detected the active state of dozens of myrosinases, glucosidases, xylosidases, and galactosidases representing seven different retaining glycosidase families. The method is simple and applicable for different organs and different plant species, in living cells and in subproteomes. We display the active state of previously uncharacterized glycosidases, one of which was encoded by a previously declared pseudogene. Interestingly, glycosidase activity profiling also revealed the active state of a diverse range of putative xylosidases, galactosidases, glucanases, and heparanase in the cell wall of Nicotiana benthamiana. Our data illustrate that this powerful approach displays a new and important layer of functional proteomic information on the active state of glycosidases.Carbohydrates are present in all kingdoms of life and are particularly prominent in plants (1). Plants produce carbohydrates as one of their major constituents through their photosynthetic activity. The simplest synthesized forms of carbohydrates are monosaccharide sugars such as glucose, which provides energy for various cellular activities. Carbohydrates also exist in very complex forms. Monosaccharide sugars are attached to one another through covalent glycosidic linkage, which generates di-, oligo-, and polysaccharides. Carbohydrates also attach to non-carbohydrate species (lipids, proteins, hormones) through a glycosidic linkage to form glycoconjugates (2).Glycosidic bonds are hydrolyzed by a group of enzymes termed glycosyl hydrolases (GHs)¹ or glycosidases (3). Because of the tremendous carbohydrate diversity, there are a vast variety of glycosidases, including glucosidases, xylosidases, and galactosidases, that preferentially hydrolyze their respective glycoside substrates. In general, the number of glycosidase-related genes in plants (for instance, Arabidopsis) is relatively high when compared with that in other sequenced organisms (for instance, human) (4). This signifies the unique importance of glycosidases in plants as opposed to other organisms. Based on protein sequence similarities, glycosidases are classified into different GH families. Members of the same GH family share a common mechanism of glycosidic bond cleavage (5).Mechanistically, glycosidases are classified as retaining or inverting enzymes (6). To hydrolyze the glycosidic bond, both retaining and inverting enzymes carry two catalytic glutamate or aspartate residues (or both) (7). Of these two catalytic residues, one acts as a proton donor and the other as a nucleophile/base. The distance between these catalytic residues in the active site of the glycosidases determines the mechanism of hydrolysis. Retaining enzymes have two catalytic residues separated by a distance of ∼5.5 Å, and their hydrolysis mechanism retains the net anomeric configuration of the C1 atom in the sugar molecule. In contrast, inverting enzymes have catalytic residues that are ∼10 Å apart, and these enzymes invert the overall anomeric configuration of the C1 carbon atom in the released sugar (8).Both retaining and inverting glycosidases are present abundantly in plants. The genome of Arabidopsis thaliana encodes for 400 glycosidases, of which 260 are retaining enzymes and 140 are inverting enzymes. Genetic, molecular, and biochemical approaches revealed that glycosidases are localized in different cellular compartments and are important for various biological processes. The majority of plant glycosidases reside in the cell wall, and these enzymes can play major roles in cell wall restructuring (9). Other characterized glycosidases reside in other compartments to regulate glycosylation of proteins and hormones. Despite the importance of GH enzymes, physiological and biochemical functions are assigned to only a few glycosidases (9).Activity-based protein profiling (ABPP) is a powerful tool for monitoring the active state of multiple enzymes without knowledge of their natural substrates (10, 11). ABPP involves chemical probes that react with active site residues in an activity-dependent manner. Thus ABPP displays the availability and reactivity of active site residues in proteins, which are hallmarks for enzyme activity (12). ABPP is particularly attractive because the profiling can be done without purifying the enzymes and can be performed in cell extracts or in living cells. Another key advantage of ABPP is that the activities of large multigene enzyme families can be monitored using broad-range probes. ABPP has had a significant impact on plant science. After the introduction of probes for papain-like cysteine proteases (13, 14), these probes revealed increased protease activities in the tomato and maize apoplasts during immune responses (15, 16) and that these immune proteases are targeted by unrelated inhibitors secreted by fungi, oomycetes, and nematodes (17–24). Likewise, probes for the proteasome displayed unexpected increased proteasome activity during immune responses (25) and revealed that the bacterial effector molecule syringolin A targets the nuclear proteasome (26). We anticipate that more regulatory mechanisms will be discovered through the use of probes introduced for serine hydrolases, metalloproteases, vacuolar processing enzymes, ATP binding proteins, and glutathione transferases (27–32).Cyclophellitol-aziridine-based probes were previously used in animal proteomes to target retaining glucosidases (33). Here we established and applied glycosidase profiling in plants. We discovered that cyclophellitol-aziridine-based probes targeted an unexpectedly broad range of glycosidases representing members of at least seven different GH families. We used these probes to study the active state of glycosidases present in living cells, in different organs and plant species, and in the apoplast of Nicotiana benthamiana.     

The 1.9 Å Structure of Human α-N-Acetylgalactosaminidase: The Molecular Basis of Schindler and Kanzaki Diseases

α-N-acetylgalactosaminidase (α-NAGAL; E.C. 3.2.1.49) is a lysosomal exoglycosidase that cleaves terminal α-N-acetylgalactosamine residues from glycopeptides and glycolipids. In humans, a deficiency of α-NAGAL activity results in the lysosomal storage disorders Schindler disease and Kanzaki disease. To better understand the molecular defects in the diseases, we determined the crystal structure of human α-NAGAL after expressing wild-type and glycosylation-deficient glycoproteins in recombinant insect cell expression systems. We measured the enzymatic parameters of our purified wild-type and mutant enzymes, establishing their enzymatic equivalence. To investigate the binding specificity and catalytic mechanism of the human α-NAGAL enzyme, we determined three crystallographic complexes with different catalytic products bound in the active site of the enzyme. To better understand how individual defects in the α-NAGAL glycoprotein lead to Schindler disease, we analyzed the effect of disease-causing mutations on the three-dimensional structure.     

Enzyme-catalyzed synthesis of carbohydrates

Several new enzymes of utility in the synthesis of carbohydrates have been reported during the past year. Additionally, the utility of several well studied enzymes has been expanded. Pyruvate aldolases, aldolase abzymes and both wild-type and mutated glycosidases have found increasing acceptance in the community. Preliminary reports suggest that thermophilic enzymes may possess significant advantages compared to their mesophilic counterparts for carbohydrate synthesis.     

Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion

Crustaceans are a diverse group, distributed in widely variable environmental conditions for which they show an equally extensive range of biochemical adaptations. Some digestive enzymes have been studied by purification/characterization approaches. However, global analysis is crucial to understand how digestive enzymes interplay. Here, we present the first proteomic analysis of the digestive fluid from a crustacean (Homarus americanus) and identify glycosidases and peptidases as the most abundant classes of hydrolytic enzymes. The digestion pathway of complex carbohydrates was predicted by comparing the lobster enzymes to similar enzymes from other crustaceans. A novel and unbiased substrate profiling approach was used to uncover the global proteolytic specificity of gastric juice and determine the contribution of cysteine and aspartic acid peptidases. These enzymes were separated by gel electrophoresis and their individual substrate specificities uncovered from the resulting gel bands. This new technique is called zymoMSP. Each cysteine peptidase cleaves a set of unique peptide bonds and the S2 pocket determines their substrate specificity. Finally, affinity chromatography was used to enrich for a digestive cathepsin D1 to compare its substrate specificity and cold-adapted enzymatic properties to mammalian enzymes. We conclude that the H. americanus digestive peptidases may have useful therapeutic applications, due to their cold-adaptation properties and ability to hydrolyze collagen.     

Study at the level of the jejunal mucosa of the effects of various carbohydrate compounds on the activity of various enzymes localized in brush borders. Augmentation of phosphatase activity]

On rat jejunum the effects of sorbitol or NaH2PO4 on the activity of various enzymes localized in brush borders was investigated: glycosidases, amino-peptidase, alkaline phosphatase. The activity of phosphatase varies under the influence of these compounds (and others carbohydrates) in the same sense and the same extact than they induce variations of Ca absorption.     

PFIT and PFRIT: bioinformatic algorithms for detecting glycosidase function from structure and sequence

The identification of the enzymes involved in the metabolism of simple and complex carbohydrates presents one bioinformatic challenge in the post-genomic era. Here, we present the PFIT and PFRIT algorithms for identifying those proteins adopting the alpha/beta barrel fold that function as glycosidases. These algorithms are based on the observation that proteins adopting the alpha/beta barrel fold share positions in their tertiary structures having equivalent sets of atomic interactions. These are conserved tertiary interaction positions, which have been implicated in both structure and function. Glycosidases adopting the alpha/beta barrel fold share more conserved tertiary interactions than alpha/beta barrel proteins having other functions. The enrichment pattern of conserved tertiary interactions in the glycosidases is the information that PFIT and PFRIT use to predict whether any given alpha/beta barrel will function as a glycosidase or not. Using as a test set a database of 19 glycosidase and 45 nonglycosidase alpha/beta barrel proteins with low sequence similarity, PFIT and PFRIT can correctly predict glycosidase function for 84% of the proteins known to function as glycosidases. PFIT and PFRIT incorrectly predict glycosidase function for 25% of the nonglycosidases. The program PSI-BLAST can also correctly identify 84% of the 19 glycosidases, however, it incorrectly predicts glycosidase function for 50% of the nonglycosidases (twofold greater than PFIT and PFRIT). Overall, we demonstrate that the structure-based PFIT and PFRIT algorithms are both more selective and sensitive for predicting glycosidase function than the sequence-based PSI-BLAST algorithm.     

Design of new reaction conditions for characterization of a mutant thermophilic α-l-fucosidase

Glycosynthases are mutant glycosidases, which in the presence of activated glycosides and suitable reaction conditions, synthesize oligosaccharides without hydrolysing them. This feature makes these catalysts promising tools for the large scale synthesis of carbohydrates. However, despite the popularity of the glycosynthetic approach, the number of enzymes effecting glycosynthetic reactions is still limited. We report here on the design of novel reaction conditions for a thermophilic α-l-fucosidase mutant, which might provide a route for the production of novel glycosynthases.     

Computational modeling of carbohydrate processing enzymes reactions

Carbohydrate processing enzymes are of biocatalytic interest. Glycoside hydrolases and the recently discovered lytic polysaccharide monooxygenase for their use in biomass degradation to obtain biofuels or valued chemical entities. Glycosyltransferases or engineered glycosidases and phosphorylases for the synthesis of carbohydrates and glycosylated products. Quantum mechanics-molecular mechanics (QM/MM) methods are highly contributing to establish their different chemical reaction mechanisms. Other computational methods are also used to study enzyme conformational changes, ligand pathways, and processivity, e.g. for processive glycosidases like cellobiohydrolases. There is still a long road to travel to fully understand the role of conformational dynamics in enzyme activity and also to disclose the variety of reaction mechanisms these enzymes employ. Additionally, computational tools for enzyme engineering are beginning to be applied to evaluate substrate specificity or aid in the design of new biocatalysts with increased thermostability or tailored activity, a growing field where molecular modeling is finding its way.     

A 2.08 Å resolution structure of HLB5, a novel cellulase from the anaerobic gut bacterium Parabacteroides johnsonii DSM 18315

Cellulases play a significant role in the degradation of complex carbohydrates. In the human gut, anaerobic bacteria are essential to the well‐being of the host by producing these essential enzymes that convert plant polymers into simple sugars that can then be further metabolized by the host. Here, we report the 2.08 Å resolution structure of HLB5, a chemically verified cellulase that was identified previously from an anaerobic gut bacterium and that has no structural cellulase homologues in PDB nor possesses any conserved region typical for glycosidases. We anticipate that the information presented here will facilitate the identification of additional cellulases for which no homologues have been identified to date and enhance our understanding how these novel cellulases bind and hydrolyze their substrates.