Towards Splicing Therapy for Lysosomal Storage Disorders: Methylxanthines and Luteolin Ameliorate Splicing Defects in Aspartylglucosaminuria and Classic Late Infantile Neuronal Ceroid Lipofuscinosis.

Splicing defects caused by mutations in the consensus sequences at the borders of introns and exons are common in human diseases. Such defects frequently result in a complete loss of function of the protein in question. Therapy approaches based on antisense oligonucleotides for specific gene mutations have been developed in the past, but they are very expensive and require invasive, life-long administration. Thus, modulation of splicing by means of small molecules is of great interest for the therapy of genetic diseases resulting from splice-site mutations. Using minigene approaches and patient cells, we here show that methylxanthine derivatives and the food-derived flavonoid luteolin are able to enhance the correct splicing of the AGA mRNA with a splice-site mutation c.128-2A>G in aspartylglucosaminuria, and result in increased AGA enzyme activity in patient cells. Furthermore, we also show that one of the most common disease causing TPP1 gene variants in classic late infantile neuronal ceroid lipofuscinosis may also be amenable to splicing modulation using similar substances. Therefore, our data suggest that splice-modulation with small molecules may be a valid therapy option for lysosomal storage disorders.

The T99K variant of glycosylasparaginase shows a new structural mechanism of the genetic disease aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is an inherited disease caused by mutations in a lysosomal amidase called aspartylglucosaminidase (AGA) or glycosylasparaginase (GA). This disorder results in an accumulation of glycoasparagines in the lysosomes of virtually all cell types, with severe clinical symptoms affecting the central nervous system, skeletal abnormalities, and connective tissue lesions. GA is synthesized as a single-chain precursor that requires an intramolecular autoprocessing to form a mature amidase. Previously, we showed that a Canadian AGU mutation disrupts this obligatory intramolecular autoprocessing with the enzyme trapped as an inactive precursor. Here, we report biochemical and structural characterization of a model enzyme corresponding to a new American AGU allele, the T99K variant. Unlike other variants with known 3D structures, this T99K model enzyme still has autoprocessing capacity to generate a mature form. However, its amidase activity to digest glycoasparagines remains low, consistent with its association with AGU. We have determined a 1.5-Å-resolution structure of this new AGU model enzyme and built an enzyme-substrate complex to provide a structural basis to analyze the negative effects of the T99K point mutation on KM and kcat of the amidase. It appears that a "molecular clamp" capable of fixing local disorders at the dimer interface might be able to rescue the deficiency of this new AGU variant.

Biochemical and structural insights into an allelic variant causing the lysosomal storage disorder - aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by defects of the hydrolase glycosylasparaginase (GA). Previously, we showed that a Canadian AGU mutation disrupts an obligatory intramolecular autoprocessing with the enzyme trapped as an inactive precursor. Here, we report biochemical and structural characterizations of a model enzyme corresponding to a Finnish AGU allele, the T234I variant. Unlike the Canadian counterpart, the Finnish variant is capable of a slow autoprocessing to generate detectible hydrolyzation activity of the natural substrate of GA. We have determined a 1.6 Å-resolution structure of the Finnish AGU model and built an enzyme-substrate complex to provide a structural basis for analyzing the negative effects of the point mutation on KM and kcat of the mature enzyme. ENZYME: Glycosylasparaginase or aspartylglucosaminidase, EC3.5.1.26.

Biochemical characterization and comparison of aspartylglucosaminidases secreted in venom of the parasitoid wasps Asobara tabida and Leptopilina heterotoma.

Aspartylglucosaminidase (AGA) is a low-abundance intracellular enzyme that plays a key role in the last stage of glycoproteins degradation, and whose deficiency leads to human aspartylglucosaminuria, a lysosomal storage disease. Surprisingly, high amounts of AGA-like proteins are secreted in the venom of two phylogenetically distant hymenopteran parasitoid wasp species, Asobara tabida (Braconidae) and Leptopilina heterotoma (Cynipidae). These venom AGAs have a similar domain organization as mammalian AGAs. They share with them key residues for autocatalysis and activity, and the mature α- and ß-subunits also form an (αß)2 structure in solution. Interestingly, only one of these AGAs subunits (α for AtAGA and ß for LhAGA) is glycosylated instead of the two subunits for lysosomal human AGA (hAGA), and these glycosylations are partially resistant to PGNase F treatment. The two venom AGAs are secreted as fully activated enzymes, they have a similar aspartylglucosaminidase activity and are both also efficient asparaginases. Once AGAs are injected into the larvae of the Drosophila melanogaster host, the asparaginase activity may play a role in modulating their physiology. Altogether, our data provide new elements for a better understanding of the secretion and the role of venom AGAs as virulence factors in the parasitoid wasps' success.

Crystal structure of a mutant glycosylasparaginase shedding light on aspartylglycosaminuria-causing mechanism as well as on hydrolysis of non-chitobiose substrate.

Glycosylasparaginase (GA) is an amidase that cleaves Asn-linked glycoproteins in lysosomes. Deficiency of this enzyme causes accumulation of glycoasparagines in lysosomes of cells, resulting in a genetic condition called aspartylglycosaminuria (AGU). To better understand the mechanism of a disease-causing mutation with a single residue change from a glycine to an aspartic acid, we generated a model mutant enzyme at the corresponding position (named G172D mutant). Here we report a 1.8Å resolution crystal structure of mature G172D mutant and analyzed the reason behind its low hydrolase activity. Comparison of mature G172D and wildtype GA models reveals that the presence of Asp 172 near the catalytic site affects substrate catabolism in mature G172D, making it less efficient in substrate processing. Also recent studies suggest that GA is capable of processing substrates that lack a chitobiose (Glycan, N-acetylchiobios, NAcGlc) moiety, by its exo-hydrolase activity. The mechanism for this type of catalysis is not yet clear. l-Aspartic acid ß-hydroxamate (ß-AHA) is a non-chitobiose substrate that is known to interact with GA. To study the underlying mechanism of non-chitobiose substrate processing, we built a GA-ß-AHA complex structure by comparing to a previously published G172D mutant precursor in complex with a ß-AHA molecule. A hydrolysis mechanism of ß-AHA by GA is proposed based on this complex model.

Molecular characterization of aspartylglucosaminidase, a lysosomal hydrolase upregulated during strobilation in the moon jellyfish, Aurelia aurita.

The life cycle of the moon jellyfish, Aurelia aurita, alternates between a benthic asexual polyp stage and a planktonic sexual medusa (jellyfish) stage. Transition from polyp to medusa is called strobilation. To investigate the molecular mechanisms of strobilation, we screened for genes that are upregulated during strobilation using the differential display method and we identified aspartylglucosaminidase (AGA), which encodes a lysosomal hydrolase. Similar to AGAs from other species, Aurelia AGA possessed an N-terminal signal peptide and potential N-glycosylation sites. The genomic region of Aurelia AGA was approximately 9.8 kb in length and contained 12 exons and 11 introns. Quantitative RT-PCR analysis revealed that AGA expression increased during strobilation, and was then decreased in medusae. To inhibit AGA function, we administered the lysosomal acidification inhibitors, chloroquine or bafilomycin A1, to animals during strobilation. Both inhibitors disturbed medusa morphogenesis at the oral end, suggesting involvement of lysosomal hydrolases in strobilation.

Functional Analysis of the Ser149/Thr149 Variants of Human Aspartylglucosaminidase and Optimization of the Coding Sequence for Protein Production.

Aspartylglucosaminidase (AGA) is a lysosomal hydrolase that participates in the breakdown of glycoproteins. Defects in the AGA gene result in a lysosomal storage disorder, aspartylglucosaminuria (AGU), that manifests mainly as progressive mental retardation. A number of AGU missense mutations have been identified that result in reduced AGA activity. Human variants that contain either Ser or Thr in position 149 have been described, but it is unknown if this affects AGA processing or activity. Here, we have directly compared the Ser149/Thr149 variants of AGA and show that they do not differ in terms of relative specific activity or processing. Therefore, Thr149 AGA, which is the rare variant, can be considered as a neutral or benign variant. Furthermore, we have here produced codon-optimized versions of these two variants and show that they are expressed at significantly higher levels than AGA with the natural codon-usage. Since optimal AGA expression is of vital importance for both gene therapy and enzyme replacement, our data suggest that use of codon-optimized AGA may be beneficial for these therapy options.

Identification of Small Molecule Compounds for Pharmacological Chaperone Therapy of Aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder that is caused by genetic deficiency of the enzyme aspartylglucosaminidase (AGA) which is involved in glycoprotein degradation. AGU is a progressive disorder that results in severe mental retardation in early adulthood. No curative therapy is currently available for AGU. We have here characterized the consequences of a novel AGU mutation that results in Thr122Lys exchange in AGA, and compared this mutant form to one carrying the worldwide most common AGU mutation, AGU-Fin. We show that T122K mutated AGA is expressed in normal amounts and localized in lysosomes, but exhibits low AGA activity due to impaired processing of the precursor molecule into subunits. Coexpression of T122K with wildtype AGA results in processing of the precursor into subunits, implicating that the mutation causes a local misfolding that prevents the precursor from becoming processed. Similar data were obtained for the AGU-Fin mutant polypeptide. We have here also identified small chemical compounds that function as chemical or pharmacological chaperones for the mutant AGA. Treatment of patient fibroblasts with these compounds results in increased AGA activity and processing, implicating that these substances may be suitable for chaperone mediated therapy for AGU.

Crystallographic snapshot of glycosylasparaginase precursor poised for autoprocessing.

Glycosylasparaginase belongs to a family of N-terminal nucleophile hydrolases that autoproteolytically generate their mature enzymes from single-chain protein precursors. Previously, based on a precursor structure paused at pre-autoproteolysis stage by a reversible inhibitor (glycine), we proposed a mechanism of intramolecular autoproteolysis. A key structural feature, a highly strained conformation at the scissile peptide bond, had been identified and was hypothesized to be critical for driving autoproteolysis through an N-O acyl shift. To examine this "twist-and-break" hypothesis, we report here a 1. 9-Å-resolution structure of an autoproteolysis-active precursor (a T152C mutant) that is free of inhibitor or ligand and is poised to undergo autoproteolysis. The current crystallographic study has provided direct evidence for the natural conformation of the glycosylasparaginase autocatalytic site without influence from any inhibitor or ligand. This finding has confirmed our previous proposal that conformational strain is an intrinsic feature of an active precursor.

Knowledge-based computational mutagenesis for predicting the disease potential of human non-synonymous single nucleotide polymorphisms.

Certain genetic variations in the human population are associated with heritable diseases, and single nucleotide polymorphisms (SNPs) represent the most common form of such differences in DNA sequence. In particular, substantial interest exists in determining whether a non-synonymous SNP (nsSNP), leading to a single residue replacement in the translated protein product, is neutral or disease-related. The nature of protein structure-function relationships suggests that nsSNP effects, either benign or leading to aberrant protein function possibly associated with disease, are dependent on relative structural changes introduced upon mutation. In this study, we characterize a representative sampling of 1790 documented neutral and disease-related human nsSNPs mapped to 243 diverse human protein structures, by quantifying environmental perturbations in the associated proteins with the use of a computational mutagenesis methodology that relies on a four-body, knowledge-based, statistical contact potential. These structural change data are used as attributes to generate a vector representation for each nsSNP, in combination with additional features reflecting sequence and structure of the corresponding protein. A trained model based on the random forest supervised classification algorithm achieves 76% cross-validation accuracy. Our classifier performs at least as well as other methods that use significantly larger datasets of nsSNPs for model training, and the novelty of our attributes differentiates the model as an orthogonal approach that can be utilized in conjunction with other techniques. A dedicated server for obtaining predictions, as well as supporting datasets and documentation, is available at http://proteins.gmu.edu/automute.

Molecular and biochemical analysis of an aspartylglucosaminidase from the venom of the parasitoid wasp Asobara tabida (Hymenoptera: Braconidae).

The most abundant venom protein of the parasitoid wasp Asobara tabida was identified to be an aspartylglucosaminidase (hereafter named AtAGA). The aim of the present work is the identification of: 1) its cDNA and deduced amino acid sequences, 2) its subunits organization and 3) its activity. The cDNA of AtAGA coded for a proalphabeta precursor molecule preceded by a signal peptide of 19 amino acids. The gene products were detected specifically in the wasp venom gland (in which it could be found) under two forms: an (active) heterotetramer composed of two alpha and two beta subunits of 30 and 18 kDa respectively and a homodimer of 44 kDa precursor. The activity of AtAGA enzyme showed a limited tolerance toward variations of pH and temperatures. Since the enzyme failed to exhibit any glycopeptide N-glycosidase activity toward entire glycoproteins, its activity seemed to be restricted to the deglycosylation of free glycosylasparagines like human AGA, indicating AtAGA did not evolve a broader function in the course of evolution. The study of this enzyme may allow a better understanding of the functional evolution of venom enzymes in hymenopteran parasitoids.

Structural basis of aspartylglucosaminuria.

To elucidate the basis of aspartylglucosaminuria (AGU) from the viewpoint of enzyme structure, we constructed structural models of mutant aspartylglucosaminidase (AGA) proteins using molecular modeling software, TINKER. We classified the amino acid substitutions responsible for AGU and divided them into three groups based on the biochemical phenotype. Then, we examined the structural changes in the AGA protein for each group by calculating the solvent-accessible surface area (ASA), the number of atoms affected, and the root-mean-square deviation (RMSD). Our results revealed that the structural changes in group 1, which exhibits folding/transport defects and a complete deficiency of AGA activity, were generally large and located in the core region of the enzyme molecule. In group 2, exhibiting the mature AGA protein but no AGA activity, the functionally important region of the enzyme molecule was seriously affected. In group 3 exhibiting residual AGA activity, the structural changes in AGA were small and localized near the surface of the enzyme molecule. Coloring of affected atoms based on the distances between the wild-type and mutant ones revealed the characteristic structural changes in the AGA protein geographically and semi-quantitatively. Structural investigation provides us with a deeper insight into the basis of AGU.

Crystallographic snapshot of a productive glycosylasparaginase-substrate complex.

Glycosylasparaginase (GA) plays an important role in asparagine-linked glycoprotein degradation. A deficiency in the activity of human GA leads to a lysosomal storage disease named aspartylglycosaminuria. GA belongs to a superfamily of N-terminal nucleophile hydrolases that autoproteolytically generate their mature enzymes from inactive single chain protein precursors. The side-chain of the newly exposed N-terminal residue then acts as a nucleophile during substrate hydrolysis. By taking advantage of mutant enzyme of Flavobacterium meningosepticum GA with reduced enzymatic activity, we have obtained a crystallographic snapshot of a productive complex with its substrate (NAcGlc-Asn), at 2.0 A resolution. This complex structure provided us an excellent model for the Michaelis complex to examine the specific contacts critical for substrate binding and catalysis. Substrate binding induces a conformational change near the active site of GA. To initiate catalysis, the side-chain of the N-terminal Thr152 is polarized by the free alpha-amino group on the same residue, mediated by the side-chain hydroxyl group of Thr170. Cleavage of the amide bond is then accomplished by a nucleophilic attack at the carbonyl carbon of the amide linkage in the substrate, leading to the formation of an acyl-enzyme intermediate through a negatively charged tetrahedral transition state.

Expression, purification and catalytic activity of Lupinus luteus asparagine beta-amidohydrolase and its Escherichia coli homolog.

We describe the expression, purification, and biochemical characterization of two homologous enzymes, with amidohydrolase activities, of plant (Lupinus luteus potassium-independent asparaginase, LlA) and bacterial (Escherichia coli, ybiK/spt/iaaA gene product, EcAIII) origin. Both enzymes were expressed in E. coli cells, with (LlA) or without (EcAIII) a His-tag sequence. The proteins were purified, yielding 6 or 30 mg.L(-1) of culture, respectively. The enzymes are heat-stable up to 60 degrees C and show both isoaspartyl dipeptidase and l-asparaginase activities. Kinetic parameters for both enzymatic reactions have been determined, showing that the isoaspartyl peptidase activity is the dominating one. Despite sequence similarity to aspartylglucosaminidases, no aspartylglucosaminidase activity could be detected. Phylogenetic analysis demonstrated the relationship of these proteins to other asparaginases and aspartylglucosaminidases and suggested their classification as N-terminal nucleophile hydrolases. This is consistent with the observed autocatalytic breakdown of the immature proteins into two subunits, with liberation of an N-terminal threonine as a potential catalytic residue.

Structure of the isoaspartyl peptidase with L-asparaginase activity from Escherichia coli.

The crystal structure of the Escherichia coli enzyme (EcAIII) with isoaspartyl dipeptidase and L-asparaginase activity has been solved and refined to a resolution of 1.65 angstroms, with crystallographic R-factor and Rfree values of 0.178 and 0.209, respectively. EcAIII belongs to the family of N-terminal hydrolases. The amino-acid sequence of EcAIII is homologous to those of putative asparaginases from plants. The structure of EcAIII is similar to the structures of glycosylasparaginases. The mature and catalytically active form of EcAIII is a heterotetramer consisting of two alpha-subunits and two beta-subunits. Both of the equivalent active sites present in the EcAIII tetramer is assisted by a metal-binding site. The metal cations, modelled here as Na+, have not previously been observed in glycosylasparaginases. This reported structure helps to explain the inability of EcAIII and other plant-type asparaginases to hydrolyze N4-(beta-N-acetylglucosaminyl)-L-asparagine, the substrate of glycosylasparaginases.

Autoproteolytic activation of human aspartylglucosaminidase.

Aspartylglucosaminidase (AGA) belongs to the N-terminal nucleophile (Ntn) hydrolase superfamily characterized by an N-terminal nucleophile as the catalytic residue. Three-dimensional structures of the Ntn hydrolases reveal a common folding pattern and equivalent stereochemistry at the active site. The activation of the precursor polypeptide occurs autocatalytically, and for some amidohydrolases of prokaryotes, the precursor structure is known and activation mechanisms are suggested. In humans, the deficient AGA activity results in a lysosomal storage disease, aspartylglucosaminuria (AGU) resulting in progressive neurodegeneration. Most of the disease-causing mutations lead to defective molecular maturation of AGA, and, to understand the structure-function relationship better, in the present study, we have analysed the effects of targeted amino acid substitutions on the activation process of human AGA. We have evaluated the effect of the previously published mutations and, in addition, nine novel mutations were generated. We could identify one novel amino acid, Gly258, with an important structural role on the autocatalytic activation of human AGA, and present the molecular mechanism for the autoproteolytic activation of the eukaryotic enzyme. Based on the results of the present study, and by comparing the available information on the activation of the Ntn-hydrolases, the autocatalytic processes of the prokaryotic and eukaryotic enzymes share common features. First, the critical nucleophile functions both as the catalytic and autocatalytic residue; secondly, the side chain of this nucleophile is oriented towards the scissile peptide bond; thirdly, conformational strain exists in the precursor at the cleavage site; finally, water molecules are utilized in the activation process.

A dual role for an aspartic acid in glycosylasparaginase autoproteolysis.

Glycosylasparaginase uses an autoproteolytic processing mechanism, through an N-O acyl shift, to generate a mature/active enzyme from a single-chain precursor. Structures of glycosylasparaginase precursors in complex with a glycine inhibitor have revealed the backbone in the immediate vicinity of the scissile peptide bond to be in a distorted trans conformation, which is believed to be the driving force for the N-O acyl shift to break the peptide bond. Here we report the effects of point mutation D151N. In addition to the loss of the base essential in autoproteolysis, this mutation also eradicates the backbone distortion near the scissile peptide bond. Binding of the glycine inhibitor to the autoproteolytic site of the D151N mutant does not restore the backbone distortion. Therefore, Asp151 plays a dual role, acting as the general base to activate the nucleophile and holding the distorted trans conformation that is critical for initiating an N-O acyl shift.

Acylation is rate-limiting in glycosylasparaginase-catalyzed hydrolysis of N4-(4'-substituted phenyl)-L-asparagines.

Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond between N-acetyl-D-glucosamine and L-asparagine in the catabolism of glycoproteins. The mechanism has been proposed to resemble that of serine proteases involving an acylation step where a nucleophilic attack by a catalytic Thr residue on the carbonyl carbon of the N-glycosylic bond gives rise to a covalent beta-aspartyl-enzyme intermediate, and a deacylation step to give the final products. The question posed in this study was: Is the acylation step the rate-limiting step in the hydrolysis reaction as in serine proteases? To answer this question a series of mostly new substituted anilides was synthesized and characterized, and their hydrolysis reactions catalyzed by glycosylasparaginase from human amniotic fluid were studied. Five N4-(4'-substituted phenyl)-L-asparagine compounds were synthesized and characterized: 4'-hydrogen, 4'-ethyl, 4'-bromo, 4'-nitro, and 4'-methoxy. Each of these anilides was a substrate for the enzyme. Hammett plots of the kinetic parameters showed that acylation is the rate-limiting step in the reaction and that upon binding the electron distribution of the substrate is perturbed toward the transition state. This is the first direct evidence that acylation is the rate-limiting step in the enzyme-catalyzed reaction. A Brønsted plot indicates a small, negative charge (-0.25) on the nitrogen atom of the leaving group anilines containing electron-withdrawing groups, and a small, positive charge (0.43) on the nitrogen atom of the leaving group anilines containing electron-donating groups. The free energy (incremental) change of binding (delta deltaGb) in the enzyme-substrate transition state complexes shows that substitution of a substituted phenyl group for the pyranosyl group in the natural substrate results in an overall loss of binding energy equivalent to a weak hydrogen bond, the magnitude of which is dependent on the substituent group. The data are consistent with a mechanism for glycosylasparaginase involving rapid formation of a tetrahedral structure upon substrate binding, and a rate-limiting breakdown of the tetrahedral structure to a covalent beta-aspartyl-enzyme intermediate that is dependent on the electronic properties of the substituent group and on the degree of protonation of the leaving group in the transition state by a general acid.

Two-step dimerization for autoproteolysis to activate glycosylasparaginase.

Glycosylasparaginase (GA) is an amidase and belongs to a novel family of N-terminal nucleophile hydrolases that use a similar autoproteolytic processing mechanism to generate a mature/active enzyme from a single chain protein precursor. From bacteria to eukaryotes, GAs are conserved in primary sequences, tertiary structures, and activation of amidase activity by intramolecular autoproteolysis. An evolutionarily conserved His-Asp-Thr sequence is cleaved to generate a newly exposed N-terminal threonine, which plays a central role in both autoproteolysis and in its amidase activity. We have recently determined the crystal structure of the bacterial GA precursor at 1.9-A resolution, which reveals a highly distorted and energetically unfavorable conformation at the scissile peptide bond. A mechanism of autoproteolysis via an N-O acyl shift was proposed to relieve these conformational strains. However, it is not understood how the polypeptide chain distortion was generated and preserved during the folding of GA to trigger autoproteolysis. An obstacle to our understanding of GA autoproteolysis is the uncertainty concerning its quaternary structure in solution. Here we have revisited this question and show that GA forms dimers in solution. Mutants with alterations at the dimer interface cannot form dimers and are impaired in the autoproteolytic activation. This suggests that dimerization of GA plays an essential role in autoproteolysis to activate the amidase activity. Comparison of the melting temperatures of GA dimers before and after autoproteolysis suggests two states of dimerization in the process of enzyme maturation. A two-step dimerization mechanism to trigger autoproteolysis is proposed to accommodate the data presented here as well as those in the literature.

Isoaspartyl dipeptidase activity of plant-type asparaginases.

Recombinant plant-type asparaginases from the cyanobacteria Synechocystis sp. PCC (Pasteur culture collection) 6803 and Anabaena sp. PCC 7120, from Escherichia coli and from the plant Arabidopsis thaliana were expressed in E. coli with either an N-terminal or a C-terminal His tag, and purified. Although each of the four enzymes is encoded by a single gene, their mature forms consist of two protein subunits that are generated by autoproteolytic cleavage of the primary translation products at the Gly-Thr bond within the sequence GTI/VG. The enzymes not only deamidated asparagine but also hydrolysed a range of isoaspartyl dipeptides. As various isoaspartyl peptides are known to arise from proteolytic degradation of post-translationally altered proteins containing isoaspartyl residues, and from depolymerization of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin), plant-type asparaginases may not only function in asparagine catabolism but also in the final steps of protein and cyanophycin degradation. The properties of these enzymes are compared with those of the sequence-related glycosylasparaginases.