Glycosylasparaginase Research Articles

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.

Release, Separation, and Recovery of Monomeric Reducing N-Glycans with Pronase E Combined with 9-Chloromethyl Chloroformate and Glycosylasparaginase.

The glycan moiety of glycoproteins plays key roles in various biological processes. However, there are few versatile methods for releasing, separating, and recovering monomeric reducing N-glycans for further functional analysis. In this study, we developed a new method to achieve the release, separation, and recovery of monomeric reducing N-glycans using enzyme E (Pronase E) combined with 9-chloromethyl chloroformate (Fmoc-Cl) and glycosylasparaginase (GA). Ovalbumin, ribonuclease B, ginkgo, and pine nut glycoproteins were used as materials and sequentially enzymatically hydrolyzed with Pronase E, derivatized with Fmoc-Cl, and enzymatically hydrolyzed with GA. The products produced by this method were then detected by electrospray ionization mass spectrometry, high-performance liquid chromatography (HPLC), and online hydrophilic interaction chromatography (HILIC-MS) separation. The results showed that all N-glycans with essentially one amino acid obtained with Pronase E were labeled with Fmoc-Cl and could be efficiently separated and detected via HPLC and HILIC-MS. Finally, the isolated Asn-glycan derivatives were digested with GA, enabling the recovery of all monomeric reducing N-glycans modified by core α-1,3 fucose. This method was simple, inexpensive, and broadly applicable and could therefore be quite important for analysis of the structure-function relationships of glycans.

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

Glycosylasparaginase or aspartylglucosaminidase, EC3.5.1.26.

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 β-hydroxamate (β-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-β-AHA complex structure by comparing to a previously published G172D mutant precursor in complex with a β-AHA molecule. A hydrolysis mechanism of β-AHA by GA is proposed based on this complex model.

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 Å 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 α-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.

Esterase activity of glycosylasparaginase

Glycosylasparaginase (GA) catalyzes the hydrolysis of the N-glycosylic bond in β-N-acetylglucosaminyl-L-asparagine to give N-acetylglucosamine, aspartic acid and ammonium ion in the catabolism of N-linked glycoproteins. A deficiency or absence of activity of GA gives rise to the most common inherited disorder of glycoprotein metabolism, aspartylglycosaminuria. The mechanism for GA has been proposed to be analogous to that of serine proteases, involving an acylation reaction to an acyl-enzyme intermediate, followed by a deacylation reaction. Studies of the mechanism thus far show that some properties of GA are similar to properties of serine proteases, although differences exist in some of the properties. Serine proteases have esterase activity where the rate-limiting step in the hydrolysis of substituted phenyl esters is the deacylation step. The esterase activity of GA was investigated using O4-(parasubstituted phenyl)-L-aspartate esters synthesized for the first time. The release of the parasubstituted phenolate was measured upon incubation with GA from human amniotic fluid. The rates of hydrolysis of the esters were dependent on the electronic properties of the parasubstitution. The rate-determining step in the hydrolysis of the esters by GA is the acylation step, opposite to that of serine proteases. The analogy of the mechanisms for serine proteases and GA is not absolute. The research was supported in part by funds provided by The University of North Carolina at Charlotte.

Massive accumulation of Man2GlcNAc2-Asn in nonneuronal tissues of glycosylasparaginase-deficient mice and its removal by enzyme replacement therapy.

Aspartylglycosaminuria (AGU) is caused by deficient enzymatic activity of glycosylasparaginase (GA). The disease is characterized by accumulation of aspartylglucosamine (GlcNAc-Asn) and other glycoasparagines in tissues and body fluids of AGU patients and in an AGU mouse model. In the current study, we characterized a glycoasparagine carrying the tetrasaccharide moiety of alpha-D-Man-(1-->6)-beta-D-Man-(1-->4)-beta-D-GlcNAc-(1-->4)-beta-D-GlcNAc-(1-->N)-Asn (Man2GlcNAc2-Asn) in urine of an AGU patient and also in the tissues of the AGU mouse model. Quantitative analysis demonstrated a massive accumulation of the compound especially in nonneuronal tissues of the AGU mice, in which the levels of Man2GlcNAc2-Asn were typically 30-87% of those of GlcNAc-Asn. The highest level of Man2GlcNAc2-Asn was found in the liver, spleen, and heart tissues of the AGU mice, the respective amounts being 87%, 76%, and 57% of the GlcNAc-Asn levels. In the brain tissue of AGU mice the Man2GlcNAc2-Asn storage was only 9% of that of GlcNAc-Asn. In contrast to GlcNAc-Asn, the storage of Man2GlcNAc2-Asn markedly increased in the liver and spleen tissues of AGU mice as they grew older. Enzyme replacement therapy with glycosylasparaginase for 3.5 weeks reduced the amount of Man2GlcNAc2-Asn by 66-97% in nonneuronal tissues, but only by 13% in the brain tissue of the AGU mice. In conclusion, there is evidence for a role for storage of glycoasparagines other than aspartylglucosamine in the pathogenesis of AGU, and this possibility should be taken into consideration in the treatment of the disease.

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.

Human leukocyte glycosylasparaginase: cell-to-cell transfer and properties in correction of aspartylglycosaminuria

Aspartylglycosaminuria (AGU), a severe lysosomal storage disease, is caused by the deficiency of the lysosomal enzyme, glycosylasparaginase (GA), and accumulation of aspartylglucosamine (GlcNAc-Asn) in tissues. Here we show that human leukocyte glycosylasparaginase can correct the metabolic defect in Epstein–Barr virus (EBV)-transformed AGU lymphocytes rapidly and effectively by mannose-6-phosphate receptor-mediated endocytosis or by contact-mediated cell-to-cell transfer from normal EBV-transformed lymphocytes, and that 2–7% of normal activity is sufficient to correct the GlcNAc-Asn metabolism in the cells. Cell-to-cell contact is obligatory for the transfer of GA since normal transformed lymphocytes do not excrete GA into extracellular medium. The combined evidence indicates that cell-to-cell transfer of GA plays a main role in enzyme replacement therapy of AGU by normal lymphocytes.

Purification and crystallization of precursors and autoprocessed enzymes of Flavobacterium glycosylasparaginase: an N-terminal nucleophile hydrolase.

Glycosylasparaginase (GA) represents a novel group of proteins that are activated by self-catalyzed peptide-bond cleavage from a single-chain precursor to yield the two subunits required for hydrolase activity. The wild-type GA precursor autoproteolyzes spontaneously into alpha and beta subunits. Strategies are reported here for purification to homogeneity of GA from Flavobacterium meningosepticum in both single-chain precursor and mature (autoprocessed) forms. The recombinant proteins crystallize in different space groups: P1 and P2(1) for the precursor and mature enzymes, respectively. The precursor crystals diffract to 1.9 A resolution with laboratory X-ray radiation.

Crystal Structures of FlavobacteriumGlycosylasparaginase: AN N-TERMINAL NUCLEOPHILE HYDROLASE ACTIVATED BY INTRAMOLECULAR PROTEOLYSIS

Glycosylasparaginase (GA) is a member of a novel family of N-terminal nucleophile hydrolases that catalytically use an N-terminal residue as both a polarizing base and a nucleophile. These enzymes are activated from a single chain precursor by intramolecular autoproteolysis to yield the N-terminal nucleophile. A deficiency of GA results in the human genetic disorder known as aspartylglycosaminuria. In this study, we report the crystal structure of recombinant GA from Flavobacterium meningosepticum. Similar to the human structure, the bacterial GA forms an alphabetabetaalpha sandwich. However, some significant differences are observed between the Flavobacterium and human structures. The active site of Flavobacterium glycosylasparaginase is in an open conformation when compared with the human structure. We also describe the structure of a mutant wherein the N-terminal nucleophile Thr152 is substituted by a cysteine. In the bacterial GA crystals, we observe a heterotetrameric structure similar to that found in the human structure, as well as that observed in solution for eukaryotic glycosylasparaginases. The results confirm the suitability of the bacterial enzyme as a model to study the consequences of mutations in aspartylglycosaminuria patients. They also suggest that further studies are necessary to understand the detail mechanism of this enzyme. The presence of the heterotetrameric structure in the crystals is significant because dimerization of precursors has been suggested in the human enzyme to be a prerequisite to trigger autoproteolysis.

Recombinant glycosylasparaginase and in vitro correction of aspartylglycosaminuria

Aspartylglycosaminuria (AGU) is the most common disorder of glycoprotein degradation. AGU patients are deficient in glycosylasparaginase (GA), which results in accumulation of aspartylglucosamine in body fluids and tissues. Human glycosylasparaginase was stably overexpressed in NIH-3T3 mouse fibroblasts, in which the unusual posttranslational processing and maturation of the enzyme occurred in a high degree. The recombinant enzyme was isolated as two isoforms, which were both phosphorylated, and actively transported into AGU fibroblasts and lymphoblasts through mannose-6-phosphate receptor-mediated endocytosis. The rate of uptake into fibroblasts was half-maximal when the concentration of GA in the medium was 5 x 10(-8) M. Immunofluorescence microscopy suggested compartmentalization of the recombinant enzyme in the lysosomes. Supplementation of culture medium with either isoform cleared AGU lymphoblasts of stored aspartylglucosamine when glycosylasparaginase activity in the cells reached 3-4% of that in normal lymphoblasts. A relatively small amount of recombinant GA in the culture medium was sufficient to reverse pathology in the target cells, indicating high corrective quality of the enzyme preparations. The combined evidence indicates that enzyme replacement therapy with the present recombinant glycosylasparaginase might reverse pathology at least in somatic cells of AGU patients.

Finnish-type aspartylglucosaminuria detected by oligonucleotide ligation assay

Aspartylglycosaminuria (AGU) is a recessively inherited lysosomal storage disease that occurs with much higher frequency in Finland than elsewhere. AGU is caused by a deficiency in glycosylasparaginase (GA), which results in the accumulation of glycoasparagines in lysosomes. In the Finnish population, a single nucleotide change in the gene encoding GA is responsible for the disease. We have used the oligonucleotide ligation assay (OLA) to detect the mutation in polymerase chain reaction (PCR)-amplified DNA samples from normal, carrier, and affected individuals. Screening for AGU among 415 random Finnish DNA samples with PCR/OLA revealed five carriers of the mutant allele and demonstrated the potential of the method for use in carrier screening. PCR/OLA provides a rapid, reliable, nonisotopic method to detect the mutation responsible for AGU that can readily be applied to large population screening.

The isolation and structure of the core oligosaccharide sequences of IgM.

Methods are presented for separating the three IgM heavy chain sialoglycopeptides associated with asparagines 170, 332, and 395. The core glycopeptide units containing the disaccharide fucosyl-N-acetylglucosamine were obtained through the use of an endo-beta-N-acetylglucosamindase from Diplococcus pneumoniae, following exoglycosidase treatment of the sialoglycopeptides. In addition to the core glycopeptides, high yields of a tetrasaccharide, (Man)3GlcNAc, were obtained. The fucose in the core disaccharide is glycosidically linked to the 6-O position of the N-acetylglucosamine residue in Asn-GlcNAc. This core unit is resistant to glycosyl asparaginase, but becomes susceptible to hydrolysis on removal of the fucosyl residue by a purified hen oviduct alpha-L-fucosidase. The core sequence of the immunoglobulin M sialoglycopeptides appears to be similar to that of most other asparagine-linked oligosaccharides in consisting of a basic unit composed of beta-D-Man-(1 leads to 4)beta-D-GlcNAc(1 leads to 4)beta-D-GlcNAc(1 leads to 4), but with L fucose linked alpha-(1 leads to 6) to the proximal GlcNAc. The two nonreducing terminal ends of (Man)3GlcNAc are linked to beta-D-Man by alpha-(1 leads to 3) and alpha(1 leads to 6) bonds, respectively.

Studies on the Carbohydrate Units of Thyroglobulin: STRUCTURE OF THE MANNOSE-N-ACETYLGLUCOSAMINE UNIT (UNIT A) OF THE HUMAN AND CALF PROTEINS

Abstract An investigation of the structure and peptide attachment of the mannose-N-acetylglucosamine unit (unit A) from human and calf thyroglobulin has been carried out on glycopeptides containing large (8 to 10 mannose plus 2 glucosamine residues) and small (5 to 6 mannose plus 2 glucosamine residues) variants of this unit, as well as on an oligosaccharide fraction (8 mannose plus 2 glucosamine residues). All of the mannose residues could be released by α-mannosidase, after which 1 N-acetylglucosamine could be removed by β-N-acetyglucosaminidase. This permitted the isolation from the glycopeptides of 2-acetamido-4-O-(2acetamido-2-deoxy-β-d-glucopyranosyl)-1-N-β-l-aspartyl-2deoxy-β-d-glucopyranosylamine and 2-acetamido-1-N-β-laspartyl-2-deoxy-β-d-glucopyranosylamine, which could be split to aspartic acid plus di-N-acetylchitobiose or N-acetylglucosamine, respectively, by the action of glycosyl asparaginase. Incubation of the unit A oligosaccharide with α-mannosidase resulted in the production of a di-N-acetylglucosamine disaccharide in high yield which was shown to be identical with di-N-acetylchitobiose. Serial periodate oxidation of the glycopeptides caused destruction of all but approximately 2 of the mannose residues, but spared both N-acetylglucosamine residues in the first Smith degradation. These 2 surviving mannose residues were destroyed in the second degradation. One glucosamine was oxidized in the third treatment, while the other glucosamine still remained intact. Glycerol was the only alcohol released during the three Smith degradations. Periodate oxidation and sodium borohydride reduction of the reduced unit A oligosaccharide, in which the terminal glucosamine had been converted to glucosaminitol, yielded 1 residue of xylosaminitol. Methylation of the glycopeptides by the method of Hakamori yielded tetra- and di-O-methyl mannose ethers from glycopeptides with small carbohydrate units, while tetra-, tri-, and di-O-methyl ethers were obtained from the glycopeptides with large carbohydrate units. All glycopeptides yielded only 1 O-methyl ether of N-methylglucosamine, which was identified by ion exchange chromatography to be 3,6-di-O-methyl-N-methylglucosamine. The results of these studies suggest the following structure for carbohydrate unit A. [see PDF for sequence] The smallest unit found was a tightly branched structure containing 5 mannose residues, and the larger units are believed to contain additional mannose residues, designated as (Man)x linked to the terminal residues of the three chains by α1-2 bonds. No significant species variations were observed between the calf and human proteins.

Studies on the Oligosaccharide Sequence of Ribonuclease B

Abstract α-Mannosidase treatment of the asparaginyl oligosaccharide derivative from ribonuclease B released 5 eq of mannose and yielded a product containing asparagine, N-acetylglucosamine, and mannose in a ratio of 1:2:1. The remaining mannose residue was resistant to hydrolysis by α-mannosidase, but could be removed by a Smith degradation, a procedure which did not affect the N-acetylglucosamine residues. The resultant product chromatographed identically with 2-acetamido-4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-N-(4-l-aspartyl)-2-deoxy-β-d-glucopyranosylamine prepared chemically by Spinola and Jeanloz (J. Biol. Chem., 245, 4158 (1970)). In addition, both synthetic and isolated compounds reacted similarly on treatment with β-N-acetylglucosaminidase and glycosyl asparaginase. Evidence is presented showing the mannose residue in the asparaginyl trisaccharide to be linked to the 3-O position of the terminal nonreducing N-acetylglucosamine and that the N-acetylglucosamines form a disaccharide identical with di-N-acetylchitobiose.