Recombinant human glycosylasparaginase catalyzes hydrolysis of <scp>l</scp>‐asparagine

Noronkoski, Tiina; Stoineva, Ivanka; Petkov, Dimiter D.; Mononen, Ilkka

doi:10.1016/s0014-5793(97)00761-8

Cited by 16 publications

(16 citation statements)

References 23 publications

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“…3 Glycosylasparaginase is transported in active form into various non-neuronal cell types, including Epstein-Barr virus-transformed (EBV) glycosylasparaginase-deficient lymphoblasts, and it effectively corrects the metabolic defects in glycosylasparaginase-deficient cell lines 4 and mice. 5 The finding that human glycosylasparaginase also hydrolyzes L-asparagine to L-aspartic acid and ammonia-like bacterial L-asparaginases 6 without any L-glutaminase activity 3 led us to investigate its potential cytotoxic activity toward leukemia cells that are dependent on their external supply of L-asparagine.…”

Section: Depletion Of L-asparagine Supply and Apoptosis Of Leukemia Cmentioning

confidence: 99%

“…The pelleted cells were washed twice with ice-cold phosphate-buffered saline (PBS) and then lysed in 50 mM sodium-potassium phosphate buffer, pH 7.5, through three freezing-thawing cycles and sonication in an ice bath for 30 s. The enzyme activity was measured by a fluorometric assay 4 and the Asn concentration was analyzed with high-performance liquid chromatography. 6 Letters to the Editor GA or two bacterial L-asparaginases used in chemotherapy, Erwinase s (ErAII) and Elspar s (E. coli L-asparaginase, EcAII). The sensitivity of the cells to each enzyme was tested with the MTT assay.…”

Section: Depletion Of L-asparagine Supply and Apoptosis Of Leukemia Cmentioning

confidence: 99%

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Depletion of L-asparagine supply and apoptosis of leukemia cells induced by human glycosylasparaginase

2009

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ReferencesA lysosomal enzyme glycosylasparaginase (GA; aspartylglucosaminidase; EC 3.5.1.26) hydrolyzes the N-glycosidic carbohydrate-to-protein linkage region, aspartylglucosamine, to L-aspartic acid and l-amino-N-acetylglucosamine through a reaction mechanism similar to L-asparaginase. 3 Glycosylasparaginase is transported in active form into various non-neuronal cell types, including Epstein-Barr virus-transformed (EBV) glycosylasparaginase-deficient lymphoblasts, and it effectively corrects the metabolic defects in glycosylasparaginase-deficient cell lines 4 and mice. 5 The finding that human glycosylasparaginase also hydrolyzes L-asparagine to L-aspartic acid and ammonia-like bacterial L-asparaginases 6 without any L-glutaminase activity 3 led us to investigate its potential cytotoxic activity toward leukemia cells that are dependent on their external supply of L-asparagine. We investigated the ability of human recombinant GA to deplete the intra-and extracellular Asn reservoirs in vitro using EBVtransformed glycosylasparaginase-deficient lymphoblasts from an Letters to the Editor 1167 Leukemia aspartylglycosaminuria patient. 4 Lymphoblasts were continuously grown in an RPMI-1640 (HyClone, Logan, Utah, USA) medium supplemented with 15% of fetal calf serum (BioClear, Wiltshire, UK), 2 mM L-glutamine (HyClone) and 1.5-2.0 mM L-asparagine (Fluka Chemie AG, Buchs, Switzerland) and various concentrations of either GA or Erwinia L-asparaginase (Erwinase, ErAII) for 24 h. The kinetic parameters of the enzymes for the hydrolysis of L-asparagine were determined by a spectrophotometric method as described. 7,8 At pH 7.5, the K m of glycosylasparaginase, Erwinia L-asparaginase and Escherichia coli L-asparaginase was 538, 90 and 130 mM and V max 1.49, 16 and 23 mM/min, respectively. One unit of enzyme causes hydrolysis of 1 mmol of L-asparagine per minute under standard conditions.The depletion of the extracellular Asn concentration in the culture medium from 2.3 to 0.2 mmol/l in the presence of 10 or 40 U/l of glycosylasparaginase or 10 U/l of ErAII took 18, 4 and 4 h, respectively. In the presence of 1500 U/l of ErAII, similar Asn concentration decrease occurred within 5 min. The Asn level in the cell culture medium remained unchanged at approximately 2 mmol/l in the absence of either glycosylasparaginase or ErAII (Figure 1a).To determine whether the enzymes can deplete the intracellular Asn reservoirs of EBV-transformed lymphoblasts, we analyzed both the Asn concentration and the enzyme activity inside the cells during the incubation. In the presence of 10 or 40 U/l of glycosylasparaginase or 10 U/l of ErAII in the cell culture medium, the Asn concentration inside the GA-deficient cells first increased within the first 2 h of incubation from the initial approximately 80 nmol/mg protein and then decreased below 6 nmol/mg protein during the next 16-18, 6 or 10-12 h, respectively (Figure 1b). In the presence of 1500 U/l of ErAII in the culture medium, the intracellular Asn concentration decreased from 89 to 14 nmol/m...

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Section: Depletion Of L-asparagine Supply and Apoptosis Of Leukemia Cmentioning

confidence: 99%

Section: Depletion Of L-asparagine Supply and Apoptosis Of Leukemia Cmentioning

confidence: 99%

Depletion of L-asparagine supply and apoptosis of leukemia cells induced by human glycosylasparaginase

2009

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“…2. We propose the name glycosylasparaginase/␤-aspartyltransferase for the multifunctional enzyme that catalyzes the enzymatic synthesis and degradation of ␤-aspartyl peptides, cleaves the Nglycosidic bond during degradation of glycoproteins (26), and possesses L-asparaginase activity (12). Because GGT is also an Ntn hydrolase (7) and there are many biochemical similarities between GGT and GA, we predict that the binding mode of the nucleophile amino acids and peptides into the active site of GGT resembles the nucleophile binding to GA.…”

Section: Discussionmentioning

confidence: 99%

“…The enzyme uses the ␥-hydroxyl and ␣-amino group of its ␤-chain N-terminal threonine as an active site nucleophile and general base in the formation of ␤-aspartyl enzyme, which is subsequently deacylated by water to L-aspartic acid (10,11). The GA-catalyzed hydrolysis of L-asparagine occurs in a similar manner, resulting in the formation of ␤-aspartyl enzyme and ammonia (12).…”

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Glycosylasparaginase-catalyzed Synthesis and Hydrolysis of β-Aspartyl Peptides

Noronkoski¹,

Stoineva²,

Ivanov³

et al. 1998

Journal of Biological Chemistry

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␤-Aspartyl di-and tripeptides are common constituents of mammalian metabolism, but their formation and catabolism are not fully understood. In this study we provide evidence that glycosylasparaginase (aspartylglucosaminidase), an N-terminal nucleophile hydrolase involved in the hydrolysis of the N-glycosidic bond in glycoproteins, catalyzes the hydrolysis of ␤-aspartyl peptides to form L-aspartic acid and amino acids or peptides. The enzyme also effectively catalyzes the synthesis of ␤-aspartyl peptides by transferring the ␤-aspartyl moiety from other ␤-aspartyl peptides or ␤-aspartylglycosylamine to a variety of amino acids and peptides. Furthermore, the enzyme can use L-asparagine as the ␤-aspartyl donor in the formation of ␤-aspartyl peptides. The data show that synthesis and degradation of ␤-aspartyl peptides are new, significant functions of glycosylasparaginase and suggest that the enzyme could have an important role in the metabolism of ␤-aspartyl peptides.␤-Aspartyl and ␥-glutamyl peptides are normal constituents of mammalian urine (1, 2) and tissues (3). Although ␥-glutamyltransferase (EC 2.3.2.2, GGT) (1) 1 is the key enzyme in the synthesis and hydrolysis of ␥-glutamyl compounds such as glutathione in the ␥-glutamyl cycle (4), little is known about the metabolism of ␤-aspartyl peptides.Glycosylasparaginase (GA; aspartylglucosaminidase;asparagine; ␤-aspartylglucosamine; GlcNAc-Asn) during degradation of glycoproteins. Genetic deficiency of glycosylasparaginase causes a lysosomal storage disease aspartylglycosaminuria (McKusick 208400) that is the most common disorder of glycoprotein degradation in humans and is clinically characterized by severe mental and motor retardation (5, 6).Glycosylasparaginase is a member of the recently described structural superfamily of enzymes termed as N-terminal nucleophile (Ntn) hydrolases (7). The hydrolysis of ␤-aspartylglycosylamines catalyzed by glycosylasparaginase is initiated by the binding of the ␤-aspartyl moiety into the active site of the enzyme through its free ␣-amino and ␣-carboxyl groups (8, 9). The enzyme uses the ␥-hydroxyl and ␣-amino group of its ␤-chain N-terminal threonine as an active site nucleophile and general base in the formation of ␤-aspartyl enzyme, which is subsequently deacylated by water to L-aspartic acid (10, 11). The GA-catalyzed hydrolysis of L-asparagine occurs in a similar manner, resulting in the formation of ␤-aspartyl enzyme and ammonia (12). The mechanism of action of glycosylasparaginase and the structural properties of its substrate-binding site (13) led us to consider that the enzyme might have a role in the metabolism of ␤-aspartyl peptides. In the present study, we demonstrate that synthesis and degradation of ␤-aspartyl peptides are new significant functions of glycosylasparaginase. This suggests that glycosylasparaginase could have an important role in the metabolism of ␤-aspartyl peptides present in body fluids and tissues. EXPERIMENTAL PROCEDURESMaterials-GA was purified to homogeneity from an NIH-3T3 cell line overexpre...

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“…Recent studies have demonstrated that mammalian tissues contain a lysosomal enzyme, glycosylasparagi-nase (glycoasparaginase, aspartylglucosaminidase; EC 3.5.1.26), which can catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia (8). Glycosylasparaginase has a mechanism of action similar to that of L-asparaginase (9 -11) and its activity can be assayed fluorometrically using L-aspartic acid ␤-(7-amido-4-methylcoumarin) (AspAMC) as substrate (12).…”

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confidence: 99%