Aspartylglycosaminuria (AGU) is a lysosomal storage disease caused by deficient activity of glycosylasparaginase (AGA), and characterized by motor and mental retardation. Enzyme replacement therapy (ERT) in adult AGU mice with AGA removes the accumulating substance aspartylglucosamine from and reverses pathology in many somatic tissues, but has only limited efficacy in the brain tissue of the animals. In the current work, ERT of AGU mice was initiated at the age of 1 week with three different dosage schedules of recombinant glycosylasparaginase. The animals received either 3.4 U of AGA/kg every second day for 2 weeks (Group 1), 1.7 U/kg every second day for 9 days followed by an enzyme injection once a week for 4 weeks (Group 2) or 17 U/kg at the age of 7 and 9 days (Group 3). In the Group 1 and Group 3 mice, ERT reduced the amount of aspartylglucosamine by 34 and 41% in the brain tissue, respectively. No therapeutic effect was observed in the brain tissue of Group 2 mice. As in the case of adult AGU mice, the AGA therapy was much more effective in the somatic tissues than in the brain tissue of the newborn AGU mice. The combined evidence demonstrates that a high dose ERT with AGA in newborn AGU mice is up to twofold more effective in reducing the amount of the accumulated storage material from the brain tissue than ERT in adult AGU animals, indicating the importance of early detection and treatment of the disease.

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.

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L-Asparaginase is known to catalyze the hydrolysis of L-asparagine to L-aspartic and ammonia, but little is known about its action on peptides. When we incubated L-asparaginases puri¢ed either from Escherichia coli or Erwinia chrysanthemi^commonly used as chemotherapeutic agents because of their antitumour activity^with eight smallL-aspartylglycine, we found that both L-asparaginases could catalyze the hydrolysis of ¢ve of them yielding L-aspartic acid and amino acids or peptides. Our data show that L-asparaginases can hydrolyze L L-aspartylpeptides and suggest that L-asparaginase therapy may a¡ect the metabolism of L L-aspartylpeptides present in human body. ß

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...