2 Laboratory Centre, Kuopio University Hospital, FIN-70210 Kuopio, Finland and 3 Department of Clinical Chemistry, University of Turku and TUCH Laboratories, PO Box 52, FIN-20521 Turku, Finland
Received on June 21, 2004; revised on August 27, 2004; accepted on August 30, 2004
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Abstract |
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Key words: aspartylglucosaminuria / enzyme replacement / lysosomal enzymes / lysosomal storage / N-linked oligosaccharide
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Introduction |
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GlcNAc-Asn has long been regarded as the main accumulating glycoasparagine in AGU (Beaudet and Thomas, 1989). The AGU patients' urine and tissues contain also unknown amounts of larger glycoasparagines with a variety of sugar structures (Beaudet and Thomas, 1989
; Lundblad et al., 1976
; Maury, 1979
; Mononen and Mononen, 1997
; Sugahara et al., 1977
). The accumulating glycoasparagines with a di-N-acetylchitobiose moiety have been regarded as partial degradation products of N-linked oligosaccharides. These include a glycoasparagine with the di-N-acetylchitobiose moiety and two mannose residues,
-D-Man-(1
6)-ß-D-Man-(1
4)-ß-D-GlcNAc-(1
4)-ß-D-GlcNAc-(1
N)-Asn (Man2GlcNAc2-Asn; MMGGA), which has been characterized in urine and liver tissue of AGU patients (Gordon et al., 1998
; Lundblad et al., 1976
; Maury, 1979
).
A mouse model of AGU (Kaartinen et al., 1996) has been created. These mice have no GA activity, and their condition mimics the characteristics of the human AGU disease, for example, they suffer the accumulation of large amounts of GlcNAc-Asn in tissues and urine, changes in external phenotype, motor retardation, and shortened life span. The first external signs of deterioration can be noticed in AGU mice at the age of 5 months, when the null mutant mice start to display general scruffiness that is recognizable from the disheveled coat. At the age of 18 months or more, all of the AGU mice have significant difficulty in moving, including poor coordination and balance culminating in hydronephrosis and hepatic necrosis (Gonzales-Gomez et al., 1998
). Enzyme replacement therapy with GA effectively corrects the metabolism of GlcNAc-Asn in nonneuronal tissues of AGU mice, but its effect on neuronal tissue is more limited (Dunder et al., 2000
). Little is known about the metabolism of larger glycoasparagines in AGU.
In this study, we demonstrate the presence of large amounts of Man2GlcNAc2-Asn in nonneuronal tissues of AGU mice and, furthermore, the increase of the level of Man2GlcNAc2-Asn with age. Enzyme therapy with GA effectively removes the compound from the nonneuronal tissues and reduces its excretion into urine of the mice. The combined evidence suggests that glycoasparagines with an oligosaccharide moiety may massively accumulate in AGU and the role of these compounds in the pathogenesis and treatment of AGU should be carefully investigated.
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Results |
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The mean excretion of Man2GlcNAc2-Asn into urine of untreated adult AGU mice was 104 µmol Man2GlcNAc2-Asn per mmol of creatinine (range 68133 µmol per mmol of creatinine; n = 6) corresponding to 44% of that of their GlcNAc-Asn excretion (mean 238 µmol GlcNAc-Asn per mmol of creatinine; range 147303 µmol per mmol of creatinine; n = 6) (Figure 4). The therapeutic protocol with the GA dose of 13 x 8 mg/kg reduced the mean excretion of Man2GlcNAc2-Asn in urine to 29 µmol Man2GlcNAc2-Asn per mmol of creatinine (range 2138 µmol per mmol of creatinine; n = 4) and that of GlcNAc-Asn to 37 µmol GlcNAc-Asn per mmol of creatinine (range 2648 µmol per mmol of creatinine; n = 4), that is, to 28% and 16% of the levels in untreated animals, respectively. The enzyme replacement therapy did not result in any noticeable changes in the external phenotypes of the AGU mice.
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Discussion |
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Mannose residues other than the core -(1-6)-linked mannose unit in the high-mannose type oligosaccharides are cleaved by the major lysosomal
-D-mannosidase, which is able to cleave
-(1-2)- and
-(1-3)-linked mannose residues (Haeuw et al., 1994
). The lysosomal core-specific
-(1-6)-mannosidase has been described to require removal of Asn from glycoasparagines by GA before the hydrolysis of the core
-(1-6)-linked mannose unit in Asn-linked glycoprotein in lysosomes can proceed (Haeuw et al., 1994
), and this is the obvious explanation for the accumulation of Man2GlcNAc2-Asn in tissues and urine of AGU patients (Gordon et al., 1998
; Lundblad et al., 1976
; Maury, 1979
, 1980
). Despite the dependence of lysosomal core-specific
-(1-6)-mannosidase on GA, the major accumulating fragment in AGU is GlcNAc-Asn, not Man2GlcNAc2-Asn. This suggests that most but not all N-linked carbohydrate structures are exposed to the action of the lysosomal enzyme chitobiase (Aronson and Kuranda, 1989
). In AGU, the lysosomal enzyme GA is lacking, and thus GlcNAc-Asn, which normally is hydrolyzed to L-aspartic acid and 1-amino-N-acetylglucosamine (Makino et al., 1966
), remains unhydrolyzed, leading to its accumulation in body fluids and tissues.
Our results show marked accumulation of -D-Man-(1
6)-ß-D-Man-(1
4)-ß-D-GlcNAc-(1
4)-ß-D-GlcNAc-(1
N)-Asn in urine and most tissues of AGU mice. There were high amounts of Man2GlcNAc2-Asn especially in nonneuronal tissues, 3087% of the level of aspartylglucosamine. The highest levels of accumulation of the compound in AGU mice were found in the liver, spleen, and heart tissues, suggesting that these are the organs in which the marked turnover of the glycoproteins with N-linked sugar chains occurs in healthy mice. Additionally, asialoglycoproteins are endocytosed from the blood circulation via galactose-specific receptors (asialoglycoprotein receptors) into liver (Joziasse et al., 2000
; Park et al., 2003
), thus increasing the amount of glycoproteins to be catabolized in the organ. Liver and spleen are also severely affected in other animal models of lysosomal storage disorders related to defective glycoprotein and mucopolysaccharide metabolism (Bhaumik et al., 1999
; Kakkis et al., 2001
; Stinchi et al., 1999
; Tomatsu et al., 2003
; Vogler et al., 2001
).
In the liver and spleen tissue of the AGU mice, the Man2GlcNAc2-Asn accumulation increased with the age of the mice, whereas the GlcNAc-Asn storage remained on the same high level during the age period from 3 weeks to over 1 year. One explanation for the increasing accumulation of Man2GlcNAc2-Asn might be that the massive amount of lysosomal storage material in the cells of these organs disturbs the ability of the lysosomal chitobiase to appropriately hydrolyze the linkage between two GlcNAc residues in the di-N-acetylchitobiose unit. For some unknown reason, the level of Man2GlcNAc2-Asn accumulation in the brain tissue of AGU mice was only one-tenth that of GlcNAc-Asn, whereas in other tissues the amounts of Man2GlcNAc2-Asn and GlcNAc-Asn were close. This suggests that the accumulation of Man2GlcNAc2-Asn in brain tissue may not play any major role in the development of the central nervous system symptoms encountered in AGU.
Enzyme therapy with IP-administered GA raised the enzyme activity levels, especially in the liver and spleen of the treated AGU mice. In those tissues also the clearance of the storage products was the most effective, suggesting that the level of uptake of the enzyme in various tissues accounts for the residual storage level. In addition to the uptake rate of the enzyme, the clearance of the storage compounds is also dependent on the turnover rate of N-linked glycoproteins as well as the half-life of GA in various tissues. Our results show that IP enzyme therapy with 13 x 8 mg/kg GA effectively accelerated the degradation of both Man2GlcNAc2-Asn and GlcNAc-Asn in all the analyzed adult AGU mouse tissues, excluding the brain. With a higher enzyme dose, the uptake of the enzyme may be even more evident also in brain tissue (Dunder et al., 2000). The age of the mice at the initiation of the therapy as well as the length of the therapy may also affect the efficacy of enzyme therapy. Although no GA activity could be detected in the kidney tissue of the treated animals, the amounts of the storage products in the tissue were lower than in the kidney of untreated AGU mice, demonstrating the therapeutic effect in this tissue. This observation may suggest lower uptake and of short half-life of GA in the kidney.
The excretions of Man2GlcNAc2-Asn and GlcNAc-Asn into urine of GA-treated AGU mice were 72% and 84% lower than those of untreated animals, respectively. The undegraded glycoasparagines found in the urine of the GA-treated mice, which did not contain any GA activity, may originate from those tissues that are not accessible to the enzyme, and these compounds continue to leak into the systemic circulation (Dunder et al., 2000). GA is known to hydrolyze large glycoasparagines, including a high-mannose type glycoasparagine, at relative rates quite comparable to that of GlcNAc-Asn (Kaartinen et al., 1992
). In this study we followed the degradation rate of Man2GlcNAc2-Asn versus GlcNAc-Asn in a liver tissue extract of an AGU mouse in the presence of GA. In that preliminary experiment the hydrolysis rate of Man2GlcNAc2-Asn was
94% of that of GlcNAc-Asn (data not shown), indicating that Man2GlcNAc2-Asn containing two mannose residues fits into the active site of GA at least as well as the glycoasparagine containing five mannose residues (relative rate 85%) (Kaartinen et al., 1992
). Enzyme replacement therapy decreased the amount of Man2GlcNAc2-Asn and GlcNAc-Asn in AGU mouse brain only by 13% and 22%, respectively. That is assumed to be due the blood-brain barrier, which effectively prevents the corrective enzyme from gaining access to the central nervous system.
The external phenotype typical of AGU mice develops very slowly. The first external signs of AGU mice can be noticed at the age of 5 months culminating in difficulties in gait, hydronephoris, and hepatic necrosis at the age of 18 months or more (Gonzales-Gomez et al., 1988). The enzyme replacement therapy lasting for about 1 month in this study did not improve the phenotype of the AGU mice, although the metabolic correction of the disorder was evident, suggesting that enzyme therapy should be initiated in newborn AGU mice and continued for several months to allow the phenotypic changes of AGU mice to recover.
Accumulation of GlcNAc-Asn in body fluids and tissues of AGU patients is regarded as the main reason for the clinical features of AGU disease. However, the mechanism for the slowly progressing abnormalities in nonneuronal tissues of AGU patients is not known. Our findings suggest that the accumulation of glycoasparagines larger than GlcNAc-Asn may be massive and increase by age. Thus they may also have a role in the progressive nature of AGU and their removal should be considered in the development of future therapeutic procedures for the disease.
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Materials and methods |
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Characterization of Man2GlcNAc2-Asn
Man2GlcNAc2-Asn was isolated from the urine of an AGU patient as described (Kaartinen and Mononen, 1989). The monosaccharide content of Man2GlcNAc2-Asn was analyzed as described (Mononen, 1981
), and the structure of the oligosaccharide chain was determined by 280 MHz 1H nuclear magnetic resonance spectroscopy (Vliegenthart et al., 1983
) as described. The structure of Man2GlcNAc2-Asn in AGU mouse tissues was confirmed by sequential digestion by exoglycosidases and analysis of the degradation products by HPLC as follows.
Seventy microliters of AGU mouse liver extract (4.60 mg protein) was dried under N2 and dissolved in 70 µl 50 mM sodium citrate, pH 4.5, containing 100 mg/L bovine serum albumin and 14 U -(1-6)-mannosidase. The mixture was incubated at 37°C in the presence of 0.8 mM CmCys (internal standard) for 3.75 h. Aliquots of 6 µl were taken at 0, 0.5, 2, and 3.75 h. After the last aliquot, 0.18 U ß-mannosidase was added into the reaction mixture, and the incubation was continued overnight. During the incubation, aliquots of 6 µl were taken at 0.5, 2, 3, and 19 h. Finally, 0.30 U ß-N-acetylglucosaminidase were added into the reaction mixture, and the incubation was continued for another 7 h. Aliquots of 6 µl were taken at 1, 3, and 7 h. The aliquots were stored at 20°C until their precolumn derivatization with FMOC (Einarsson, 1985
) and analysis by HPLC. The protein concentration of the tissue lysates was determined with a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA).
To follow the effect of GA on the amount of Man2GlcNAc2-Asn and GlcNAc-Asn in the liver, AGU mouse liver homogenate samples containing 3.56 mg protein in 50 mM Na-K-phosphate buffer, pH 7.5, were incubated in the presence of GA (9.1 U/L) and an internal standard CmCys (0.8 mM) at 37°C in a total volume of 200 µl for 24 h. Aliquots of 10 µl were withdrawn periodically from the incubation mixture and stored at 20°C until the simultaneous quantitative analysis of Man2GlcNAc2-Asn and GlcNAc-Asn as their PITC derivatives by HPLC.
HPLC analysis of Man2GlcNAc2-Asn and GlcNAc-Asn
The accumulation of glycoasparagines Man2GlcNAc2-Asn and GlcNAc-Asn in tissues and urine was assayed by HPLC by using a Merck/Hitachi L-6200 liquid chromatograph (Hitachi, Tokyo) as their PITC derivatives as described (Kaartinen and Mononen, 1990).
The HPLC analysis of Man2GlcNAc2-Asn and its degradation products after the exoglycosidase digestions was performed with a Merck/Hitachi L-6200 liquid chromatograph equipped with an D-6000A interface, L-6200 pump, AS-4000 autosampler, and F1000 fluorescence spectrophotometer. The column was Waters Spherisorb S3 ODS2 3 mm, 15 cm x 4.6 mm, Waters, Milford, CT). The samples were derivatized with FMOC (Einarsson, 1985) and dissolved in solvent A (80% 50 mM sodium acetate, pH 4.6, and 20% acetonitrile). After the sample injection, the column was eluted to 98% solvent A and 2% solvent B (80% acetonitrile and 20% 50 mM sodium acetate) with a linear gradient over a period of 19 min and then to 100% acetonitrile within 6 min. The flow rate of the mobile phase was 0.8 ml/min, and the eluent was monitored by fluorescence detector by using excitation and emission wavelengths of 263 nm and 313 nm, respectively.
Treatment of AGU mice and collection of samples
Three adult (over 1 year old) AGU mice (Kaartinen et al., 1996) received 8 mg/kg GA IP every second day (total of 13 injections, 4 of which 1.7 U/kg and 9 of which 3.1 U/kg). After the treatment protocol, the mice were anesthetized, killed, and perfused as described (Dunder et al., 2000
). The tissues were homogenized in 50 mM Na-K-phosphate buffer, pH 7.5, containing 0.1% Triton X-100 and then lysed by three freezing and thawing cycles and sonication. The GA activity of tissue homogenates and the concentrations of GlcNAc-Asn and Man2GlcNAc2-Asn in tissues and urine were then determined by HPLC as PITC derivatives as described (Kaartinen and Mononen, 1990
).
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Acknowledgements |
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Abbreviations |
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References |
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