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

Eira Kelo2, Ulla Dunder2 and Ilkka Mononen1,3

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


1 To whom correspondence should be addressed; e-mail: ilkka.mononen{at}tyks.fi

Received on June 21, 2004; revised on August 27, 2004; accepted on August 30, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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)-ß-D-Man-(1->4)-ß-D-GlcNAc-(1->4)-ß-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.

Key words: aspartylglucosaminuria / enzyme replacement / lysosomal enzymes / lysosomal storage / N-linked oligosaccharide


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Aspartylglycosaminuria (AGU, McKusick 208400) is the most common inherited disorder of glycoprotein degradation. The inherited deficiency of the lysosomal enzyme glycosylasparaginase (GA, aspartylglycosaminidase, EC 3.5.1.26) in AGU leads to the accumulation of aspartylglucosamine (GlcNAc-Asn) and other glycoasparagines in tissues and body fluids of these patients, causing many clinical symptoms, including skeletal abnormalities and progressive psychomotor retardation (Arvio et al., 1997Go; Beaudet and Thomas, 1989Go).

GlcNAc-Asn has long been regarded as the main accumulating glycoasparagine in AGU (Beaudet and Thomas, 1989Go). The AGU patients' urine and tissues contain also unknown amounts of larger glycoasparagines with a variety of sugar structures (Beaudet and Thomas, 1989Go; Lundblad et al., 1976Go; Maury, 1979Go; Mononen and Mononen, 1997Go; Sugahara et al., 1977Go). 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, {alpha}-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., 1998Go; Lundblad et al., 1976Go; Maury, 1979Go).

A mouse model of AGU (Kaartinen et al., 1996Go) 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., 1998Go). 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., 2000Go). 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Characterization of Man2GlcNAc2-Asn in AGU mouse liver
The structure of Man2GlcNAc2-Asn in the AGU mouse liver tissue was confirmed by demonstrating its sequential hydrolysis at first with {alpha}-(1-6)-mannosidase, followed by ß-mannosidase and finally with ß-N-acetylglucosaminidase to GlcNAc-Asn (Figure 1). The native compound was not hydrolyzed either by ß-mannosidase before its incubation in the presence of {alpha}-(1-6)-mannosidase or by ß-N-acetylglucosaminidase prior to its incubation in the presence of both mannosidases (data not shown). The compound was not hydrolyzed by {alpha}-(1-3)-mannosidase (data not shown). When a reaction mixture containing an aliquot of AGU mouse liver tissue (3.56 mg protein; 1.34 nmol Man2GlcNAc2-Asn per mg protein, and 1.73 nmol GlcNAc-Asn per mg protein), 0.8 mM of CmCys and 9.1 U/L of glycosylasparaginase, was incubated at 37°C, the relative hydrolysis rate of Man2GlcNAc2-Asn was 94% that of GlcNAc-Asn (data not shown).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1. Characterization of Man2GlcNAc2-Asn in AGU mouse liver. Man2GlcNAc2-Asn and GlcNAc-Asn were detected simultaneously from AGU mouse liver tissue by HPLC as their FMOC derivatives (See Materials and methods for further details). CmCys was used as an internal standard. AGU mouse liver tissue (4.60 mg protein) was incubated sequentially in the presence of three different exoglycosidases: 1 min after the addition of {alpha}-(1-6)-mannosidase the formation of Man2GlcNAc2-Asn can be detected (A). After 4 h in the presence of {alpha}-(1-6)-mannosidase and 15 min with ß-mannosidase, the formation of GlcNAc2-Asn can be detected (B). After another 3 h, the incubation mixture contained GlcNAc2-Asn and GlcNAc-Asn as the main compounds (C). Seven hours after the addition of ß-N-acetylglucosaminidase GlcNAc2-Asn had been totally hydrolyzed to GlcNAc-Asn (D).

 
Man2GlcNAc2-Asn in mouse tissues and urine
The concentration of Man2GlcNAc2-Asn and GlcNAc-Asn in the tissues of untreated AGU mice, AGU mice treated with GA, and wild-type mice (3 weeks, 6 weeks, or adult) was determined by high-performance liquid chromatography (HPLC). The accumulation of Man2GlcNAc2-Asn and GlcNAc-Asn in the tissues of adult mice is illustrated in Figure 2A and 2B, respectively. The highest Man2GlcNAc2-Asn storage was found in liver (mean 26 nmol/mg protein, n = 3), followed by the spleen (mean 22 nmol/mg protein, n = 3) and heart (mean 9.8 nmol/mg protein, n = 3) of the AGU mice. The highest concentration of GlcNAc-Asn was found in the brain tissue (mean 42 nmol/mg protein, n = 2), followed by the spleen (mean 39 nmol/mg protein, n = 3) and the liver (mean 30 nmol/mg protein, n = 3) tissues of the AGU mice. The amount of GlcNAc-Asn was higher than that of Man2GlcNAc2-Asn in all of the tissues examined.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. Accumulation of Man2GlcNAc2-Asn (A) and GlcNAc-Asn (B) in tissues of adult AGU mice and the effect of enzyme therapy with GA. Gray columns represent untreated AGU mice, white columns represent AGU mice treated with 13 x 8 mg/kg of glycosylasparaginase IP (see Materials and methods for details), and black columns represent untreated wild-type mice. The columns represent the mean value of two different animals unless mentioned otherwise and the bars represent the ± range of the values. An asterisk indicates that the result is from one animal and § indicates the mean of three animals. The Man2GlcNAc2-Asn or GlcNAc-Asn levels below the detection limit of the assay (0.1 nmol/mg protein) are indicated with a hash mark.

 
The effect of age of the AGU mice on the accumulation of Man2GlcNAc2-Asn and GlcNAc-Asn in their liver, spleen, brain, and kidney tissues was analyzed at the ages of 3 weeks, 6 weeks, and over 1 year. The accumulation of Man2GlcNAc2-Asn in AGU liver and spleen was age-related: When the mice grew older, the concentration of the compound increased in the tissues (Figure 3). In the brain tissue of the AGU mice, the amount of Man2GlcNAc2-Asn remained at the same low level during the age period from 3 weeks to over 1 year, and it was only 8–10% that of the amount of GlcNAc-Asn. From the age of 3 weeks to 6 weeks, the amount of Man2GlcNAc2-Asn in the liver tissue increased by threefold, and from the age of 6 weeks to 1 year it increased 60%. The accumulation of Man2GlcNAc2-Asn in the spleen tissue occurred somewhat later with an increase of four- to fivefold noted from the age of 6 weeks to 1 year. The Man2GlcNAc2-Asn level in the liver tissue was only 15% of that of GlcNAc-Asn at the age of 3 weeks, but it increased to a level of 87% by the time the mice were full adults. This kind of increase with age was not detected in the accumulation of GlcNAc-Asn; its level in tissues of the AGU mice remained relatively stable from the age of 3 weeks to 1 year.



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 3. The effect of age of the AGU mice on the accumulation of Man2GlcNAc2-Asn and GlcNAc-Asn in their liver, brain, spleen, and kidney. The columns represent the mean value of three different animals unless mentioned otherwise. The bars represent the ± range of the values. An asterisk indicates that the result is from one animal and § indicates the mean of two animals.

 
Effects of GA on Man2GlcNAc2-Asn in tissues and urine of AGU mice
A marked increase in the GA activity was observed in the liver and spleen tissues of AGU mice after 13 intraperitoneal injections of GA (Table I). After enzyme therapy, the GA activity was in the wild-type range in the liver of the treated AGU mice, whereas in spleen of the treated animals the enzyme activity was fourfold higher than in the corresponding wild-type tissue. In the lung and heart tissues of the treated animals the GA activity was only 1% that of the wild-type mice. In kidney and brain tissues the GA activity was undetectable after the treatment. GA activity could not be detected either in urine or blood.


View this table:
[in this window]
[in a new window]
 
Table I. GA activity (µU/mg protein) in tissues of mice

 
Accumulated Man2GlcNAc2-Asn and GlcNAc-Asn were effectively cleared from all other tissues except brain of adult AGU mice by 13 IP injections of GA. The elimination of both compounds was most effective in the liver tissue, in which the level of Man2GlcNAc2-Asn decreased by 97% (from 26 to 0.7 nmol of Man2GlcNAc2-Asn per mg protein) and the concentration of GlcNAc-Asn by 98% (from 30 to 0.6 nmol of GlcNAc-Asn per mg protein). In the brain tissue of the treated AGU animals, the mean concentration of Man2GlcNAc2-Asn decreased only by 13% (from 3.9 to 3.4 nmol of Man2GlcNAc2-Asn per mg protein) and that of GlcNAc-Asn by 22% (from 42 to 33 nmol GlcNAc-Asn per mg protein) (Figure 2A and B).

The mean excretion of Man2GlcNAc2-Asn into urine of untreated adult AGU mice was 104 µmol Man2GlcNAc2-Asn per mmol of creatinine (range 68–133 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 147–303 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 21–38 µmol per mmol of creatinine; n = 4) and that of GlcNAc-Asn to 37 µmol GlcNAc-Asn per mmol of creatinine (range 26–48 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.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. Effect of GA treatment on Man2GlcNAc2-Asn and GlcNAc-Asn in the adult AGU mouse urine. White columns represent untreated AGU mice (n = 6) and gray bars represent AGU mice treated with 13 x 8 mg/kg of GA as IP injections (n = 4). The bars represent the ±range of the values.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
After fulfilling their task in the body, Asn-linked glycoproteins are sequestered into lysosomes. The nonreducing end of the oligosaccharide chain of glycoasparagines or oligosaccharides liberated by lysosomal enzyme chitobiase (Aronson and Kuranda, 1989Go) is digested sequentially by exoglycosidases (Beaudet and Thomas, 1989Go). The degradation of protein and protein-to-carbohydrate region proceeds in a specific order initiated by proteolysis (Aronson and Kuranda, 1989Go), which is followed by the removal of any fucose residues by {alpha}-L-fucosidase and the hydrolysis of the linkage between GlcNAc and Asn by GA (Noronkoski and Mononen, 1997Go).

Mannose residues other than the core {alpha}-(1-6)-linked mannose unit in the high-mannose type oligosaccharides are cleaved by the major lysosomal {alpha}-D-mannosidase, which is able to cleave {alpha}-(1-2)- and {alpha}-(1-3)-linked mannose residues (Haeuw et al., 1994Go). The lysosomal core-specific {alpha}-(1-6)-mannosidase has been described to require removal of Asn from glycoasparagines by GA before the hydrolysis of the core {alpha}-(1-6)-linked mannose unit in Asn-linked glycoprotein in lysosomes can proceed (Haeuw et al., 1994Go), and this is the obvious explanation for the accumulation of Man2GlcNAc2-Asn in tissues and urine of AGU patients (Gordon et al., 1998Go; Lundblad et al., 1976Go; Maury, 1979Go, 1980Go). Despite the dependence of lysosomal core-specific {alpha}-(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, 1989Go). 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., 1966Go), remains unhydrolyzed, leading to its accumulation in body fluids and tissues.

Our results show marked accumulation of {alpha}-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, 30–87% 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., 2000Go; Park et al., 2003Go), 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., 1999Go; Kakkis et al., 2001Go; Stinchi et al., 1999Go; Tomatsu et al., 2003Go; Vogler et al., 2001Go).

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., 2000Go). 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., 2000Go). 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., 1992Go). 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., 1992Go). 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., 1988Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
GlcNAc-Asn, S-carboxymethyl-L-cysteine (CmCys), ß-mannosidase (30,000 U/L), ß-N-acetylglucosaminidase (50,000 U/L), 9-fluorenylmethyl chloroformate (FMOC), and phenylisothiocyanate (PITC) were products of Sigma Chemical (St. Louis, MO). {alpha}-(1-6)-Mannosidase (2,000,000 U/L) and bovine serum albumin were products of New England Biolabs (Beverly, MA). Human recombinant GA (913 U/L) was purified from NIH-3T3 mouse fibroblasts as described previously (Mononen et al., 1995Go). Organic solvents for HPLC were purchased from Rathburn Chemicals (Walkerburn, Scotland).

Characterization of Man2GlcNAc2-Asn
Man2GlcNAc2-Asn was isolated from the urine of an AGU patient as described (Kaartinen and Mononen, 1989Go). The monosaccharide content of Man2GlcNAc2-Asn was analyzed as described (Mononen, 1981Go), and the structure of the oligosaccharide chain was determined by 280 MHz 1H nuclear magnetic resonance spectroscopy (Vliegenthart et al., 1983Go) 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 {alpha}-(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, 1985Go) 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, 1990Go).

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, 1985Go) 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., 1996Go) 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., 2000Go). 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, 1990Go).


    Acknowledgements
 
This work was supported by grants from the Sigrid Jusélius Foundation.


    Abbreviations
 
AGU, aspartylglycosaminuria; GA, glycosylasparaginase, aspartylglucosaminidase; CmCys, S-carboxymethyl-L-cysteine; FMOC, 9-fluorenylmethyl chloroformate; HPLC, high-performance liquid chromatography; MMGGA, Man2GlcNAc2-Asn; PITC, phenylisothiocyanate


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Aronson, N. Jr. and Kuranda, M. (1989) Lysosomal degradation of Asn-linked glycoproteins. FASEB J., 3, 2615–2622.[Abstract/Free Full Text]

Arvio, M., Autio, S., and Mononen, T. (1997) Clinical manifestations of aspartylglycosaminuria. In Mononen, I. and Aronson, N. (Eds.), Lysosomal storage disease: aspartylglycosaminuria. Landes/Springer/Verlag, Austin, TX, pp. 19–31.

Beaudet, A. and Thomas, G. (1989) Disorders of glycoprotein degradation: mannosidosis, fucosidosis, sialidosis, and aspartylglycosaminuria. In Scriver, C., Beaudet, A., Sly, W., and Valle, D. (Eds.), The metabolic basis of inherited disease II. McGraw-Hill, New York, pp. 1603–1621.

Bhaumik, M., Muller, V., Rozaklis, T., Johnson, L., Dobrenis, K., Brattacharyya, R., Wurzelmann, S., Finamore, P., Hopwood, J., Walkley, S., and Stanley, P. (1999) A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome). Glycobiology, 9, 1389–1396.[Abstract/Free Full Text]

Dunder, U., Kaartinen, V., Valtonen, P., Väänänen, E., Kosma, V.-M., Heisterkamp, N., Groffen, J., and Mononen, I. (2000) Enzyme replacement therapy in a mouse model of aspartylglucosaminuria. FASEB J., 14, 361–367.[Abstract/Free Full Text]

Einarsson, S. (1985) Selective determination of secondary amino acids using precolumn derivatization with 9-fluorenylmethylchloroformate and reversed-phase high-performance liquid chromatography. J. Chromatogr., 348, 213–220.[CrossRef]

Gonzales-Gomez, I., Mononen, I., Heisterkamp, N., Groffen, J., and Kaartinen, V. (1998) Progressive neurodegeneration in aspartylglycosaminuria mice. Am. J. Pathol., 153, 1293–1300.[Abstract/Free Full Text]

Gordon, B., Rupar, C., Rip, J., Haust, M., Coulter-Mackie, M., Scott, E., and Hinton, G. (1998) Aspartylglucosaminuria in a Canadian family. Clin. Invest. Med., 21, 114–123.[ISI][Medline]

Haeuw, J.-F., Grard, T., Alonso, C., Strecker, G., and Michalski, J.-C. (1994) The core-specific lysosomal {alpha}(1-6)-mannosidase activity depends on aspartaminohydrolase activity. Biochem. J., 297, 463–466.[ISI][Medline]

Joziasse, D., Lee, R., Lee, Y., Biessen, E., Schiphorst, W., Koeleman, C., and van den Eijnden, D. (2000) {alpha}3-Galactosylated glycoproteins can bind to the hepatic asialoglycoprotein receptor. Eur. J. Biochem., 267, 6501–6508.[Abstract/Free Full Text]

Kaartinen, V. and Mononen, I. (1989) Analysis of aspartylglucosamine at the picomole level by high-performance liquid chromatography. J. Chromatogr., 490, 293–299.[Medline]

Kaartinen, V. and Mononen, I. (1990) Assay of aspartylglycosylaminase by high-performance liquid chromatography. Anal. Biochem., 190, 98–101.[ISI][Medline]

Kaartinen, V., Mononen, T., Laatikainen, R., and Mononen, I. (1992) Substrate specificity and reaction mechanism of human glycosylasparaginase. The N-glycosidic linkage of various glycoasparagines is cleaved through a reaction mechanism similar to L-asparaginase. J. Biol. Chem., 267, 6855–6858.[Abstract/Free Full Text]

Kaartinen, V., Mononen, I., Voncken, J., Noronkoski, T., Gonzalez-Gomez, I., Heisterkamp, N., and Groffen, J. (1996) A mouse model for the human lysosomal disease aspartylglycosaminuria. Nat. Med., 2, 1375–1378.[ISI][Medline]

Kakkis, E., Schuchman, E., He, X., Wan, Q., Kania, S., Wiemelt, S., Hasson, C., O'Malley, T., Weil, M., Aguirre, G., and others. (2001) Enzyme replacement therapy in feline mucopolysaccharidosis I. Mol. Genet. Metab., 72, 199–208.[CrossRef][ISI][Medline]

Lundblad, A., Masson, P., Nordén, N., Svensson, S., Öckerman, P.-A., and Palo, J. (1976) Structural determination of three glycoasparagines isolated from the urine of a patient with aspartylglycosaminuria. Eur. J. Biochem., 67, 209–214.[Abstract]

Makino, M., Kojima, T., and Yamashina, I. (1966) Enzymatic cleavage of glycopeptides. Biochem. Biophys. Res. Commun., 24, 961–966.[CrossRef][ISI][Medline]

Maury, P. (1979) Accumulation of two glycoasparaginases in the liver in aspartylglycosaminuria. J. Biol. Chem., 254, 1513–1515.[Abstract]

Maury, C. (1980) Accumulation of glycoprotein-derived metabolites in neural and visceral tissues in aspartylglucosaminuria. J. Lab. Clin. Med., 96, 838–844.[ISI][Medline]

Mononen, I. (1981) Quantitative analysis, by gas-liquid chromatography and mass fragmentography, of monosaccharides after methanolysis and deamination. Carbohyd. Res., 88, 39–50.[CrossRef][ISI]

Mononen, T. and Mononen, I. (1997) Biochemistry and biochemical diagnosis of aspartylglycosaminuria. In Mononen, I. and Aronson, N. (Eds.), Lysosomal storage disease: aspartylglycosaminuria, R.G. Landes Company/Springer-Verlag, Austin, TX, pp. 41–54.

Mononen, I., Heisterkamp, N., Dunder, U., Romppanen, E.-L., Noronkoski, T., Kuronen, I., and Groffen, J. (1995) Recombinant glycosylasparaginase and in vitro correction of aspartylglycosaminuria. FASEB J., 9, 428–433.[Abstract/Free Full Text]

Noronkoski, T. and Mononen, I. (1997) Influence of L-fucose attached {alpha}1 ->6 to the asparagine-linked N-acetylglucosamine on the hydrolysis of the N-glycosidic linkage by human glycosylasparaginase. Glycobiology, 7, 217–220.[Abstract]

Park, E., Manzella, S., and Baenziger, J. (2003) Rapid clearance of sialylated glycoproteins by the asialoglycoprotein receptor. J. Biol. Chem., 278, 4597–4602.[Abstract/Free Full Text]

Stinchi, S., Lüllmann-Rauch, R., Hartmann, D., Coenen, R., Beccari, T., Orlacchio, A., von Figura, K., and Saftig, P. (1999) Targeted disruption of the lysosomal {alpha}-mannosidase gene results in mice resembling a mild form of human {alpha}-mannosidosis. Hum. Mol. Genet., 8, 1365–1372.[Abstract/Free Full Text]

Sugahara, K., Akasaki, M., Funakoshi, I., Aula, P., and Yamashina, I. (1977) Structure of two glycoasparagines isolated from the urine of patients with aspartylglycosaminuria (AGU). J. Biochem., 82, 1499–1501.[Abstract]

Tomatsu, S., Orii, K., Vogler, C., Nakayama, J., Levy, B., Grubb, J., Gutierrez, M., Shim, S., Yamaguchi, S., Nishioka, T., and others. (2003) Mouse model of N-acetylgalactosamine-6-sulfate sulfatase deficiency (Galns–/–) produced by targeted disruption of the gene defective in Morquio A disease. Hum. Mol. Genet., 12, 3349–3358.[Abstract/Free Full Text]

Vliegenthart, J., Dorland, L., and Van Halbeek, H. (1983) High resolution H-1 nuclear magnetic resonance spectroscopy as a tool in the structural analysis of carbohydrates related to glycoproteins. Adv. Carbohydr. Chem. Biochem., 41, 209–374.[ISI]

Vogler, C., Barker, J., Sands, M., Levy, B., Galvin, N., and Sly, W. (2001) Murine mucopolysaccharidosis VII: Impact of therapies on the phenotype, clinical course, and pathology in a model of a lysosomal storage disease. Pediatr. Dev. Pathol., 4, 421–433.[CrossRef][ISI][Medline]





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
15/1/79    most recent
cwh145v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Kelo, E.
Articles by Mononen, I.
PubMed
PubMed Citation
Articles by Kelo, E.
Articles by Mononen, I.