28
Biochimica et Biophysica Acta, 1097 (1991) 28-30 © 1991 Elsevier Science Publishers B.V. 0925-4439/91/$03.50 ADONIS 092544399100095D
BBADIS 61054
Glycoasparaginase in human urine V e s a K a a r t i n e n 1,2 1 Department of Clinical Chemistry, Kuopio Unicersity Central Hospital, Kuopio (Finland) and : Department of Chemistry, UniL,ersity of Kuopio, Kuopio (Finland) (Received 22 January 1991)
Key words: Aspartylglycosaminuria; Glycoasparaginase; (Human urine)
Glycoasparaginase was purified 15 000-fold from human urine. The enzyme is a tetrameric protein of 86 kDa, composed of two heavy chains (25 kDa) and two light chains (18 kDa). Its structure and properties are very similar to those of human leukocyte glycoasparaginase. Glycoasparaginase activity is totally absent from urine of aspartylglycosaminuria patients.
Introduction
In glycoprotein degradation, glycoasparaginase ( N 4(/3-glycosyl)-L-asparaginase, aspartylglucosaminidase, EC 3.5.1.26) catalyses the hydrolytic cleavage of the N-glycosidic linkage between asparagine and Nacetylglucosamine [1]. The deficient glycoasparaginase activity leads to a lysosomal storage disease called aspartylglycosaminuria (AGU, McKusick 20840), in which large amounts of various glycoasparagines accumulate in body fluids and tissues [2]. AGU is usually diagnosed by measuring the excreted aspartylglucosamine (GlcNAc-Asn) in urine samples and confirmed by glycoasparaginase assay in lymphocytes [2,3]. The only structurally characterized glycoasparaginases are those purified from rat and human liver and human leukocytes [4-6]. The heterodimeric rat hepatic glycoasparaginase (m = 43 kDa) is composed of a 20 kDa light (L) chain and a 24 kDa heavy (H) chain [4]. Human hepatic enzyme has been proposed to be heterotrimeric with a molecular mass of 64 kDa [5], whereas human leukocyte glycoasparaginase (m = 88 kDa) has a heterotetrameric structure containing two 19 kDa L-chains and two 25 kDa H-chains [6]. Cloning and nucleotide sequence analysis of cDNA of human leukocyte glycoasparaginase has indicated that the enzyme is encoded as a single 34.6-kDa polypeptide that is post-translationally cleaved to two subunits and glycosylated [7] similar to an otherwise uncharacterized placenta enzyme [8]. In this paper, glycoasparaginase
Correspondence: V. Kaartinen, Department of Clinical Chemistry, Kuopio University Central Hospital, SF-70210 Kuopio, Finland.
was purified from human urine and some of its properties were characterized. Application of urine samples for glycoasparaginase assay in diagnosis of aspartylglycosaminuria will be described. Materials and Methods
For glycoasparaginase activity in human urine, each sample (40 ml) was concentrated 20-fold and dialyzed against 50 mM Tris-HCl buffer (pH 8.0) using Amicon 8200 concentrator and YM-10 membranes (Danvers, MA, U.S.A.). The enzyme activity was determined by HPLC [9] except the kinetic studies, which were carried out using the photometric assay described by Tarentino and Maley [10]. Glycoasparaginase activity in normal human urine ranged from 243 to 640 /~U/mg
TABLE I
The characteristics of glycoasparaginase in human urine Activity controls AGU-patients Molecular weight light-chain heavy-chain N-terminal sequence light-chain heavy-chain pI Optimal pH K,1 for GlcNAc-Asn
243-640 p,U/mg prot. (mean 433 p~U/mg prot.; n = 10) 0 / z U / m g prot. (n = 6) 18 000 25 000 NH 2-T-I-G-M-V-V-I-H NH 2-S-X *-P-L-P-L-V-V 4.9-5.0 8.0 90 p~M
* X indicates that no amino acid signal above the background was detected in protein sequence analysis.
29 TABLE II
Purification of glycoasparaginase from human urine Step
Protein (mg)
Total activity (U)
Specific activity (mU/mg)
Yield (%)
Fold
1 Concentrate 2 Caprylic acid 3 DEAE-Sephadex A-50 4 DEAE-Sepharose CI-6B 5 Concanavalin A-Sepharose 6 Sephacryl S-200 HR 7 Alkyl-Superose 8 Mono-Q
15 000 12 000 6 750 1750 83 2.8 0.25 0.03
3.0 3.0 2.7 2.1 1.5 0.45 0.27 0.09
0.2 0.25 0.4 1.2 18 160 1 100 3 000
100 100 90 70 50 15 9 3
1 1.25 2.0 6.0 90 800 5 500 15 000
protein mean 433 ~ g / m g prot. (n = 10) (Table I), which is higher than in many human tissues [9]. Glycoasparaginase activity in urine from aspartylglycoaminuria patients was totally deficient (Table I). One unit indicates the amount of enzyme causing the loss of 1 p.mol substrate per min at 37°C. For isolation of glycoasparaginase in human urine, 100 1 of urine was collected from several healthy volunteers and concentrated to a vol. of 1 1 using a Pellicon ultrafiltration system (Millipore, Bedford, MA, U.S.A.) equipped with a PTTK00001 membrane (cut-off 30000). The retentate contained 15 g protein and 3.0 units of glycoasparaginase corresponding to a specific activity of 0.2 m U / m g protein meanwhile the filtrate did not contain any detectable amount of glycoasparaginase activity. The purification was continued by caprylic acid precipitation, anion-exchange chromatography (DEAE-Sephadex A-50 and DEAE-Sepharose CI-6B), affinity chromatography (Concanavalin A-Senharose) followed by HPLC using gel filtration (Se-
A
Mr x
(_)
phacryl S-200 HR, 1.6 × 50 cm, i.d.) and hydrophobic interaction chromatography (Alkyl Superose, 0.5 × 5.0 cm, i.d.) techniques [6] (Table II). At the final step, a broad protein peak with glycoasparaginase activity was eluted from the Mono-Q ion-exchange column (0.5 × 5.0 cm, i.d.) [6]. The enzyme preparation was dialyzed against 50 mM phosphate buffer (pH 7.5) containing 30% glycerol (v/v) and stored frozen. The specific activity of the final preparation was 3.0 U / m g prot., the purification was 15 000-fold and the recovery of the purification procedure was 3%. For the sequencing studies, the subunits of the enzyme were isolated by reverse-phase HPLC [6]. Glycoasparaginase of human leukocytes was isolated as described [6]. Results and Discussion
In native polyacrylamide gel electrophoresis, the purified urinary glycoasparaginase ran as a single major band and it was slightly smaller (m = 80 kDa; Fig.
B
Mr
10 -3
x 10 .3
(_)
C
(_)
pH
•| ~i
669 440
45.0 30.0
7.00
20.1
6.50
232 --"
140
5.10
14.4
1 2
(÷)
i~3
67
1
2 (÷)
4.65
3 (÷)
Fig. 1. The electrophoretic properties ot purlhecl lauman urinary and leukocyte glycoasparaginases. (A) Polyacrylamide gel electrophoresis of purified urinary glycoasparaginase (lane 1), leukocyte glycoasparaginase (lane 2) and the molecular weight markers (lane 3) on an 8-25% gradient gel. The electrophoresis was carried out on PhastSystemT M (Pharmacia LKB Biotechnology, Uppsala, Sweden) according to the manufacturer's instructions. (B) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of urinary glycoasparaginase (lane 1), leukocyte glycoasparaginase (lane 2) and the molecular weight markers (lane 3) on a 12% gel under reducing conditions. SDS-PAGE was carried out according to Laemmli [12]. (C) Isoelectric focusing (IEF) of purified urinary glycoasparaginase (lane 1), leukocyte glycoasparaginase (lane 2) and the marker proteins (lane 3) on a 3.0-9.0 gradic " ~el. IEF was performed on PhastSystem TM.All gels were stained with silver.
30 1A, lane 1) than glycoasparaginase from leukocytes (m = 83 kDa; Fig. 1A, lane 2). When the enzyme was treated with SDS under reducing conditions at 100°C for 3 min, the protein was dissociated to subunits of 18 and 25 kDa (Fig. 1B, lane 1). The size of the H-chains of the urinary and the leukocyte enzymes was similar, but the L-chain of the urinary enzyme was slightly smaller than that of the leukocyte enzyme. The Nterminal sequences of the subunits of urinary glycoasparaginase were determined using an Applied Biosysterns 477A protein sequencer equipped with on-line 120A PTH-analyzer (Foster City, CA, U.S.A.). The sequences (Table I) were identical to those of the subunits of human leukocyte glycoasparaginases [6]. In the second sequencing cycle of the heavy chain, no amino acid signal above the background was detected; based on the cDNA sequence analysis [8], the amino acid residue at this position is cysteine. The isoelectric point of the urinary enzyme was at pH 4.9-5.0 (Fig. 1C) and it showed considerably less charge heterogeneity than the leukocyte enzyme. These findings along with the different electrophoretic mobilities on both SDS- and native-PAGE may reflect different glycosylation pattern in the L-chain of the urinary and leukocyte enzymes. Like glycoasparaginase from leukocytes, the urinary enzyme had a broad pH-optimum around pH 8.0 and retained approx. 95% of its initial activity after incubation for 60 min at 60°C. The Michaelis constant (K m) of the urinary glycoasparaginase for GlcNAc-Asn was 90 /xM that corresponds to 110 /xM of the leukocyte enzyme [6]. The lysosomal hydrolases in urine originate mainly from the proximal tube epithelial cells of kidneys and to a smaller extent from plasma, and their excretion rate is determined by genetic factors [11]. Human urine contains a considerable amount of glycoasparaginase activity with a specific activity higher than that in plasma, blood cells and tissues studied so far. The structure and other characteristics of the urinary gly-
coasparaginase are very similar to those of the leukocyte enzyme [6] indicating a general expression of the gene encoding tetrameric form of glycoasparaginase. The glycoasparaginase activity is totally deficient in urine from aspartylglycosaminuria patients. Therefore, urine can be used for determination of both GlcNAcAsn excretion and glycoasparaginase activity in diagnostics of aspartylglycosaminuria.
Acknowledgements This work was financially supported by the Finnish Academy of Sciences. I thank Dr. Ilkka Mononen, M.D., from the Division of Medical Genetics, Childrens Hospital of Los Angeles, for comments on the manuscript and Dr. John Tomich, Ph.D., from the same institute, for discussions.
References 1 Makino, M., Kojima, T. and Yamashina, I. (1966) Biochem. Biophys. Res. Commun. 24, 961-966. 2 Beaudet, A.L. and Thomas, G.H. (1989) in The metabolic basis of inherited disease (Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D., eds.), pp. 1603-1621, McGraw-Hill, New York. 3 Mononen, I., Kaartinen, V. and Mononen, T. (1988) Scand. J. Clin. Lab. Invest. 48 Suppl 191, 7-11. 4 Tollersrud, O.K. and Aronson, N.N. (1989) Biochem. J. 260, 101-108. 5 Baumann, M., Peltonen, L., Aula, P. and Kalkkinen, N. (1989) Biochem. J. 262,189-194. 6 Kaartinen, V., Williams, J.C., Tomich, J., Yates, J.R., III, Hood, L.E. and Mononen. I. (1991) J. Biol. Chem. 266, 5860-5869. 7 Mononen, I., Heisterkamp, N., Kaartinen, V., Williams, J.C., Yates J.R., III, Griffin, P.R., Hood, L.E. and Groffen, J. (1991) Proc. Natl, Acad. Sci. USA 88, 2941-2945. 8 Fisher, K.J., Tollersrud, O.K. and Aronson, N.N. (1990) Febs. Lett. 269, 440-444. 9 Kaartinem V. and Mononen, I. (1990) Anal. Biochem. 190, 98101. 10 Tarentino, A.L. and Maley, F. (1969) Arch. Biochem. Biophys. 130, 295-303. 11 Paigen, K., Peterson, J. and Ward, E.A. (1984) Biochem. Genet. 22, 517-527. 12 Laemmli, U.K. (1970) Nature 227, 68(I-685.
Biochimica et Biophysica Acta, 1097 (1991) 28-30 © 1991 Elsevier Science Publishers B.V. 0925-4439/91/$03.50 ADONIS 092544399100095D
BBADIS 61054
Glycoasparaginase in human urine V e s a K a a r t i n e n 1,2 1 Department of Clinical Chemistry, Kuopio Unicersity Central Hospital, Kuopio (Finland) and : Department of Chemistry, UniL,ersity of Kuopio, Kuopio (Finland) (Received 22 January 1991)
Key words: Aspartylglycosaminuria; Glycoasparaginase; (Human urine)
Glycoasparaginase was purified 15 000-fold from human urine. The enzyme is a tetrameric protein of 86 kDa, composed of two heavy chains (25 kDa) and two light chains (18 kDa). Its structure and properties are very similar to those of human leukocyte glycoasparaginase. Glycoasparaginase activity is totally absent from urine of aspartylglycosaminuria patients.
Introduction
In glycoprotein degradation, glycoasparaginase ( N 4(/3-glycosyl)-L-asparaginase, aspartylglucosaminidase, EC 3.5.1.26) catalyses the hydrolytic cleavage of the N-glycosidic linkage between asparagine and Nacetylglucosamine [1]. The deficient glycoasparaginase activity leads to a lysosomal storage disease called aspartylglycosaminuria (AGU, McKusick 20840), in which large amounts of various glycoasparagines accumulate in body fluids and tissues [2]. AGU is usually diagnosed by measuring the excreted aspartylglucosamine (GlcNAc-Asn) in urine samples and confirmed by glycoasparaginase assay in lymphocytes [2,3]. The only structurally characterized glycoasparaginases are those purified from rat and human liver and human leukocytes [4-6]. The heterodimeric rat hepatic glycoasparaginase (m = 43 kDa) is composed of a 20 kDa light (L) chain and a 24 kDa heavy (H) chain [4]. Human hepatic enzyme has been proposed to be heterotrimeric with a molecular mass of 64 kDa [5], whereas human leukocyte glycoasparaginase (m = 88 kDa) has a heterotetrameric structure containing two 19 kDa L-chains and two 25 kDa H-chains [6]. Cloning and nucleotide sequence analysis of cDNA of human leukocyte glycoasparaginase has indicated that the enzyme is encoded as a single 34.6-kDa polypeptide that is post-translationally cleaved to two subunits and glycosylated [7] similar to an otherwise uncharacterized placenta enzyme [8]. In this paper, glycoasparaginase
Correspondence: V. Kaartinen, Department of Clinical Chemistry, Kuopio University Central Hospital, SF-70210 Kuopio, Finland.
was purified from human urine and some of its properties were characterized. Application of urine samples for glycoasparaginase assay in diagnosis of aspartylglycosaminuria will be described. Materials and Methods
For glycoasparaginase activity in human urine, each sample (40 ml) was concentrated 20-fold and dialyzed against 50 mM Tris-HCl buffer (pH 8.0) using Amicon 8200 concentrator and YM-10 membranes (Danvers, MA, U.S.A.). The enzyme activity was determined by HPLC [9] except the kinetic studies, which were carried out using the photometric assay described by Tarentino and Maley [10]. Glycoasparaginase activity in normal human urine ranged from 243 to 640 /~U/mg
TABLE I
The characteristics of glycoasparaginase in human urine Activity controls AGU-patients Molecular weight light-chain heavy-chain N-terminal sequence light-chain heavy-chain pI Optimal pH K,1 for GlcNAc-Asn
243-640 p,U/mg prot. (mean 433 p~U/mg prot.; n = 10) 0 / z U / m g prot. (n = 6) 18 000 25 000 NH 2-T-I-G-M-V-V-I-H NH 2-S-X *-P-L-P-L-V-V 4.9-5.0 8.0 90 p~M
* X indicates that no amino acid signal above the background was detected in protein sequence analysis.
29 TABLE II
Purification of glycoasparaginase from human urine Step
Protein (mg)
Total activity (U)
Specific activity (mU/mg)
Yield (%)
Fold
1 Concentrate 2 Caprylic acid 3 DEAE-Sephadex A-50 4 DEAE-Sepharose CI-6B 5 Concanavalin A-Sepharose 6 Sephacryl S-200 HR 7 Alkyl-Superose 8 Mono-Q
15 000 12 000 6 750 1750 83 2.8 0.25 0.03
3.0 3.0 2.7 2.1 1.5 0.45 0.27 0.09
0.2 0.25 0.4 1.2 18 160 1 100 3 000
100 100 90 70 50 15 9 3
1 1.25 2.0 6.0 90 800 5 500 15 000
protein mean 433 ~ g / m g prot. (n = 10) (Table I), which is higher than in many human tissues [9]. Glycoasparaginase activity in urine from aspartylglycoaminuria patients was totally deficient (Table I). One unit indicates the amount of enzyme causing the loss of 1 p.mol substrate per min at 37°C. For isolation of glycoasparaginase in human urine, 100 1 of urine was collected from several healthy volunteers and concentrated to a vol. of 1 1 using a Pellicon ultrafiltration system (Millipore, Bedford, MA, U.S.A.) equipped with a PTTK00001 membrane (cut-off 30000). The retentate contained 15 g protein and 3.0 units of glycoasparaginase corresponding to a specific activity of 0.2 m U / m g protein meanwhile the filtrate did not contain any detectable amount of glycoasparaginase activity. The purification was continued by caprylic acid precipitation, anion-exchange chromatography (DEAE-Sephadex A-50 and DEAE-Sepharose CI-6B), affinity chromatography (Concanavalin A-Senharose) followed by HPLC using gel filtration (Se-
A
Mr x
(_)
phacryl S-200 HR, 1.6 × 50 cm, i.d.) and hydrophobic interaction chromatography (Alkyl Superose, 0.5 × 5.0 cm, i.d.) techniques [6] (Table II). At the final step, a broad protein peak with glycoasparaginase activity was eluted from the Mono-Q ion-exchange column (0.5 × 5.0 cm, i.d.) [6]. The enzyme preparation was dialyzed against 50 mM phosphate buffer (pH 7.5) containing 30% glycerol (v/v) and stored frozen. The specific activity of the final preparation was 3.0 U / m g prot., the purification was 15 000-fold and the recovery of the purification procedure was 3%. For the sequencing studies, the subunits of the enzyme were isolated by reverse-phase HPLC [6]. Glycoasparaginase of human leukocytes was isolated as described [6]. Results and Discussion
In native polyacrylamide gel electrophoresis, the purified urinary glycoasparaginase ran as a single major band and it was slightly smaller (m = 80 kDa; Fig.
B
Mr
10 -3
x 10 .3
(_)
C
(_)
pH
•| ~i
669 440
45.0 30.0
7.00
20.1
6.50
232 --"
140
5.10
14.4
1 2
(÷)
i~3
67
1
2 (÷)
4.65
3 (÷)
Fig. 1. The electrophoretic properties ot purlhecl lauman urinary and leukocyte glycoasparaginases. (A) Polyacrylamide gel electrophoresis of purified urinary glycoasparaginase (lane 1), leukocyte glycoasparaginase (lane 2) and the molecular weight markers (lane 3) on an 8-25% gradient gel. The electrophoresis was carried out on PhastSystemT M (Pharmacia LKB Biotechnology, Uppsala, Sweden) according to the manufacturer's instructions. (B) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of urinary glycoasparaginase (lane 1), leukocyte glycoasparaginase (lane 2) and the molecular weight markers (lane 3) on a 12% gel under reducing conditions. SDS-PAGE was carried out according to Laemmli [12]. (C) Isoelectric focusing (IEF) of purified urinary glycoasparaginase (lane 1), leukocyte glycoasparaginase (lane 2) and the marker proteins (lane 3) on a 3.0-9.0 gradic " ~el. IEF was performed on PhastSystem TM.All gels were stained with silver.
30 1A, lane 1) than glycoasparaginase from leukocytes (m = 83 kDa; Fig. 1A, lane 2). When the enzyme was treated with SDS under reducing conditions at 100°C for 3 min, the protein was dissociated to subunits of 18 and 25 kDa (Fig. 1B, lane 1). The size of the H-chains of the urinary and the leukocyte enzymes was similar, but the L-chain of the urinary enzyme was slightly smaller than that of the leukocyte enzyme. The Nterminal sequences of the subunits of urinary glycoasparaginase were determined using an Applied Biosysterns 477A protein sequencer equipped with on-line 120A PTH-analyzer (Foster City, CA, U.S.A.). The sequences (Table I) were identical to those of the subunits of human leukocyte glycoasparaginases [6]. In the second sequencing cycle of the heavy chain, no amino acid signal above the background was detected; based on the cDNA sequence analysis [8], the amino acid residue at this position is cysteine. The isoelectric point of the urinary enzyme was at pH 4.9-5.0 (Fig. 1C) and it showed considerably less charge heterogeneity than the leukocyte enzyme. These findings along with the different electrophoretic mobilities on both SDS- and native-PAGE may reflect different glycosylation pattern in the L-chain of the urinary and leukocyte enzymes. Like glycoasparaginase from leukocytes, the urinary enzyme had a broad pH-optimum around pH 8.0 and retained approx. 95% of its initial activity after incubation for 60 min at 60°C. The Michaelis constant (K m) of the urinary glycoasparaginase for GlcNAc-Asn was 90 /xM that corresponds to 110 /xM of the leukocyte enzyme [6]. The lysosomal hydrolases in urine originate mainly from the proximal tube epithelial cells of kidneys and to a smaller extent from plasma, and their excretion rate is determined by genetic factors [11]. Human urine contains a considerable amount of glycoasparaginase activity with a specific activity higher than that in plasma, blood cells and tissues studied so far. The structure and other characteristics of the urinary gly-
coasparaginase are very similar to those of the leukocyte enzyme [6] indicating a general expression of the gene encoding tetrameric form of glycoasparaginase. The glycoasparaginase activity is totally deficient in urine from aspartylglycosaminuria patients. Therefore, urine can be used for determination of both GlcNAcAsn excretion and glycoasparaginase activity in diagnostics of aspartylglycosaminuria.
Acknowledgements This work was financially supported by the Finnish Academy of Sciences. I thank Dr. Ilkka Mononen, M.D., from the Division of Medical Genetics, Childrens Hospital of Los Angeles, for comments on the manuscript and Dr. John Tomich, Ph.D., from the same institute, for discussions.
References 1 Makino, M., Kojima, T. and Yamashina, I. (1966) Biochem. Biophys. Res. Commun. 24, 961-966. 2 Beaudet, A.L. and Thomas, G.H. (1989) in The metabolic basis of inherited disease (Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D., eds.), pp. 1603-1621, McGraw-Hill, New York. 3 Mononen, I., Kaartinen, V. and Mononen, T. (1988) Scand. J. Clin. Lab. Invest. 48 Suppl 191, 7-11. 4 Tollersrud, O.K. and Aronson, N.N. (1989) Biochem. J. 260, 101-108. 5 Baumann, M., Peltonen, L., Aula, P. and Kalkkinen, N. (1989) Biochem. J. 262,189-194. 6 Kaartinen, V., Williams, J.C., Tomich, J., Yates, J.R., III, Hood, L.E. and Mononen. I. (1991) J. Biol. Chem. 266, 5860-5869. 7 Mononen, I., Heisterkamp, N., Kaartinen, V., Williams, J.C., Yates J.R., III, Griffin, P.R., Hood, L.E. and Groffen, J. (1991) Proc. Natl, Acad. Sci. USA 88, 2941-2945. 8 Fisher, K.J., Tollersrud, O.K. and Aronson, N.N. (1990) Febs. Lett. 269, 440-444. 9 Kaartinem V. and Mononen, I. (1990) Anal. Biochem. 190, 98101. 10 Tarentino, A.L. and Maley, F. (1969) Arch. Biochem. Biophys. 130, 295-303. 11 Paigen, K., Peterson, J. and Ward, E.A. (1984) Biochem. Genet. 22, 517-527. 12 Laemmli, U.K. (1970) Nature 227, 68(I-685.
