Hum. Genet. 44, 79--87 (1978) © by Springer-Verlag 1978
Lysosomal Acid Hydrolases in Established Lymphoblastoid Cell Lines, Transformed by Epstein-Barr Virus, From Patients With Genetic Lysosomal Storage Diseases R. Minami, Y. Watanabe, T. Kudoh, M. Suzuki, K. Oyanagi, T. Orii, and T. Nakao Department of Pediatrics, Sapporo Medical College, S.1 W.16 Chuo-Ku, Sapporo, Japan
Summary. Lysosomal acid hydrolases were determined in established lymphoblastoid cell lines, transformed in vitro by Epstein-Barr virus (EBV) from lymphocyte-rich cell populations isolated from the peripheral blood of patients with genetic lysosomal storage diseases--Hurler syndrome, Scheie syndrome, GMl-gangliosidosis type 1 and type 2, Tay-Sachs disease, and I-cell disease--and from obligate heterozygotes for these diseases. The respective enzyme activity was undectectable in lymphoblastoid cells from the patients, but not from controls. Obligate heterozygotes could not always be distinguished from controls in lymphoblastoid cells as well as in leukocytes. These results suggest that established lymphoblastoid cell lines are useful material for the enzymatic study of genetic lysosomal storage diseases.
Introduction Long-term lymphoblastoid cell lines have been established from patients with genetic metabolic disorders, and the activity of the underlying deficient enzyme in the cells has been assayed (Turner et al., 1974; Beratis et al., 1975; Glade and Beratis, 1976). Lymphoblastoid cell lines originating from B-lymphocytes were easily established from peripheral blood by incubation with EBV (Katsuki and Hinuma, 1976). Lymphoblastoid cell lines have several advantages over skin fibroblast lines, such as a more readily obtainable material, far less cytogenetic variation, more rapid cell proliferation without senescence, more massive production of cellular material, and easier storage in the frozen state followed by reestablishment in culture (Povey et al., 1973; Beratis et al., 1975; Glade and Beratis, 1976). Minami et al. (1977) found that a-L-iduronidase activity in lymphoblastoid cells from patients with Hurler and Scheie syndrome was undetectable, while activity in control cells was clearly detectable. In the present study we investigated the levels of activity of lysosomal hydrolases in lymphoblastoid cell lines from patients with genetic lysosomal storage diseases (Hurler
0340-6717/78/0044/0079/$ 01.80
80
R. Minami et al.
s y n d r o m e , S c h e i e s y n d r o m e , G M l - g a n g l i o s i d o s i s t y p e 1 a n d t y p e 2, T a y - S a c h s disease, a n d I-cell d i s e a s e ) , f r o m o b l i g a t e h e t e r o z y g o t e s , a n d f r o m c o n t r o l subjects.
Materials and Methods Subjects Six patients with Hurler syndrome, Scheie syndrome, GMi-gangliosidosis type 1 or type 2, TaySachs disease, or I-cell disease, ten obligate heterozygotes (all parents except GMl-gangliosidosis type 2), and four control subjects. The diagnosis in all patients had been determined by typical clinical pictures and biochemical findings.
Methods Lymphoblastoid Cell Lines. Long-term lymphoblastoid cell lines were established by incubation with EBV as described previously (Minami et al., 1977). Each lymphoblastoid cell-suspension culture was centrifuged. Cells were washed twice with 0.9% saline and stored as cell pellets at -70 ° C until analysis.
Plasma andLeukocytes. Plasma was prepared from freshly drawn heparinized peripheral blood and stored at - 7 0 ° C. Leukocytes were obtained by the method described by Orii et al. (1976) and stored as cell pellets at - 7 0 ° C until required.
Sample Preparation. To the cell pellets of lymphoblastoid cells or leukocytes was added an aliquot of ice-cold distilled water, and the mixture was then treated with an sonicator (Model 200M, Kubota) for 2 min. This sonically disrupted suspension was used for the enzyme assay.
Assay of Enzymes a-L-iduronidase activity was determined in the manner described previously (Minami et al., 1977). Other lysosomal hydrolases were assayed in the following incubation mixtures, using the fluorimetric technique with 4-methylumbelliferyl (4-MU) substrates (Koch-Light): fl-D-galactosidase: 0.1 ml of enzyme preparation, 0.5 ml of 0.8 mM 4-MU-fl-D-galactopyranoside .in 0.1 M citrate-phosphate buffer containing 50 mM NaC1, pH4.0; N-acetyl-fl-D-glucosaminidase (total hexosaminidase): 0.1 ml of enzyme preparation, 0.5 ml of 1 mM 4-MU-N-acetyl-fl-Dglucosaminide in 0.1 M citrate-phosphate buffer, pH 4.0; a-D-mannosidase: 0.1 ml of enzyme preparation, 0.5 ml of 1 mM 4-MU-a-D-mannopyranoside in 0.1 M citrate-phosphate buffer, pH 4.5; a-L-fucosidase: 0.1 ml of enzyme preparation 0.5 ml of 1 mM 4-MU-a-L-fucopyranoside in 0.1 M citrate-phosphate buffer, pH 5.5; fl-D-glucuronidase: 0.1 ml of enzyme preparation, 0.5 ml of 1 mM 4-MU-fl-D-glucuronide in 0.1 M citrate-phosphate buffer, pH 5.0. Each incubation was carried out at 37° C for 60 min. The reaction was stopped with 3 ml of 0.25 M glycine buffer, pH 10.7. Fluorescence was read immediately on a Hitachi spectrophotofluorometer (Model MPF-4) with an excitation wavelength 365 mla, emission wavelength 450 mg. Protein was assayed according to the method of Lowry et al. (1951).
Isoelectric Focusing The LKB 2117 Multiphor Apparatus (LKB Instruments, Stockholm, Sweden) was used. Isoelectric focusing was carried out in thin-layer acrylamide gel using a pH gradient of 3.5--9.0 (Ampholine PAG plate) on a ground-glass cooling plate through which water at 4°C was
Lysosomal Hydrolases in Lymphoblastoid Cells
81
circulated. Samples were inserted on small pieces of Whatman No. 3 filter paper. Filter paper strips that connected the gel to the cathodal and anodaI electrodes were soaked in 1 M NaOH and 1 M phosphoric acid, respectively. Focusing was continued for 105 min, during which time the voltage was increased stepwise from 210 to 1000 V (Turner et al., 1975).
Staining The staining mixture consisted of 1.0 mM 4-MU-fl-D-galactopyranoside or N-acetyl-fl-Dglucosaminide in 0.1 M citrate-phosphate buffer, pH 4.0. After a piece of Whatman No. 3 filter paper was removed, the gel was thoroughly soaked in each buffered substrate solution and incubated at 37°C for 60 min. The gel surface was then flooded with 0.25 M glycine buffer, pH 10.7. Bands of fl-D-galactosidase or N-acetyl-fl-D-glucosaminidase could be seen under longwave ultraviolet light (Manaslu-Light).
Results On the basis of protein content, control lymphoblastoid cells had activity similar to control leukocytes in a-L-fucosidase and a-D-mannosidase, about half activity in N-acetyl-/LDglucosaminidase, about 25% in fl-D-galactosidase, and about 10% in fl-D-glucuronidase and a-Liduronidase, although the number of controls was too small to have statistical significance.
Hurler and Seheie Syndromes a-L-iduronidase activity in lymphoblastoid cells and in leukocytes from a patient with Hurler syndrome was undetectable. N o overlap between controls and the obligate heterozygotes was observed on assays of a-L-iduronidase in lymphoblastoid cells and leukocytes (Table 1). In the Scheie syndrome, a-L-iduronidase activity was f o u n d to be deficient in lymphoblastoid cells and markedly reduced in leukocytes. The obligate heterozygotes could not be distinguished from controls by assay of a-L-iduronidase activity in leukocytes. However, the activity in lymphoblastoid cells from the father was within the carrier range, although not in those f r o m the m o t h e r (Table 2).
GMl-Gangliosidosis Type 1 and Type 2 fl-D-galactosidase activity in lymphoblastoid cells and leukocytes from the patient with GMi-gangliosidosis type 1 was markedly reduced. The activity of the obligate heterozygotes was within heterozygote range in both lymphoblastoid cells and leukocytes (Table 3). fl-D-galactosidase activity at p H 4,0 and 6.5 in lymphoblastoid cells f r o m patients with GM~-gangliosidosis type 1 and type 2 was f o u n d to be markedly reduced, and ratios o f p H 4 . 0 / p H 6.5 were also significantly lower than those in controls. The activity at p H 4.0 was higher than at p H 6.5 in type 1, while the activity at p H 6.5 was higher than at p H 4.0 in type 2 and in controls (Table 4). On isoelectric focusing in thin-layer acrylamide gel, fl-Dgalactosidase in lymphoblastoid cells from controls showed four different isozyme components (A', A, B, and C). In the patient with type 1, three c o m p o n e n t s (A', B, and C) were completely absent, while in the patient with type 2, B and C c o m p o n e n t s were completely absent and A' c o m p o n e n t was distinctly present (Fig. 1).
82
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Lysosomal Acid Hydrolases in Established Lymphoblastoid Cell Lines, Transformed by Epstein-Barr Virus, From Patients With Genetic Lysosomal Storage Diseases R. Minami, Y. Watanabe, T. Kudoh, M. Suzuki, K. Oyanagi, T. Orii, and T. Nakao Department of Pediatrics, Sapporo Medical College, S.1 W.16 Chuo-Ku, Sapporo, Japan
Summary. Lysosomal acid hydrolases were determined in established lymphoblastoid cell lines, transformed in vitro by Epstein-Barr virus (EBV) from lymphocyte-rich cell populations isolated from the peripheral blood of patients with genetic lysosomal storage diseases--Hurler syndrome, Scheie syndrome, GMl-gangliosidosis type 1 and type 2, Tay-Sachs disease, and I-cell disease--and from obligate heterozygotes for these diseases. The respective enzyme activity was undectectable in lymphoblastoid cells from the patients, but not from controls. Obligate heterozygotes could not always be distinguished from controls in lymphoblastoid cells as well as in leukocytes. These results suggest that established lymphoblastoid cell lines are useful material for the enzymatic study of genetic lysosomal storage diseases.
Introduction Long-term lymphoblastoid cell lines have been established from patients with genetic metabolic disorders, and the activity of the underlying deficient enzyme in the cells has been assayed (Turner et al., 1974; Beratis et al., 1975; Glade and Beratis, 1976). Lymphoblastoid cell lines originating from B-lymphocytes were easily established from peripheral blood by incubation with EBV (Katsuki and Hinuma, 1976). Lymphoblastoid cell lines have several advantages over skin fibroblast lines, such as a more readily obtainable material, far less cytogenetic variation, more rapid cell proliferation without senescence, more massive production of cellular material, and easier storage in the frozen state followed by reestablishment in culture (Povey et al., 1973; Beratis et al., 1975; Glade and Beratis, 1976). Minami et al. (1977) found that a-L-iduronidase activity in lymphoblastoid cells from patients with Hurler and Scheie syndrome was undetectable, while activity in control cells was clearly detectable. In the present study we investigated the levels of activity of lysosomal hydrolases in lymphoblastoid cell lines from patients with genetic lysosomal storage diseases (Hurler
0340-6717/78/0044/0079/$ 01.80
80
R. Minami et al.
s y n d r o m e , S c h e i e s y n d r o m e , G M l - g a n g l i o s i d o s i s t y p e 1 a n d t y p e 2, T a y - S a c h s disease, a n d I-cell d i s e a s e ) , f r o m o b l i g a t e h e t e r o z y g o t e s , a n d f r o m c o n t r o l subjects.
Materials and Methods Subjects Six patients with Hurler syndrome, Scheie syndrome, GMi-gangliosidosis type 1 or type 2, TaySachs disease, or I-cell disease, ten obligate heterozygotes (all parents except GMl-gangliosidosis type 2), and four control subjects. The diagnosis in all patients had been determined by typical clinical pictures and biochemical findings.
Methods Lymphoblastoid Cell Lines. Long-term lymphoblastoid cell lines were established by incubation with EBV as described previously (Minami et al., 1977). Each lymphoblastoid cell-suspension culture was centrifuged. Cells were washed twice with 0.9% saline and stored as cell pellets at -70 ° C until analysis.
Plasma andLeukocytes. Plasma was prepared from freshly drawn heparinized peripheral blood and stored at - 7 0 ° C. Leukocytes were obtained by the method described by Orii et al. (1976) and stored as cell pellets at - 7 0 ° C until required.
Sample Preparation. To the cell pellets of lymphoblastoid cells or leukocytes was added an aliquot of ice-cold distilled water, and the mixture was then treated with an sonicator (Model 200M, Kubota) for 2 min. This sonically disrupted suspension was used for the enzyme assay.
Assay of Enzymes a-L-iduronidase activity was determined in the manner described previously (Minami et al., 1977). Other lysosomal hydrolases were assayed in the following incubation mixtures, using the fluorimetric technique with 4-methylumbelliferyl (4-MU) substrates (Koch-Light): fl-D-galactosidase: 0.1 ml of enzyme preparation, 0.5 ml of 0.8 mM 4-MU-fl-D-galactopyranoside .in 0.1 M citrate-phosphate buffer containing 50 mM NaC1, pH4.0; N-acetyl-fl-D-glucosaminidase (total hexosaminidase): 0.1 ml of enzyme preparation, 0.5 ml of 1 mM 4-MU-N-acetyl-fl-Dglucosaminide in 0.1 M citrate-phosphate buffer, pH 4.0; a-D-mannosidase: 0.1 ml of enzyme preparation, 0.5 ml of 1 mM 4-MU-a-D-mannopyranoside in 0.1 M citrate-phosphate buffer, pH 4.5; a-L-fucosidase: 0.1 ml of enzyme preparation 0.5 ml of 1 mM 4-MU-a-L-fucopyranoside in 0.1 M citrate-phosphate buffer, pH 5.5; fl-D-glucuronidase: 0.1 ml of enzyme preparation, 0.5 ml of 1 mM 4-MU-fl-D-glucuronide in 0.1 M citrate-phosphate buffer, pH 5.0. Each incubation was carried out at 37° C for 60 min. The reaction was stopped with 3 ml of 0.25 M glycine buffer, pH 10.7. Fluorescence was read immediately on a Hitachi spectrophotofluorometer (Model MPF-4) with an excitation wavelength 365 mla, emission wavelength 450 mg. Protein was assayed according to the method of Lowry et al. (1951).
Isoelectric Focusing The LKB 2117 Multiphor Apparatus (LKB Instruments, Stockholm, Sweden) was used. Isoelectric focusing was carried out in thin-layer acrylamide gel using a pH gradient of 3.5--9.0 (Ampholine PAG plate) on a ground-glass cooling plate through which water at 4°C was
Lysosomal Hydrolases in Lymphoblastoid Cells
81
circulated. Samples were inserted on small pieces of Whatman No. 3 filter paper. Filter paper strips that connected the gel to the cathodal and anodaI electrodes were soaked in 1 M NaOH and 1 M phosphoric acid, respectively. Focusing was continued for 105 min, during which time the voltage was increased stepwise from 210 to 1000 V (Turner et al., 1975).
Staining The staining mixture consisted of 1.0 mM 4-MU-fl-D-galactopyranoside or N-acetyl-fl-Dglucosaminide in 0.1 M citrate-phosphate buffer, pH 4.0. After a piece of Whatman No. 3 filter paper was removed, the gel was thoroughly soaked in each buffered substrate solution and incubated at 37°C for 60 min. The gel surface was then flooded with 0.25 M glycine buffer, pH 10.7. Bands of fl-D-galactosidase or N-acetyl-fl-D-glucosaminidase could be seen under longwave ultraviolet light (Manaslu-Light).
Results On the basis of protein content, control lymphoblastoid cells had activity similar to control leukocytes in a-L-fucosidase and a-D-mannosidase, about half activity in N-acetyl-/LDglucosaminidase, about 25% in fl-D-galactosidase, and about 10% in fl-D-glucuronidase and a-Liduronidase, although the number of controls was too small to have statistical significance.
Hurler and Seheie Syndromes a-L-iduronidase activity in lymphoblastoid cells and in leukocytes from a patient with Hurler syndrome was undetectable. N o overlap between controls and the obligate heterozygotes was observed on assays of a-L-iduronidase in lymphoblastoid cells and leukocytes (Table 1). In the Scheie syndrome, a-L-iduronidase activity was f o u n d to be deficient in lymphoblastoid cells and markedly reduced in leukocytes. The obligate heterozygotes could not be distinguished from controls by assay of a-L-iduronidase activity in leukocytes. However, the activity in lymphoblastoid cells from the father was within the carrier range, although not in those f r o m the m o t h e r (Table 2).
GMl-Gangliosidosis Type 1 and Type 2 fl-D-galactosidase activity in lymphoblastoid cells and leukocytes from the patient with GMi-gangliosidosis type 1 was markedly reduced. The activity of the obligate heterozygotes was within heterozygote range in both lymphoblastoid cells and leukocytes (Table 3). fl-D-galactosidase activity at p H 4,0 and 6.5 in lymphoblastoid cells f r o m patients with GM~-gangliosidosis type 1 and type 2 was f o u n d to be markedly reduced, and ratios o f p H 4 . 0 / p H 6.5 were also significantly lower than those in controls. The activity at p H 4.0 was higher than at p H 6.5 in type 1, while the activity at p H 6.5 was higher than at p H 4.0 in type 2 and in controls (Table 4). On isoelectric focusing in thin-layer acrylamide gel, fl-Dgalactosidase in lymphoblastoid cells from controls showed four different isozyme components (A', A, B, and C). In the patient with type 1, three c o m p o n e n t s (A', B, and C) were completely absent, while in the patient with type 2, B and C c o m p o n e n t s were completely absent and A' c o m p o n e n t was distinctly present (Fig. 1).
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