Vol.
172,
November
No.
BIOCHEMICAL
3, 1990
15,
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1990
GALACTOSIALIDOSIS: CARBOXY-TERMINAL
SIMULTANEOUS DEAMIDASE
AND
Ryoichi Kase’, Kohji Itoh’,
ACID
Nobuaki
Hitoshi Sakuraba’,
DEFICIENCY
and
1175-1179
OF ESTERASE,
CARBOXYPEPTIDASE
ACTIVITIES
Takiyama 12 9 , Akihiro Oshimal, Yoshiyuki Suzuki’
‘Department of Clinical Genetics The Tokyo Metropolitan Institute of Medical Science Honkomagome, Bunkyo-ku, Tokyo 113, Japan ‘Department
Received
August
30,
of Pediatrics, Saiseikai Utsunomiya Hospital Utsunomiya 320, Japan
1990
Esterase and deamidase activities at pH 7.0 and carboxypeptidase activity at pH 5.7 were markedly low or deficient in seven galactosialidosis fibroblast strains with deficient activity of “protective protein” for lysosomal Bgalactosidase and neuraminidase. No simultaneous deficiency of these three enzyme activities was observed in other lysosomal disease fibroblasts examined in this study. This result strongly suggests that “protective protein” is identical with a multifunctional protein with esterase/deamidase/carboxypeptidase activities and its mutation in galactosialidosis results in deficiency of these three enzyme activities. 0 19’30 Academrc Press, Inc.
Summary:
Galactosialidosis is an inherited lysosomal storage disease caused by deficiency of “protective protein”, which stabilizes lysosomal Bgalactosidase (EC 3.2.1.23) and activates lysosomal neuraminidase (EC 3.2.1.18) (l-4). Its molecular mechanism to regulate these two lysosomal enzymes in a high molecular weight complex is not known at present. Recently, human and murine “protective protein” cDNAs
ficant
have
been
cloned,
and the deduced amino acid sequence showed a signi-
homology to yeast carboxypeptidase Y and the KEXI
Furthermore,
an enzyme with
gene product (5,6).
esterase, peptidase and deamidase activities
purified from human platelets, and the sequences of the NH2-terminal
25 amino
acids in each of its two polypeptide chains were found to be identical those deduced for two fragments of “protective In this report, we describe deficient
was with
protein” (7).
activities
of esterase, carboxy-terminal
deamidase and acid carboxypeptidase in fibroblasts derived from patients with galactosialidosis, and their pathophysiological significance is discussed. Materials
and Methods
Materials Skin fibroblasts were obtained from seven galactosialidosis patients and normal and pathological control subjects. One of the control cell strains was pur-
Vol. 172, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
chased from the NIGMS Human Genetic Mutant Cell- Repository (Camden, NJ, U.S.A.). The diagnosis of galactosialidosis was established by clinical manifestations and biochemical data (8). Benzoyl-tyrosine ethyl ester (BTEE) and N-carbobenzoxy-L-phenylalanyl-L-leucine (N-CBZ-Phe-Lsu) yere purchased from Sigma MO U.S.A.); D-Ala -Met -enkephalinamide (DME-NH2), ~~~~~-ze~en~~h~~is~DME) and leupeptin from Peptide Institute (Osaka, N-acetyl-&glucosaminide from Nacalai Tesque Japan); and ii-methylumbellif&yl (Kyoto, Japan). Cell culture and enzyme preparation Fibroblasts were cultured in Ham’s F-10 medium supplemented with 10% fetal calf serum and antibiotics. The confluent cells were washed three times with phosphate-buffered saline, harvested and suspended in 1 mM by scraping, EDTA (pH 5.6) containing 0.25 mM leupeptin. The suspension was homogenized by sonication, and centrifuged at 10,000 x g for 15 min at 4’C. The supernatant was used for enzyme assays. Assay of esterase Esterase activity was measured by the method of Walsh and Wilcox using BTEE as substrate (9). The reaction mixture contained 0.5 mM BTEE, 100 mM sodium phosphate buffer (pH 7.0), and enzyme solution (!5-30 ug protein) in a final volume of 1 ml. The increase of absorbance at 256 nm was monitored in a thermo-regulated cuvette at 37’C using a spectrophotometer (Hitachi Model U3200; Tokyo, Japan). Assay of deamidase Deamidase activity was determined by measuring the amount of DME liberated from DME-NH (7). The enzyme preparation (15-20 ug protein) was incubated at 25’C in 33 0 uM DME-NH2 and 100 mM HEPES buffer (pH 7.0) in a final volume of 50 ul. After incubation for 15 min, the reaction was terminated by addition of 10 1.11of 0.76 N HCI. Product analysis was performed by reversephase high performance liquid chromatography using a LiChrospher RP-18(e) column (Cida-Merck 4x250 mm, particle size 5 urn; Kanto Chemical Co., Tokyo, The column was Japan) with a 4x4 mm guard column of the same material. isocratically developed with 0.1% trifluoroacetic acid-acetonitrile (80:20, v/v) at a flow rate of 1 ml/min at 40°C. The absorbance at 275 nm was monitored. The retention time was 11.4 min for DME-NH2 and 16.2 min for DME. Assay of acid carboxypeptidase Carboxypeptidase activity was measured using N-CBZ-Phe-Leu as substrate The reaction mixture contained 0.75 mM N-CBZ-Phe-Leu, 50 mM sodium (10). acetate buffer (pH 5.7), and enzyme solution (5-10 ug protein) in a final volume of 0.1 ml. After incubation at 25’C for 30 min, 0.1 ml of distilled water was added and the reaction was terminated by boiling for 3 min, and the released Lleucine was determined according to the method of Stevens et al. (11). Assay of &hexosaminidase BHexosaminidase activity was assayed using 4-methylumbelliferyl N-acetyl-@glucosaminide as substrate, as described previously (12). Protein determination Protein determination was performed by the method of Bradford (13) using bovine serum albumin as standard.
Results
and Discussion
Galactosialidosis was first recognized as a new disease with 6-galactosidase deficiency (14), followed by demonstrations of neuraminidase deficiency (15), “protective protein” deficiency (l), and carboxypeptidase deficiency (10) in patients’ fibroblasts. We also confirmed the carboxypeptidase deficiency in lymphoblastoid cells as well as in fibroblasts from galactosialidosis patients (Itoh, K., et al., unpublished data).
Furthermore, a recent study demonstrated that a purified human platelet enzyme with esterase, peptidase and deamidase activities has the same 1176
Vol.
172,
amino
No.
acid
sequence
investigate
in Table activity
RESEARCH
(71, and this
COMMUNICATIONS
report
prompted
were
low
or deficient at
activities
of esterase,
in galactosialidosis
observed
at pH 5.7 was
the
5-10%
levei
de-
The
fibroblasts.
of control
cells,
and
at pH 7.0 were below the limit of detection in
our assay methods for most of the cases examined.
One of the normal control
cell strains from a 5-year-old boy (GM05381) showed relatively both esterase and deamidase.
us to
in galactosialidosis.
1, markedly
esterase and deamidase activities
BIOPHYSICAL
protein”
activities
and carboxypeptidase
carboxypeptidase
AND
as “protective
these enzyme
As shown amidase,
BIOCHEMICAL
3, 1990
low activities
of
They may represent age-dependent changes, but
the condition of the culture cells should also be considered in this case. Deficient with
other
esterase or deamidase activities
were not specifically
lysosomal diseases in our present study.
#369) and siaiidosis (#319) showed normal or high
Table
associated
GMl-gangliosidosis
enzyme activities,
(#34,
excluding
1
Esrerase,deamidase,carboxypeptidase,and Bhexosaminidase
activities
in fibroblasts
Cell
strain
Age/Sex
Esterase-z
Deamidasr+
Peptidase#
a-Hex#
Galactosialidosis # 89 # 83 #186 tt a2 #274
18ylF
20ylM 23ylM
27y/F 32y/M
172,
November
No.
BIOCHEMICAL
3, 1990
15,
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1990
GALACTOSIALIDOSIS: CARBOXY-TERMINAL
SIMULTANEOUS DEAMIDASE
AND
Ryoichi Kase’, Kohji Itoh’,
ACID
Nobuaki
Hitoshi Sakuraba’,
DEFICIENCY
and
1175-1179
OF ESTERASE,
CARBOXYPEPTIDASE
ACTIVITIES
Takiyama 12 9 , Akihiro Oshimal, Yoshiyuki Suzuki’
‘Department of Clinical Genetics The Tokyo Metropolitan Institute of Medical Science Honkomagome, Bunkyo-ku, Tokyo 113, Japan ‘Department
Received
August
30,
of Pediatrics, Saiseikai Utsunomiya Hospital Utsunomiya 320, Japan
1990
Esterase and deamidase activities at pH 7.0 and carboxypeptidase activity at pH 5.7 were markedly low or deficient in seven galactosialidosis fibroblast strains with deficient activity of “protective protein” for lysosomal Bgalactosidase and neuraminidase. No simultaneous deficiency of these three enzyme activities was observed in other lysosomal disease fibroblasts examined in this study. This result strongly suggests that “protective protein” is identical with a multifunctional protein with esterase/deamidase/carboxypeptidase activities and its mutation in galactosialidosis results in deficiency of these three enzyme activities. 0 19’30 Academrc Press, Inc.
Summary:
Galactosialidosis is an inherited lysosomal storage disease caused by deficiency of “protective protein”, which stabilizes lysosomal Bgalactosidase (EC 3.2.1.23) and activates lysosomal neuraminidase (EC 3.2.1.18) (l-4). Its molecular mechanism to regulate these two lysosomal enzymes in a high molecular weight complex is not known at present. Recently, human and murine “protective protein” cDNAs
ficant
have
been
cloned,
and the deduced amino acid sequence showed a signi-
homology to yeast carboxypeptidase Y and the KEXI
Furthermore,
an enzyme with
gene product (5,6).
esterase, peptidase and deamidase activities
purified from human platelets, and the sequences of the NH2-terminal
25 amino
acids in each of its two polypeptide chains were found to be identical those deduced for two fragments of “protective In this report, we describe deficient
was with
protein” (7).
activities
of esterase, carboxy-terminal
deamidase and acid carboxypeptidase in fibroblasts derived from patients with galactosialidosis, and their pathophysiological significance is discussed. Materials
and Methods
Materials Skin fibroblasts were obtained from seven galactosialidosis patients and normal and pathological control subjects. One of the control cell strains was pur-
Vol. 172, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
chased from the NIGMS Human Genetic Mutant Cell- Repository (Camden, NJ, U.S.A.). The diagnosis of galactosialidosis was established by clinical manifestations and biochemical data (8). Benzoyl-tyrosine ethyl ester (BTEE) and N-carbobenzoxy-L-phenylalanyl-L-leucine (N-CBZ-Phe-Lsu) yere purchased from Sigma MO U.S.A.); D-Ala -Met -enkephalinamide (DME-NH2), ~~~~~-ze~en~~h~~is~DME) and leupeptin from Peptide Institute (Osaka, N-acetyl-&glucosaminide from Nacalai Tesque Japan); and ii-methylumbellif&yl (Kyoto, Japan). Cell culture and enzyme preparation Fibroblasts were cultured in Ham’s F-10 medium supplemented with 10% fetal calf serum and antibiotics. The confluent cells were washed three times with phosphate-buffered saline, harvested and suspended in 1 mM by scraping, EDTA (pH 5.6) containing 0.25 mM leupeptin. The suspension was homogenized by sonication, and centrifuged at 10,000 x g for 15 min at 4’C. The supernatant was used for enzyme assays. Assay of esterase Esterase activity was measured by the method of Walsh and Wilcox using BTEE as substrate (9). The reaction mixture contained 0.5 mM BTEE, 100 mM sodium phosphate buffer (pH 7.0), and enzyme solution (!5-30 ug protein) in a final volume of 1 ml. The increase of absorbance at 256 nm was monitored in a thermo-regulated cuvette at 37’C using a spectrophotometer (Hitachi Model U3200; Tokyo, Japan). Assay of deamidase Deamidase activity was determined by measuring the amount of DME liberated from DME-NH (7). The enzyme preparation (15-20 ug protein) was incubated at 25’C in 33 0 uM DME-NH2 and 100 mM HEPES buffer (pH 7.0) in a final volume of 50 ul. After incubation for 15 min, the reaction was terminated by addition of 10 1.11of 0.76 N HCI. Product analysis was performed by reversephase high performance liquid chromatography using a LiChrospher RP-18(e) column (Cida-Merck 4x250 mm, particle size 5 urn; Kanto Chemical Co., Tokyo, The column was Japan) with a 4x4 mm guard column of the same material. isocratically developed with 0.1% trifluoroacetic acid-acetonitrile (80:20, v/v) at a flow rate of 1 ml/min at 40°C. The absorbance at 275 nm was monitored. The retention time was 11.4 min for DME-NH2 and 16.2 min for DME. Assay of acid carboxypeptidase Carboxypeptidase activity was measured using N-CBZ-Phe-Leu as substrate The reaction mixture contained 0.75 mM N-CBZ-Phe-Leu, 50 mM sodium (10). acetate buffer (pH 5.7), and enzyme solution (5-10 ug protein) in a final volume of 0.1 ml. After incubation at 25’C for 30 min, 0.1 ml of distilled water was added and the reaction was terminated by boiling for 3 min, and the released Lleucine was determined according to the method of Stevens et al. (11). Assay of &hexosaminidase BHexosaminidase activity was assayed using 4-methylumbelliferyl N-acetyl-@glucosaminide as substrate, as described previously (12). Protein determination Protein determination was performed by the method of Bradford (13) using bovine serum albumin as standard.
Results
and Discussion
Galactosialidosis was first recognized as a new disease with 6-galactosidase deficiency (14), followed by demonstrations of neuraminidase deficiency (15), “protective protein” deficiency (l), and carboxypeptidase deficiency (10) in patients’ fibroblasts. We also confirmed the carboxypeptidase deficiency in lymphoblastoid cells as well as in fibroblasts from galactosialidosis patients (Itoh, K., et al., unpublished data).
Furthermore, a recent study demonstrated that a purified human platelet enzyme with esterase, peptidase and deamidase activities has the same 1176
Vol.
172,
amino
No.
acid
sequence
investigate
in Table activity
RESEARCH
(71, and this
COMMUNICATIONS
report
prompted
were
low
or deficient at
activities
of esterase,
in galactosialidosis
observed
at pH 5.7 was
the
5-10%
levei
de-
The
fibroblasts.
of control
cells,
and
at pH 7.0 were below the limit of detection in
our assay methods for most of the cases examined.
One of the normal control
cell strains from a 5-year-old boy (GM05381) showed relatively both esterase and deamidase.
us to
in galactosialidosis.
1, markedly
esterase and deamidase activities
BIOPHYSICAL
protein”
activities
and carboxypeptidase
carboxypeptidase
AND
as “protective
these enzyme
As shown amidase,
BIOCHEMICAL
3, 1990
low activities
of
They may represent age-dependent changes, but
the condition of the culture cells should also be considered in this case. Deficient with
other
esterase or deamidase activities
were not specifically
lysosomal diseases in our present study.
#369) and siaiidosis (#319) showed normal or high
Table
associated
GMl-gangliosidosis
enzyme activities,
(#34,
excluding
1
Esrerase,deamidase,carboxypeptidase,and Bhexosaminidase
activities
in fibroblasts
Cell
strain
Age/Sex
Esterase-z
Deamidasr+
Peptidase#
a-Hex#
Galactosialidosis # 89 # 83 #186 tt a2 #274
18ylF
20ylM 23ylM
27y/F 32y/M
