(Received for publication, February 5, 1997, and in revised form, April 3, 1997)
From the Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba R3E OW3, Canada
Two benign mutations, C739T(R247W) and
C745T(R249W), in the -subunit of
-hexosaminidase A (Hex A)
have been found in all but one of the currently identified Hex
A-pseudodeficient subjects. To confirm the relationship of the benign
mutations and Hex A pseudodeficiency and to determine how the benign
mutations reduce Hex A activity, we transiently expressed each of the
benign mutations, and other mutations associated with infantile,
juvenile, and adult onset forms of GM2
gangliosidosis, as Hex S (
) and Hex A (
) in COS-7 cells.
The benign mutations decreased the expressed Hex A and Hex S activity
toward the synthetic substrate
4-methylumbelliferyl-6-sulfo-
-N-acetylglucosaminide (4-MUGS) by 60-80%, indicating that they are the primary cause of Hex A pseudodeficiency. Western blot analysis showed that the benign
mutations decreased the enzymatic activity by reducing the
-subunit
protein level. No change in heat sensitivity, catalytic activity, or
the substrate specificity to the synthetic substrates, 4-methylumbelliferyl-
-N-acetylglucosaminide or
4-methylumbelliferyl-6-sulfo-
-N-acetylglucosaminide, was detected. The effects of the benign mutations on Hex A were further analyzed in fibroblasts, and during transient expression, using
pulse-chase metabolic labeling. These studies showed that the
benign mutations reduced the
-subunit protein by affecting its
stability in vivo, not by affecting the processing of the
-subunit, i.e. phosphorylation, targeting, or secretion.
Our studies also demonstrated that these benign mutations could be readily differentiated from disease-causing mutations using a transient
expression system.
The lysosomal hydrolase, -hexosaminidase
(
-N-acetylhexosaminidase, EC 3.2.1.52), has two major
isoenzyme forms, A (Hex A)1 and B (Hex B),
and a minor form, S (Hex S) (reviewed in Ref. 1). These isozymes are
dimers formed from the
-subunit encoded by HEXA and/or
the
-subunit encoded by HEXB. The primary natural substrate of this enzyme, GM2 ganglioside, is only
hydrolyzed by the
dimer, Hex A, with the help of the
GM2 activator protein (GM2A gene), which binds
and solubilizes the sphingolipid for hydrolysis (2, 3). The presence of
mutation on both alleles of any one of the genes, HEXA,
HEXB, or GM2A, can result in a loss of Hex A
(
) activity in vivo and GM2 gangliosidosis
(reviewed in Ref. 4).
HEXA mutations that cause a complete Hex A deficiency and
Tay-Sachs disease, have a higher frequency among Ashkenazi Jews (5) and
French Canadians of Eastern Quebec (6). Screening programs for the
prevention of this disease have been established (7). They utilize a
synthetic substrate,
4-methylumbelliferyl--N-acetylglucosaminide (4-MUG), in
combination with a heat denaturation step, to differentiate the
activities of the heat-stable Hex B (
) from Hex A (
) (8, 9). Through these programs, healthy individuals with a low in
vitro Hex A activity (i.e. pseudodeficient) have been
identified (10-15). Pseudodeficiency complicates prenatal diagnosis
because a pseudodeficient fetus may be wrongly diagnosed as affected
(14, 16).
Two benign mutations, C739T(R247W) and C745T(R249W), have been
identified in subjects with Hex A pseudodeficiency (17, 18). Pseudodeficient subjects are compound heterozygotes, typically having
the C739T mutation on one chromosome and a common Tay-Sachs disease
mutation on the other. Studies of the percentage of Hex A in their
samples using the synthetic substrate 4-MUG, revealed very low levels
in serum (0-15%), and higher levels in leukocyte (13-24%) and
fibroblast (8-26%) samples (10-15). Using GM2
ganglioside, fibroblast-loading studies and enzyme assays gave results
in the low normal range (10, 11, 13, 14, 17). Metabolic labeling studies in fibroblasts from two subjects harboring the benign mutation
C739T showed that the -subunit protein was processed to its mature
lysosomal size (13, 14). Although this analysis was not quantitative,
the protein level was consistent with that which might be expected in a
Tay-Sachs disease heterozygote. Some investigators proposed that the
enzyme's capacity to hydrolyze the synthetic, but not the natural,
substrate was reduced (10, 11, 17). Others suggested that there was a
reduction in the activity of the enzyme toward both synthetic and
natural substrates and that a differential tissue distribution
accounted for the very low serum levels (13, 14).
Our aims were to determine 1) if the benign mutations are the primary cause of Hex A pseudodeficiency; 2) if the benign mutations can be differentiated from disease-causing mutations using a transient expression system; and 3) how the benign mutations lead to reduced Hex A activity.
The vectors pBluescript
(pBS) and pSVL were purchased from Stratagene and Pharmacia Biotech,
Inc., respectively. The -subunit expression vector pCD43 (19) and
pTK18 plasmid (20) were gifts from Dr. Roy Gravel (McGill University,
Montreal). The
-galactosidase expression vector, pRc/CMV-
-gal,
was provided by Dr. D. Litchfield (University of Western Ontario,
London, Canada). Double-stranded DNA sequencing kits were obtained from
Pharmacia; Geneclean II kits from BIO/CAN Scientific (Mississauga,
Canada); restriction and DNA modifying enzymes from New England Biolabs
or Life Technologies, Inc.; Nucleobond AX kits from the Nest Group Inc.
(Southport, MA);
-minimum essential medium (
-MEM), phosphate-free
medium, fetal calf serum, antibiotics, and rabbit polyclonal antiserum against human fibronectin from Life Technologies, Inc.;
PansorbinTM from Calbiochem; methionine/cysteine-free
minimum essential medium (Met/Cys-free medium) from ICN
Pharmaceuticals; 4-MUG and
4-methylumbelliferyl-6-sulfo-
-N-acetylglucosaminide (4-MUGS) from Toronto Research Chemicals Inc. (Toronto, Canada); dialyzed fetal bovine serum, 4-methylumbelliferyl-
-galactoside, N-ethylmaleimide, E-64, and leupeptin from Sigma;
4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc) from Boehringer
Mannheim; and DEAE-Cellulose resin from Pharmacia. A polyclonal
antibody against human placental Hex A, provided by Dr. Don Mahuran
(University of Toronto) and prepared by Dr. Greg Lee (University of
British Columbia, Vancouver, Canada), was a gift from Dr. Roy Gravel
(McGill University, Montreal, Canada). The anti-
-subunit and
anti-Hex B specific polyclonal antibodies prepared in goat (21) were
gifts from Dr. Rick Proia (National Institutes of Health). ECL Western
blotting detection reagents and [35S]dATP were from
Amersham Corp. Tran35S-label
(L-[35S]methionine/L-[35S]cysteine)
was from ICN Pharmaceuticals. 32P and Entensify solutions
were from DuPont NEN. COS-7 cells were obtained from the American Type
Culture Collection (Rockville, MD). Fibroblast cell lines from
individuals with Hex A pseudodeficiency, TC72 (13) and GM04863 (14),
were from Dr. George Thomas (the Kennedy Institute, Baltimore, MD) and
the NIGMS (National Institutes of Health) Human Genetic Mutant Cell
Repository (Camden, NJ), respectively. These samples were identified as
subjects B (GM04863) and F (TC72) in a previous study (17), where they
were shown to be compound heterozygotes for the C739T(R247W) mutation
and a second mutation associated with Tay-Sachs disease. The second allele in GM04863 was 1278ins4 (22), and in TC72 it was 1073+1G
A (23, 24). The control fibroblast cell lines included normal WP09
(Winnipeg), normal MCH065, Tay-Sachs disease WG1881 (both from the
Repository for Mutant Human Cell Strains, Montreal, PQ) and Sandhoff
disease GM00294 (NIGMS Human Genetic Mutant Cell Repository, Camden,
NJ).
The
-subunit cDNA fragment from pTK18
(NarI/PstI) was subcloned into pBS+
(AccI/PstI) to create pHHEXA2 and into pSVL to
create
pSVL as described (25). The C739T mutation was introduced
into the
pSVL cDNA by replacing a
NdeI/SnaBI fragment with the same fragment from a
polymerase chain reaction product generated from fibroblast cDNA
containing C739T. The C745T mutation was introduced with the
oligonucleotide 5
-TACGCACGGCTCTGGGGTATCCG-3
using the
Unique Site Elimination (USE) mutagenesis method (26). The other
mutations were introduced into pHHEXA2 single-stranded DNA following
instructions from Bio-Rad, based on the procedure of Kunkel et
al. (27) and using T7 polymerase (28). The 5
-end phosphorylated
oligonucleotides 5
-TCCTGGGGACCAAGTATCCCTGGA-3
,
5
-ACGGCTCCGGGATATCCGTGTGC-3
, and
5
-GCTTTCCTCACTGGGGCTTGCTG-3
were used to create the
substitutions G805A(G269S) (29, 30), G749A(G250D) (31), and
C508T(R170W) (32), respectively. The reaction mix was separated by
electrophoresis on a 0.8% agarose gel, the newly synthesized DNA was
excised and purified using the Geneclean II kit, and 20-40% of the
product was transformed into Escherichia coli/DH5
. To
identify which colonies contained the mutant vector, the relevant
region was polymerase chain reaction-amplified and restriction
enzyme-digested with an enzyme whose site was created or destroyed by
the mutation (the base change G749A creates an EcoRV site;
C508T destroys a HpaII site; G805A creates a ScaI
site when an antisense oligonucleotide, 5
-CAAGGAGTCAGTAATCCAGAGTAC-3
containing a single base substitution and base deletion, is used in the
polymerase chain reaction). The mutagenized
-subunit cDNA
fragments were subcloned into pSVL (BamHI/XhoI)
to create the various mutant
pSVL(s). In all cases, the entire
cDNA insert was sequenced to confirm the presence of the desired,
but no additional, base changes.
COS-7 and human fibroblast cell lines were
cultured in -MEM containing 10% fetal bovine serum and penicillin
(100 units/ml)/streptomycin (100 µg/ml) at 37 °C in 5%
CO2. For some experiments, 200 µM leupeptin was added to the
-MEM when the cells were about 60% confluent.
Plasmid DNA for transfection was isolated
using Nucleobond AX columns and quantitated by gel electrophoresis and
absorbance at 260 nm. COS-7 cells, grown to 60-80% of confluence,
were collected by trypsinization, washed, and resuspended in PBS
containing 2.7 mM KCl, 1.1 mM
KH2PO4, 138 mM NaCl, 8.1 mM Na2HPO4, pH 7.6. Approximately 3 × 106 cells were mixed with the DNA in total volume
of 400 µl of PBS for transfection (6.5 µg of pSVL, or its
derivatives, alone for Hex S expression, and together with 2.3 µg of
pCD43 for Hex A plus S expression). Two µg of pRc/CMV-
-gal vector,
a concentration that did not influence the expressed Hex S and Hex A
plus S activities, was co-transfected to express
-galactosidase as a
measure of transfection efficiency. Expression vectors were introduced
into the cells by electroporation using an Electro Cell Manipulator 600 (BTX Inc., San Diego, CA) with the settings of 150 charging volts, 48 ohms, and 1200 microfarads (33). The medium was changed at 24 h,
and the cells were harvested at 72 h post-transfection.
Cell extracts for
enzyme assay were prepared differently from that for
immunoprecipitation. For enzyme assays, the cell monolayer was washed
with ice-cold PBS, scraped off into TEN buffer (40 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl) and
pelleted by centrifugation. Cells were resuspended in a 20 mM Tris-HCl, pH 7.0, and lysed by three rounds of freezing
(70 °C) and thawing (37 °C), and cell debris was removed by
centrifugation at 14,000 × g for 20 min. For
immunoprecipitation, cell and medium extracts were prepared according
to a protocol by Proia et al. (34) except that lysis buffer
and the solutions used in the preparation procedures contained 1 mM Pefabloc.
Protein
concentrations were determined by the Bradford method (35) using
-globulin as the standard. Hex S and Hex A plus S activities were
measured using 4-MUGS or 4-MUG as a substrate (9).
-Galactosidase
activity was determined using 4-methylumbelliferyl-
-galactoside as a
substrate (36).
Previously described protocols were modified to separate Hex A from Hex B and Hex S (1, 37). A DEAE-cellulose column (1.4-ml bed volume) was equilibrated with 10 mM phosphate buffer, pH 6.0, and 400-900 µl (1.2-3.0 mg of protein) of cell extract was loaded. After a 20-min incubation at 4 °C, the column was sequentially eluted with 6 ml of the phosphate buffer, 20 ml of a linear NaCl gradient (0-0.215 M), 5 ml of 0.215 M NaCl, 5 ml of 0.3 M NaCl, and 5 ml of 0.5 M NaCl, all in the phosphate buffer. One-ml fractions were collected, and the activity in each fraction was measured using 4-MUG and 4-MUGS. The fractions representing each isoenzyme form were pooled, dialyzed, and concentrated; their identity was determined by Western blot analysis. The fractions corresponding to Hex A were used for Km and Vmax determination.
Western Blot AnalysisCell extract proteins (equal amount of normalized COS-7 protein, typically 25 µg) were separated by SDS-PAGE as described (38). Fibroblast extracts were loaded (20-50 µg) on gels as controls. The proteins were transferred to a nitrocellulose membrane (MSI) according to Sambrook et al. (39). The anti-Hex A antibody (1:5,000) was used to probe the blot, and the bound antibody was detected using a 1:10,000 dilution of anti-rabbit horseradish peroxidase-conjugated antibody and the ECL solutions.
Cell Metabolic Labeling and ImmunoprecipitationProcedures
for cell labeling were based on the protocol of Hasilik and Neufeld
(40) except that PBS was used to wash the cell monolayer, Met/Cys-free
medium supplemented with antibiotics and L-glutamine was
used to deplete the intracellular pool of methionine and cysteine, and
20 µl (240 µCi) of Tran35S-label was employed for
labeling the cells in 2.9 ml of Met/Cys-free medium supplemented with
150 µl of dialyzed fetal bovine serum. For pulse-chase experiments,
3.6 ml of -MEM supplemented with 10% fetal bovine serum,
antibiotics, 0.075 mg/ml L-methionine, and 0.5 mg/ml
L-cysteine was used to replace the labeling medium. In the
experiments with protease inhibitors, the cells were cultured in the
presence of appropriate inhibitors throughout pulse and chase. In the
experiments with NH4Cl, the cells were cultured in 3.95 ml
of Met/Cys-free medium with 30 µl (360 µCi) of
Tran35S-label and 10 mM NH4Cl.
For cell labeling with 32Pi, phosphate-free medium supplemented with sodium pyruvate and L-glutamine was used to rinse the cell monolayer and to deplete the intracellular phosphate. Cells were labeled with a mix of 3.8 ml of this medium, 150 µl of dialyzed fetal bovine serum, and 1 µl of 32P (500 mCi/ml).
For immunoprecipitation, two methods were used. The first method was
according to Proia et al. (34) except that three different polyclonal antibodies, against human -subunit, Hex B, and Hex A,
were used for immunoprecipitation. In the second method, total
-subunit was first immunoprecipitated using anti-Hex A antibody and
following the method of Proia et al. (34). The
immunoprecipitated complexes were then dissociated by suspending the
antibody-antigen-Pansorbin cell pellet in 50 µl of a denaturing
solution (20 mM Tris-HCl, pH 7.4, 1% SDS, 20 mM dithiothreitol) and incubating at 100 °C for 10 min
followed by a centrifugation. This process was repeated once, and
supernatants were combined and mixed with 0.9 ml of lysis buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P40, 0.02% NaN3, 1 mM Pefabloc) containing 1%
bovine serum albumin and 4 mM N-ethylmaleimide.
N-Ethylmaleimide was used as a chelator of dithiothreitol in
the denaturing solution. The total
-subunit in the mix was then
immunoprecipitated using 4 µl of anti-
-subunit antibody and
following the method of Proia et al. (34). The process was
repeated once to completely recover the
-subunit.
High porous 5-12% gradient polyacrylamide separating gels (0.075 × 12 × 16 cm) and 4% polyacrylamide stacking gels were prepared and run as described previously (41, 42).
Fluorography and AutoradiographySignals from 35S-labeled proteins were detected by fluorography according to the instruction provided by the manufacturer of DuPont Entensify solution.
Autoradiography was used to detect the signals of the 32P-labeled proteins. The gel was incubated twice for 30 min in 50 ml of fixing solution (acetic acid:methanol:H2O = 10:20:70), dried, and exposed to Kodak X-Omat film.
To determine the effects of the benign mutations,
C739T(R247W) and C745T(R249W), on Hex A activity and the -subunit
protein, both a transient expression system and fibroblast cell lines
from normal and Hex A pseudodeficient subjects were employed. Hex S was
expressed and analyzed in this study because it is an
-subunit dimer, and if the benign mutation was present on both of the subunits in Hex S, their effects might be more obvious than on Hex A (
). Hex A was also expressed to mimic the physiological form of the enzyme.
To determine the effects of the benign mutations on the
activity of Hex S and the -subunit protein level, normal and mutant Hex S was expressed in COS-7 cells. Four mutant vectors, containing C739T(R247W), C745T(R249W), G805A(G269S), or C508T(R170W)
mutations associated with Hex A pseudodeficiency (two vectors), adult
onset and infantile forms of GM2 gangliosidosis,
respectively, were expressed. The results showed that normal Hex S
activity (1026 ± 200 nmol/h/mg of protein) was expressed to a
level more than 10 times that of the
-hexosaminidase activity in
COS-7 cells (82 ± 10 nmol/h/mg of protein). For comparison, the
wild-type Hex S activity, after subtracting the COS-7 cell background
and normalization, was expressed as 100%. The activities of the
various mutant Hex S isozymes were converted to a percentage of the
wild-type (Fig. 1). Data revealed that the activity of
Hex S harboring the benign mutations, R247W and R249W, was about
20-35% of the wild-type Hex S activity and was substantially higher
than that of Hex S containing the G269S substitution that is associated
with adult onset GM2 gangliosidosis.
The level of the mature -subunit protein containing the benign
mutations was also decreased to about 40% of the normal level (Fig.
2), consistent with its reduced enzyme activity. No
detectable mature form, but a normal level of the precursor form, of
the
-subunit corresponding to the G269S- and R170W-containing Hex S
was observed.
Analysis of Expressed Hex A plus S Activity and
To examine the effects of the benign mutations,
C739T(R247W) and C745T(R249W), on Hex A plus S activity and the
-subunit protein, normal and mutant Hex A plus S were transiently
expressed by introducing pCD43, together with
pSVL or its mutant
variants (including the G749A
pSVL construct, which contains a
mutation associated with juvenile onset GM2 gangliosidosis)
into COS-7 cells. Once again, the specific activity of the expressed
normal Hex A plus S was about 10-fold above the COS-7 cell background (Fig. 1). The specific activity of Hex A plus S harboring the benign
mutation, R247W and R249W, was about 38 and 22% of that of the normal
Hex A plus S, respectively (Fig. 1). Hex A plus S with the adult onset
disease G269S mutation in the
-subunit had about 11% of the normal
level of activity, significantly less than either benign mutation. The
activity of Hex A plus S containing the G749A(G250D) mutation was
less than 4% of the normal, consistent with a juvenile onset clinical
phenotype (43). The Tay-Sachs disease mutation R170W expressed a
specific activity similar to the COS-7 cell background (Fig. 1).
The levels of the mature -subunit protein expressed as Hex A plus S
were also correspondingly decreased by the benign mutations (Fig. 2,
B and C), but the level of the precursor
-subunit containing the benign mutations was similar to that of the
normal. The mature
-subunit was not detected in the extracts
prepared from expression of disease-causing mutations, although a
significant amount of the precursor was detected. The differences
between the benign mutations and disease-causing mutations appeared
more obvious at the level of the mature
-subunit protein than at the
activity level.
The co-transfection of pCD43 and pSVL in COS-7 cells under the
conditions used in these studies (see "Experimental Procedures") resulted in the formation of both Hex A and Hex S; Hex A was
predominant, although there was a significant level of Hex S produced
(Fig. 3). Anion exchange chromatography showed that Hex
A was formed when the
-subunit was co-expressed with the
-subunit
carrying the benign mutations (data not shown).
The level of normalized Hex S and Hex A plus S activities was found to
be influenced by the transfection efficiency. When transfection
efficiency was low, higher levels of both -hexosaminidase activity
and
-subunit protein were expressed from mutant vectors in
comparison with the normal vector. In Fig. 2B, expressed
levels of
-subunit protein were higher than those in Fig.
2C, where the transfection efficiency was higher.
The Hex A activity
and the -subunit protein in fibroblasts from Hex A pseudodeficient
subjects were compared with that from the normal. Hex A-pseudodeficient
fibroblasts had 36-41% of normal Hex A activity (Table
I) and a comparable level of mature
-subunit protein
(data not shown). This is consistent with previous studies where
pseudodeficient fibroblasts had Hex A activity that was 23-26% of the
total
-hexosaminidase activity compared with 49-65% of Hex A for
the normal (13). Leupeptin, a lysosomal protease inhibitor at 200 µM (44) did not increase the percentage of the specific
activity of Hex A (Table I) or the level of mature
-subunit protein
(data not shown) in either the normal or mutant fibroblasts.
|
Hex A was separated from Hex B and Hex S as shown in Fig. 3. The 0-0.215 M NaCl gradient, followed by 0.215 M NaCl, eluted almost all Hex A from the column, and 0.3 M NaCl eluted all of the Hex S (Fig. 3). The activity peaks corresponding to Hex A and Hex S possessed activities toward both the synthetic substrates, 4-MUG and 4-MUGS. The Hex B peak had activity only toward 4-MUG.
The identity of the eluted activities was confirmed by Western blot
(Fig. 3, inset). Only the -subunit was detected in the Hex B fractions;
- and
-subunits were in the Hex A fractions; and
only the
-subunit was in the 0.3 M NaCl-eluted Hex S
fraction. An additional peak, of unknown identity, was eluted with 0.5 M NaCl. This fraction contained both the
- and
-subunits and has previously been observed (37).
The benign mutations, R247W and R249W, do not
have a significant effect on the Km of Hex A for the
synthetic substrates, although the apparent Vmax
was substantially decreased (Table II). To determine if
the lower apparent Vmax was the result of a
change in catalytic activity of Hex A or a reduction of the -subunit
protein, the
-subunit protein levels of equal Hex A activities
toward 4-MUGS were analyzed by Western blot. The levels of protein in
normal and Hex A-pseudodeficient fibroblasts and the COS-7 cells
transfected with normal and benign mutation-containing
pSVLs were
similar (Fig. 4). This indicated that the benign
mutations affect Hex A by reducing the
-subunit protein level, not
by affecting Hex A's affinity toward the synthetic substrates and/or
Hex A's catalytic activity.
|
Heat Sensitivity of Benign Mutation-containing Hex A plus S
The activity of Hex A and S harboring the benign mutation,
R247W or R249W, in the -subunit, decreased at a rate similar to that
of the normal enzyme at 45 or 50.1 °C (data not shown). The results
from fibroblast and expressed Hex A plus S were shown to be similar
(data not shown). Similar results were also previously reported using
fibroblast extract and a treatment at 37 °C (14). Hex A plus S
containing the adult onset mutation G269S was shown to be more
sensitive than normal (data not shown; Ref. 25). These suggested that
the benign mutations do not increase the heat sensitivity of the
-subunit in vitro.
The optimal pH
for the hydrolysis of the synthetic substrate by Hex A in the
cell extracts from MCH065 and TC72 fibroblasts and the COS-7
cells, transfected with pCD43/pSVL or pCD43/C739T
pSVL or
pCD43/C745T
pSVL, was 4.0-4.4 (data not shown).
To determine the effects of the benign mutations on Hex A and S in transfected COS-7 cells and on Hex A in the fibroblasts from a normal and a Hex A-pseudodeficient subject with the C739T(R247W) on one chromosome and a null mutation on the other, pulse-chase metabolic labeling was used.
The specificity of the antibody against human Hex A, which was used for
immunoprecipitation, was analyzed; it was shown to recognize both the
free and -subunit-associated forms of the
-subunit (data not
shown). The anti-Hex B and anti-
-subunit antisera were confirmed to
have the specificity described by Proia (34). The anti-Hex B antibody
recognizes all
-subunit monomers and
-subunit containing dimers,
while the anti-
-subunit antibody recognizes only the
-monomer.
Fibroblasts from normal, Hex A-pseudodeficient,
Tay-Sachs disease and Sandoff disease subjects were pulse-labeled with
32P. The radiolabeled forms of -hexosaminidase were
immunoprecipitated using antibodies against human Hex A, Hex B, and the
-subunit, separated by gradient SDS-PAGE, and detected by
autoradiography. Three separate experiments showed a considerable
amount of precursor
-subunit in the free and
-subunit-associated
forms in both normal and the C739T benign mutation-containing
fibroblasts (data not shown), suggesting that Hex A with the benign
mutation was normally phosphorylated. The Tay-Sachs disease sample did
not show a 32P-labeled band corresponding to the
-subunit, and the Sandhoff disease sample did not exhibit a band
corresponding to the radiolabeled
-subunit.
Data derived from three repeated experiments showed that the
secretion of the precursor -subunit from both normal and Hex A-pseudodeficient fibroblasts was enhanced by growth in the presence of
NH4Cl (Fig. 5). The effect of
NH4Cl on the dissociation of Hex A and the mannose
6-phosphate receptor occurs in the prelysosomal and lysosomal
compartments (44). Normal secretion suggests that the benign mutation
did not affect the processing and targeting of Hex A.
Effect of Benign Mutations on the Stability of the
Expressed Hex S was studied
because fibroblasts containing the benign mutation C745T(R249W) were
not available and because the transient expression system
overexpressing the -subunit provided information not seen in
fibroblasts. Results (Fig. 6) showed that the normal and
mutant
-subunit precursors containing the benign mutation, C739T or
C745T, or an adult onset mutation, G805A, were present at a similar
level at 3 h of pulse. Benign mutation-containing
-subunit
precursors appeared to have levels similar to or slightly higher than
the wild type, and they were still present at 20 h of chase;
however, the G805A mutation-containing
-subunit precursor disappeared by 20 h of chase. The mature
-subunit containing the benign mutations reached its maximum level by 8 h of chase, about 3 h later than the normal, and its level, at 20 h of
chase, was significantly less than normal. The mature
-subunit with the G805A mutation was not detectable after 8 h of chase. These results suggested that the benign mutations produced mature
-subunit less stable than the wild type but more stable than the adult onset
G805A mutation. The delay in the decrease of the benign mutation-containing precursor suggests the rate of conversion from the
precursor
-subunit to its mature form is delayed. This may reflect
an effect on folding or dimerization.
Effect of the Benign Mutation, R247W, on the Stability of the
To test the
stability of the benign mutation-containing -subunit in
vivo, the experiments were designed differently from those in the
previous reports (13, 14). The differences are that 1) equal amounts of
cell extract protein were used for immunoprecipitation, and 2) two
approaches were used in immunoprecipitation. The first approach used
three types of antibodies to immunoprecipitate both precursors and the
mature forms of the
- and
-subunit; the second method used
anti-
-subunit antibodies in combination with anti-Hex A antibodies
to immunoprecipitate only
-subunit. Results derived using the first
method (data not shown) and the second method (Fig. 7)
appeared similar. The benign mutation-containing fibroblasts, at 3 h of pulse, produced about half the level of the
-subunit precursor
produced from normal fibroblasts. The benign mutation-containing mature
-subunit reached its maximal level at about 14 h of chase, similar to the normal. However, by 20 h of chase, the level of benign mutation-containing
-subunit was much lower than that of the
normal consistent with a defect in stability.
Effect of Protease Inhibitors, E-64 and Leupeptin, on the Stability of the
Fibroblasts from normal and Hex
A-pseudodeficient subjects were analyzed by pulse-chase metabolic
labeling in the presence of 280 µM E-64 or 105 µM leupeptin. The lysosomal protease inhibitors had no
apparent effect on the stability of the -subunit containing the
benign mutation R247W (data not shown). Leupeptin (105 µM) has successfully been used to increase the activity
and protein levels of arylsulfatase A in the fibroblasts from adults
with metachromatic leukodystrophy (46).
The benign mutations, C739T(R247W) and C745T(R249W), were originally identified in subjects with Hex A pseudodeficiency and in enzyme-defined Tay-Sachs disease carriers (6, 16, 17, 42). These mutations were clearly associated with enzyme deficiency, but we could not rule out the possibility that additional mutations, i.e. regulatory mutations, on the same allele were the cause of the enzyme deficiency.
Using a COS-7 expression system, the relationship between the benign
mutations and Hex A pseudodeficiency was confirmed. The levels of
expressed Hex S and Hex A plus S activities and their corresponding
-subunit protein were significantly decreased by the benign
mutations C739T and C745T (Figs. 1 and 2). Hex A-pseudodeficient fibroblasts also demonstrated similar results (Table I). This is the
first direct evidence that the benign mutations are the primary cause
of the Hex A pseudodeficiency.
Benign mutations, C739T and C745T, were readily differentiated from the
mutations associated with adult onset and other forms of
GM2 gangliosidosis using transient expression (Figs. 1 and 2). Normally there is an overlap between the percent Hex A activity that is associated with various phenotypes, including pseudodeficiency, when Hex A activity is determined in serum, leukocytes, or fibroblasts (10, 13-15). Our results extended a previous study by Brown and Mahuran (25), who showed that -/
-subunit co-expression could distinguish the adult onset mutation from those associated with more
severe forms of GM2 gangliosidosis. Our studies showed that the disease-causing mutations resulted in a lower percentage of Hex A
plus S, compared with normal, than that previously reported for the
-/
-subunit co-expression (25). This may be because 1) whole cell
extracts were used, instead of immunoprecipitated enzyme, to determine
Hex A plus S activity, and (2) there were differences in the ratio of
pSVL:pCD43, the DNA concentration, and the method used for DNA
transfection.
Data derived from the current studies and previous studies suggest that the benign mutations do not cause disease. The levels of Hex A activity in fibroblasts and leukocytes of Hex A-pseudodeficient subjects with the R247W and R249W amino acid changes (10-15, 17-18) are usually slightly higher than that found in a patient with juvenile or adult onset GM2 gangliosidosis (15). Results from in vitro and in situ GM2 ganglioside hydrolysis assays (13, 14), together with the results from the current transient expression studies, suggest that the level of the enzyme associated with benign mutation is above the critical threshold required for normal GM2 ganglioside hydrolysis. Indeed, several pseudodeficient subjects are presently more than 40 years old (17-18), and patients with the adult onset mutation G805A(G269S) typically show symptoms before the age of 40 years (15, 47). However, one cannot rule out the possibility of a late onset phenotype in these subjects.
The pH optimum of Hex A was not altered by the benign mutations,
indicating that the Arg247 and Arg249 residues
are not required for proton transfer in the catalytic reaction and are
not involved in the catalytic reaction. This is consistent with the
prediction that these residues are located on the surface of the
-subunit molecule (48), not in the active site where the substrate
binding and catalytic reaction occur.
Because the benign mutation did not alter Hex A's affinity for the
synthetic substrates and there was clearly a reduction in the
-subunit protein level, we attempted to determine the basis of the
decreased protein level. Triggs-Raine et al. (17) had
previously shown the
-subunit cDNA could be amplified after reverse transcription of mRNA isolated from Hex
A-pseudodeficient fibroblasts. This indicated that the
HEXA gene with the benign mutation, C739T(R247W), was
normally transcribed, because the other allele in the subject had the
1278ins4 mutation that does not produce a stable
-subunit mRNA
(49).
Previous studies (13, 14) also showed an apparently normal amount of
the precursor -subunit protein in Hex A-pseudodeficient fibroblasts. This was also observed in our Western blot analysis (Fig.
2), and pulse-labeling analysis (Figs. 6 and 7). Together these results
suggest that the benign mutations do not affect the
-subunit
biosynthesis.
The benign mutation, C739T(R247W), did not affect the phosphorylation
of the -subunit or the secretion of Hex A (Fig. 5), indicating that
1) the benign mutations did not cause Hex A to be trapped in the
endoplasmic reticulum or the Golgi apparatus, and 2) the mutant Hex A
could be normally targeted to the lysosome or to the prelysosomal
compartments. These results did not explain the absence of serum Hex A
in pseudodeficient subjects.
Further studies using both Hex A in fibroblasts and Hex S expressed in
COS-7 cells demonstrated that the benign mutations significantly
decreased the stability of the mature -subunit (Figs. 6 and 7). A
slight delay in the precursor conversion to its mature
-subunit as
expressed Hex S was also indicated, but this was not seen in the
fibroblasts. The difference might have resulted from the effect of Hex
S overexpression in COS-7 cells. These indicated that effects of the
benign mutations on the
-subunit at the prelysosomal level may also
exist.
The protease inhibitors, leupeptin and E-64, did not have an effect on
the decreased level of the mature -subunit by the benign mutations.
This may be because the benign mutations render the
-subunit
susceptible to a lysosomal protease(s) that cannot be inhibited by
these protease inhibitors, or the mutant
-subunit may be susceptible
to nonenzymatic factors.
The residues Arg247 and Arg249 fall in a highly
conserved region of exon 7 of -hexosaminidase from mammals,
bacteria, and yeast, suggesting that these residues are important in
maintaining normal Hex A function. They have been predicted to locate
on the surface of the
-subunit molecule and might be involved in
maintaining the stability of the
-subunit and/or in interacting with
the
-subunit or GM2 activator protein.
We are grateful to Dr. Hans Jacobs for critical review of the manuscript.