From the The Research Institute, The Hospital for
Sick Children, Toronto, Ontario M5G 1X8, Canada, the
§ Department of Laboratory Medicine and Pathobiology,
University of Toronto, Toronto, Ontario M5G 2C4, Canada, and the
¶ Department of Neurology and Neurosurgery, McGill University and
Montreal Neurological Institute,
Montreal, Quebec H3A 2B4, Canada
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ABSTRACT |
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The GM2 gangliosidoses are
caused by mutations in the genes encoding the - (Tay-Sachs) or
-
(Sandhoff) subunits of heterodimeric
-hexosaminidase A (Hex A), or
the GM2 activator protein (AB variant), a
substrate-specific co-factor for Hex A. Although the active site
associated with the hydrolysis of GM2 ganglioside, as well as part of the binding site for the ganglioside-activator complex, is
associated with the
-subunit, elements of the
-subunit are also
involved. Missense mutations in these genes normally result in the
mutant protein being retained in the endoplasmic reticulum and
degraded. The mutations associated with the B1-variant of Tay-Sachs are
rare exceptions that directly affect residues in the
-active site.
We have previously reported two sisters with chronic Sandhoff disease
who were heterozygous for the common HEXB deletion allele.
Cells from these patients had higher than expected levels of mature
-protein and residual Hex A activity, ~20%. We now identify these
patients' second mutant allele as a C1510T transition encoding a
-Pro504
Ser substitution. Biochemical
characterization of Hex A from both patient cells and cotransfected CHO
cells demonstrated that this substitution (a) decreases the
level of heterodimer transport out of the endoplasmic reticulum by
~45%, (b) lowers its heat stability, (c)
does not affect its Km for neutral or charged
artificial substrates, and (d) lowers the ratio of units of
ganglioside/units of artificial substrate hydrolyzed by a factor of 3. We concluded that the
-Pro504
Ser mutation directly
affects the ability of Hex A to hydrolyze its natural substrate but not
its artificial substrates. The effect of the mutation on ganglioside
hydrolysis, combined with its effect on intracellular transport,
produces chronic Sandhoff disease.
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INTRODUCTION |
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The hydrolysis of GM2 ganglioside
(GM2)1 requires
the proper synthesis, intracellular transport, and protein-protein
interactions of three different gene products. Two of these, encoded by
the evolutionarily related HEXA (15q23-q24 (1)) and
HEXB (5q13 (2)) genes, are the - and
-subunits of
heterodimeric
-hexosaminidase A (Hex A), respectively. The third
gene product is a small heat-stable protein, the GM2
activator protein (activator), encoded by the GM2A gene
(5q31.3-33.1 (3)). Mutations in any one of these genes can result in
the storage of GM2 and one of the family of human diseases
known as the GM2 gangliosidoses. HEXA mutations are associated with Tay-Sachs disease, HEXB with Sandhoff
disease, and GM2A with the AB variant form (reviewed in Ref.
4).
The GM2 gangliosidoses show extreme variability in clinical expression. Typically, the earlier the age of onset of clinical symptoms the more severe the disease. A nomenclature based on the different clinical phenotypes and recognizing the dominance of the encephalopathy (rather than only the age of onset) has been suggested (4): acute (the classical infantile form), subacute (late infantile and juvenile forms), and chronic (adult and chronic forms). The most common, acute form is a severe neurological disorder that usually results in death within 4 years. Mutations associated with this classical phenotype prevent the formation of any functional Hex A. It is now generally believed that the broad range of less severe phenotypes result from small variations in the levels of residual Hex A activity, on the order of 0-5% (5). Healthy individuals with ~10% residual Hex A activity have been described (reviewed in Ref. 4).
While patients with the acute form of GM2 gangliosidosis
are deficient in Hex A activity, their total Hex activity, as measured by neutral artificial substrates, is significant. Tay-Sachs patients often have nearly normal levels of activity due to the presence of the
homodimeric Hex B isozyme (), and Sandhoff patients have 1-5%
of normal levels from homodimeric Hex S (
) (reviewed in Ref. 4).
Since any dimeric combination of
- and/or
-subunits produces an
active isozyme, each subunit must contain a potential active site. The
characteristics of the two sites have been examined in the two
homodimers (6) and in a novel form of Hex A with an inactive
-subunit due to an Arg211
Lys substitution (7).
These data indicate that the presence of the
-subunit affects the
Km and Vmax of the
active site toward neutral substrates (7). Whereas both subunits of Hex A are
equally capable of hydrolyzing neutral
-GlcNAc- or
-GalNAc-containing substrates, e.g.
4-methylumbelliferyl-
-N-acetylglucosamine (MUG), only
isozymes containing an
-subunit can efficiently hydrolyze negatively charged
-GlcNAc-6-sulfate-containing substrates,
e.g. methylumbelliferyl-
-N-acetylglucosamine-6-sulfate
(MUGS), i.e. the MUG/MUGS hydrolysis ratio for Hex B is
~300, for Hex A ~4, and for Hex S ~1 (7). The specificity of the
Hex isozymes for GM2 ganglioside indicates that it should
also be considered as a negatively charged substrate, presumably due to
the sialic acid residue attached to the penultimate Gal residue.
However, whereas Hex S as well as Hex A, but not Hex B, can hydrolyze
GM2 in the presence of detergents, only Hex A is functional
in vivo with the GM2 activator-GM2
ganglioside complex (reviewed in Ref. 4). Thus, some component(s) of
the
-subunit are necessary for the hydrolysis of
GM2 in vivo.
The exact role of the activator remains controversial (8-10). However,
it is generally agreed that it binds both the lipid and oligosaccharide
portions of GM2, extracting or at least lifting the
ganglioside out of the membrane, and then the complex interacts with
Hex A for hydrolysis (11). Hex B can hydrolyze the neutral, asialo
derivative of GM2, GA2, in the presence of
detergent, but it has little activity in the presence of activator.
Furthermore, it has been reported that the activator, even in the
absence of GM2, can slightly inhibit the hydrolysis of MUGS
by both Hex A and Hex S (reviewed in Refs. 12 and 13). These data
indicate that at least a portion of the binding site for the complex is also located in the -subunit. The required elements of the
-subunit may function by increasing the affinity of Hex A for the
complex and/or correctly orient the complex, allowing the efficient
hydrolysis of the terminal sugar from the ganglioside. Furthermore,
these
-elements may act directly by interacting with the complex or indirectly by affecting the conformation of the
-subunit. Other functions associated with the
-subunit include greatly increasing the stability of the resulting dimer and facilitating the transport of
the
-subunit out of the endoplasmic reticulum (ER) (reviewed in
Refs. 4 and 14).
To date, all missense mutations except those at two codons, in either
HEX gene, result in normal levels of mutant mRNA but paradoxically with a dramatic reduction in both mature - and/or
-protein and Hex B and/or Hex A activity in patient cells. This is
believed to be the result of a strict "quality control system" in
the ER that prevents the transport and increases the degradation rate
of missfolded proteins or unassembled subunits (unlike
-subunits,
-subunits have an apparently low affinity for each other) (reviewed in Refs. 4 and 14). In several cases, it has been demonstrated that
subunits with missense mutations, even those associated with the most
severe clinical phenotype, are not totally incapable of forming a
partially functional Hex A but may be prevented from doing so by their
retention and degradation in the ER (15, 16). Thus, the major
detrimental effect caused by most HEX missense mutations is
at the level of intracellular transport rather than structural changes
specifically affecting some aspect of enzyme function. The exceptions
to this conclusion are the missense mutations at
-Arg178
(17, 18) and
-Asp258 (19), which produce the B1
biochemical phenotype. Patients with the most common Arg178
His substitution were originally thought to have an activator defect, because they express normal levels of both Hex A and Hex B
activities, as assayed with neutral (common) substrates,
e.g. MUG. However, unlike the normal Hex A found in the true
AB variants, Kytzia et al. (20) found that B1 variant-Hex A
was inactive toward an
-specific GlcNAc 6-sulfate-containing
substrate (as well as GM2 ganglioside even in the presence
of added activator protein), and they suggested the presence of a
mutation at or near the active site of the
-subunit. This hypothesis
has been demonstrated to be correct for substitutions at either residue based on mutational and expression studies of the aligned
-residues, i.e.
-analogs,
-Arg211 (21, 22) and
-Asp290 (23), and on molecular modeling of human Hex
using the structure of bacterial chitobiase (24).
We described 10 years ago two sisters of French Canadian ancestry with
a chronic Sandhoff phenotype (25). We have also previously reported
that these patients are heterozygous for the common 16-kb 5'
HEXB deletion allele, which does not transcribe -mRNA
(26). In this report, we characterize the second mutant allele in these patients, a missense mutation in exon 13 of the HEXB gene
that results in a Pro504
Ser substitution. This
mutation produces a novel biochemical phenotype that impacts directly
on the ability of Hex A to hydrolyze GM2. This is the first
report of a mutation in the
-subunit that affects the ability of Hex
A to hydrolyze its natural but not its artificial substrates and
localizes essential elements of the
-chain for natural substrate
hydrolysis to its C terminus.
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MATERIALS AND METHODS |
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Preparation of Genomic DNA-- Cultured fibroblasts were lysed by directly adding 1.0 ml of DNAZOL Reagent (Life Technologies, Inc.) to the 10-cm2 culture dish. The lysate was then transferred into an Eppendorf tube, and insoluble cell debris was removed by brief centrifugation. The genomic DNA in the supernatant was precipitated with ethanol and resuspended in 10 mM Tris-HCl buffer containing 1 mM EDTA, pH 7.4 (27).
RNA Isolation and Reverse Transcription-- Total RNA was isolated by using TRIzoI Reagent (Life Technologies, Inc.), as described by Hou et al. (28). Two µg of total RNA were used to synthesize the single strand cDNA according to the SUPERSCRIPTTM II procedure (Life Technologies). Briefly, RNA was first denatured at 70 °C for 10 min and then incubated at 42 °C for 50 min with 200 units of SUPERSCRIPT II and 0.2 µg of random primers in 20 µl of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 20 mM dithiothreitol, and 0.5 mM each of four dNTPs. Two µl of this mixture were directly used for PCR to synthesize and amplify double strand cDNA.
DNA Amplification and Direct Sequencing--
Amplification of
exonic and intron/exon junctions from genomic DNA and cDNA
fragments was performed by PCR as described previously (27). The
reactions were carried out in a 100-µl volume of 0.1-0.5-µg genomic DNA or 2 µl of cDNA (by reverse transcription), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 0.2 mM
each of four dNTPs, 0.5 µg of each primer, and 2.5 units of AmpliTaqTM Taq polymerase. Amplification was
achieved by incubation in a DNA Thermal Cycler (Perkin-Elmer) for 30 cycles, each consisting of 30 s of denaturation at 94 °C,
30 s of annealing at 55-60 °C, and 1-3 min of extension at
72 °C. The region around exon 13, found to be heterozygous for
Pro504 Ser mutation in genomic DNA and homozygous in
cDNA was amplified by PCR using oligonucleotides 129 (exon 10, sense; GGTTTTGGATATTATTGCAACCATAAA) and 14A (3'-untranslated region,
antisense; TCAATCAATAAAAATATTTTATTC). The resulting PCR products from
genomic DNA and cDNA were 799 and 716 bp, respectively. PCR
products were purified by utilizing the Geneclean Kit (Bio 101, Inc.,
Vista, CA), and direct sequencing was performed with
[
-35S]dATP using a modification of the
SequenaseTM protocol (U.S. Biochemical Corp.), as described
by McInnes et al. (27).
Generation of Mutant Constructs--
The wide type constructs,
pREP4- and pEFNEO-
, have been reported (7). The mammalian
expression vectors pREP4 (Invitrogen) and pEFNEO (kindly supplied by
Dr. Anson) (29), have hygromycin B and neomycin (G418) resistance
markers, respectively. To construct the mutant cDNA into pCD
vector, a 636-bp product by reverse transcription-PCR from patient
fibroblast (as described above) containing
-Pro504
Ser was digested with PflMI at a site 5' to the mutation and BanI at a site 3' to the mutation. The middle fragment of
387 bp was purified and subcloned into pCD
43 (21) treated with PfoMI/partial BanI. To generate the mutant
pEFNEO-
-Pro504
Ser, a 2.0-kb fragment, partially
digested by BamHI from pCD-
-Pro504
Ser,
was isolated and subcloned into the BamHI site of the pEFNEO-
vector. The mutation was verified by DNA sequencing. A
construct encoding an Asp208
Asn substitution in the
-cDNA insert of pEFNEO has previously been reported (23). In
permanently transfected CHO cells, this construct produces only
soluble, monomeric, precursor
-subunits (23). We now used this
transfected clonal CHO cell line as a control for the ER-retention on
mutant
-protein.
Cell Culture and DNA Transfection-- CHO cells were grown in minimal essential medium with 10% FCS and antibiotics at 37 °C in 5% CO2. Transfections were performed using Lipofection from Life Technologies, as described previously (7). Transfected cells were also grown in serum-free medium containing 10 mM NH4Cl, which diverts proteins targeted to the lysosome to the secretory pathway, for 1 and 2 days, and the Hex activity was measured using MUG.
Hex Activity Assay--
Cells were lysed in a buffer of 10 mM Tris-HCl, pH 7.5, and 5% glycerol through five sets of
freeze-thaw cycles. Protein from cell lysate was quantitated by the
Lowry method (30). Hex activity from cell lysates was determined using
a -chain-specific substrate MUGS and the common substrate MUG
(15).
Western Blotting-- The protein (amounts loaded are indicated in each figure) from cell lysates or DEAE fractions were resolved by SDS-PAGE using a Bio-Rad minigel system (31). Proteins were transferred to nitrocellulose overnight at 4 °C. The filter was blocked in 5% skim milk and then incubated overnight with primary antibody, rabbit anti-human Hex A (7, 28). Nitrocellulose was washed four times with 1% skim milk and incubated with a secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG for 1 h. The filter was developed and exposed to Hyperfilm using the ECL system (Amersham Pharmacia Biotech).
Separation of Hex Isozymes-- Proteins (3 mg) from lysates of patient or normal fibroblasts or from control or transfected CHO cells were applied to a 1.0-ml column of DEAE CL-6B (Amersham Pharmacia Biotech). The unbound Hex B fraction was collected with 10 mM sodium phosphate, pH 6.0. Hex A was eluted by applying 0.15 M NaCl in 10 mM sodium phosphate, pH 6.0 (7). Three-ml fractions were collected and assayed for Hex activity.
Kinetic Analysis-- The Km value was determined by varying the concentration of the substrates from 0.125 to 4.0 mM for MUG and from 0.05 to 2.5 mM for MUGS. Also, 10 experimental points were used for each Km determination. The normal and mutant Hex A from transfected CHO or patient cells were purified away from the other Hex isozymes by DEAE ion exchange chromatography (see above). Kinetic constants were calculated using a computerized nonlinear least squares curve fitting program for the Macintosh, KaleidaGraphTM 3.0 (7).
GM2 Hydrolysis
Assay--
[3H]GM2 ganglioside (20 nmol),
labeled in the C-6-position of its N-acetylgalactosamine
moiety (32), was incubated in the presence of 2.0 µg of recombinant
activator protein from bacteria (33, 34) at 37 °C for 18 h in
10 mM citrate buffer (pH 4.1), 0.5% human serum albumin,
and 10 mM GlcNAc (carrier), with 0, 50, 100, and 200 units
of Hex A (nmol of MUGS hydrolyzed/h), from normal or patient
fibroblasts or produced from human cDNAs (normal with normal or
mutant
) in transfected CHO cells (final volume of 100 µl). The
hydrolyzed product from GM2, i.e.
[3H]GalNAc, was separated from the unreacted
GM2 substrate by passage through a positively charged ion
exchange minicolumn of 0.6 ml of AG3X4 (acetate form) resin. The
unbound fraction containing [3H]GalNAc was determined by
liquid scintillation counting, as described previously (7).
Thermal Stability Study-- The wild-type and mutant Hex A or Hex B isozymes, which had been separated by DEAE chromatography, were added to 700 µl of preheated citrate phosphate buffer (pH 4.1) with 0.3% human serum albumin. The heat denaturation was performed at 45 °C, and aliquots (100 µl) were removed at intervals of 0, 15, 30, 45, 60, 75, and 90 min for Hex A and 0, 30, 60, 90, 120, 150, and 180 min for Hex B, placed on ice, and assayed for enzyme activity. The wild-type and mutant Hex A from transfected CHO cells were also tested for their residual MUGS activity after incubation at 37 °C for 18 h, under conditions that mimicked the natural substrate assay above.
Intracellular Localization of -Proteins Quantified Using
Indirect Immunofluorescence--
Nontransfected CHO cells or CHO cells
transfected with constructs encoding (a) the wild-type
-cDNA (lysosomal localization control), (b) the
Pro504
Ser substitution, or (c) an
Asp208
Asn substitution (ER localization control) were
grown at 37 °C in 5% CO2 on glass slide covers in a
10-cm2 culture dish. After 24 h of incubation, the
cells were fixed and gently permeabilized with 100% cold methanol at
20 °C for 30 min. The fixed cells were then washed in
phosphate-buffered saline, blocked with 1% bovine serum albumin, and
incubated with the primary polyclonal anti-Hex B antibody (35), diluted
1:200 for 1 h. The secondary antibody, a green fluorescein-labeled
goat-anti-rabbit IgG F(ab')2), diluted 1:100, was then added for 1 h, either alone or in combination with a 1:10,000 dilution of propidium
iodide, which in addition to nuclear DNA also stains the cytoplasmic
RNA and marks the position of the ER with the red fluorescence. The cells were then washed three times with phosphate-buffered saline and
mounted with elvanol. In control cultures, the preimmune rabbit IgG
substituted for the primary antibody. The slides were analyzed, and the
proportion of
-protein present in the ER or endosome/lysosome was
determined using a fluorescent microscope (Olympus Vanox-AH-3, magnification × 800) and two narrow band filters to detect the green and red fluorescence separately. An additional broad spectrum filter was also used for the simultaneous detection of the
fluorescein-tagged green Hex B and the nucleic acids labeled with red
propidium iodide fluorescence. In this setting, the overlapping of the
red and green labels in the cytoplasm is marked by yellow fluorescence and indicates the colocalization of Hex B and ER (36). Multiple images
of the same cell obtained with all of the above mentioned filters were
captured with the CCD camera (Optronix), stored in a Macintosh 9500 computer, and quantitatively analyzed using the Image Pro Plus program
(Media Cybernetics, Silver Spring, MD) according to the manufacturer's
instructions. In each of the three experimental groups (wild type
,
-Pro504
Ser, and
-Asp208
Asn),
images of 50 cells were analyzed, and results were statistically evaluated to give quantitative measurements of the percentage of each
-protein that resides in the ER and/or lysosome.
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RESULTS |
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Direct sequencing of the exons and exon/intron junctions of the
HEXB gene revealed that the patients were heterozygous for a
C1510T transition in exon 13 (+2 bp from intron 12) at the codon for
Pro504, which results in its conversion to a Ser codon
(Fig. 1A). We have previously
reported that the patients were also heterozygous for the common 16-kb
5' partial HEXB deletion allele, 16kb (26). To confirm
that this missense mutation was not part of the deletion allele, we
also sequenced the
-cDNA. In this case, the patients appear to
be homozygous for the missense mutation (Fig. 1B). Since a
HaeIII site was predicted to be lost in the presence of the C
T transition, the direct sequencing results from both genomic DNA
(Fig. 2A) and cDNA (Fig.
2B) were confirmed by HaeIII digestions of a
strategic PCR fragment from both patients and normal individuals. In
addition to the genomic PCR fragments from the five normal individuals
shown in Fig. 2A, samples from at least 45 other normal individuals were analyzed and found not to contain this mutation (data
not shown). Thus, the C
T transition in the Pro504
codon is not present in either the 16-kb deletion or any of the 100 normal HEXB alleles we analyzed.
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Interestingly, the residual Hex A activity present in the patients'
fibroblasts, ~20%, is only about half that found in cells from an
obligate carrier of Sandhoff disease (acute form), 5-10-fold higher
than the average levels of five cell lines from subacute patients
(Table I), and even slightly higher than
those reported for asymptomatic individuals with low Hex A activity
(10-15%) (5, 37, 38). We also investigated the levels of - and
-CRM in the patient's cells and compared them to levels found in
cell lines from a normal individual, a subacute patient (2.5% residual
Hex A activity), and an acute patient (0% residual Hex A activity)
(Fig. 3). The apparent levels of mature
-CRM in these samples were consistent with the decreased Hex A and B
activities reported in Table I, indicating that the specific activity
of the mutant Hex isozymes for artificial substrates had not changed. However, it was also apparent that there was a great increase in the
ratio of precursor/mature forms of the
- and/or
-polypeptides, suggesting that the
-Pro504
Ser mutation results in
the retention of a significant amount of newly synthesized
pro-
-chains in the ER (39, 40) and probably a more rapid turnover
rate (41). To confirm that the mutant mature polypeptides were not
being degraded in the lysosome, normal and patient cells were grown in
media containing leupeptin, which has been shown to inhibit the
turnover of mutant
-chains in the lysosome (21, 22). No dramatic
increase in either Hex activity or mature
-CRM was observed (Fig.
4).
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To fully characterize the biochemical effects of the Pro504
Ser mutation, CHO cells were permanently co-transfected with two cDNAs encoding the normal
- and the mutant
-polypeptides. A high producing clone was isolated and grown (Fig. 3). The Hex isozymes
from the lysate of these cells were separated by ion exchange
chromatography (Fig. 3). Since several mutations linked to the chronic
form of GM2 gangliosidosis have been shown to produce a
less heat-stable isozyme, as well as an increased retention of the
mutant subunit in the ER (15, 16, 42, 43), the T1/2 values of
both Hex isozymes carrying the mutant
-subunit were determined at
45 °C (Table II). Consistent with
these previous observations, the
-Pro504
Ser
substitution decreases the heat stability of both the A and B isozymes
(Table II).
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The ability of the -Pro504
Ser substitution to
inhibit ER to Golgi transport was directly confirmed by
immunofluorescence microscopy (Fig. 5).
CHO cells permanently transfected with either the wild-type (Fig.
5B) or mutant
-cDNAs encoding the Pro504
Ser (Fig. 5D) or Asp208
Asn (Fig.
5C) substitutions were examined. The latter substitution results in only monomeric, precursor
-chains in transfected cells (23) and thus serves as a control for ER retention (39, 44). Quantitative measurements of the green versus yellow
(overlapping of the red and green labels in the cytoplasm,
i.e. ER) fluorescence in 50 cells from each group indicated
that virtually all of the Asp208
Asn
-protein
(97 ± 2%) is present in the ER of transfected cells, compared
with 60 ± 10% of the
-Pro504
Ser protein and
15 ± 5% of the wild-type
-chain (Fig.
6). Furthermore, CHO cells co-transfected
with the wild type
and
and those transfected with
- and
-Pro504
Ser were grown in 10 mM
NH4Cl, and the MUG activity was measured in the media.
Wild-type transfected cells secreted 1.1 × 104
nmol/h/ml on day 1 and 2.4 × 104 units on day 2. Cells expressing the mutant
-cDNA secreted 0.18 × 104 units on day 1 and 0.28 × 104 units
on day 2. Thus, diverting the mutant Hex from the lysosomes to the
secretory pathway did not result in a large increase in activity,
confirming that the loss of activity and
-CRM occurs at an early
point in protein transport, i.e. the ER.
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Due to the relatively high levels of residual, mutant Hex A activity,
i.e. -Pro504
Ser (
*, Hex A*),
that we found in both our patients' cells and in co-transfected CHO
cells, it remained difficult to explain why the patients should present
with any disease phenotype. One possibility would be that the
-mutation had some direct effect on the function of the Hex A*
isozyme. To address this question, we first examined the kinetic
behavior of Hex A* with the common and
-specific substrates, MUG and
MUGS, respectively. Kinetic analysis confirmed that Hex A* has the same
apparent Km values as the wild type isozyme for
these artificial substrates (Table II). We next tested the ability of
the Hex A* to hydrolyze its natural substrate, the GM2
activator-GM2 ganglioside complex. Using samples of Hex A
and Hex A* that contained the same number of MUGS units, we found that
the mutant isozyme is 3-fold less active toward the natural substrate
than is the wild type Hex A (Fig. 7,
Table II). Furthermore, we confirmed that the residual Hex A in the
patient's fibroblasts also had a decreased activity toward
GM2 as compared with MUGS (Table II, GM2/MUGS
ratio). Finally, we tested the stability of the wild type and mutant
Hex A over the 18-h, 37 °C incubation period used in the
GM2 hydrolysis assay. Both forms of Hex A lost some
activity toward MUGS over this time period; however, at the end of
18 h. the residual activity of the mutant form was only 15% less
than the wild type (Table II).
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DISCUSSION |
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We have previously demonstrated that two French Canadian patients
with chronic Sandhoff disease are heterozygous for the common 16kb
HEXB allele (26, 27). Since this allele produces no
-mRNA, the uncharacterized, second allele must be responsible for the 15-25% residual Hex A activity (using MUG) we reported to be
present in the patients' fibroblasts and for their mild chronic
phenotype. Western blotting with anti-
antiserum has also indicated
a similar reduction in the amount of mature
-protein (27). In this
report, we identify the second allele as a C1510T transition encoding a
Pro504
Ser substitution. We demonstrate that this
substitution is not found in either the 16-kb deletion allele or in 100 normal HEXB alleles we examined, indicating that it is the
single cause of the patients' biochemical and clinical phenotypes.
This conclusion was strengthened by studies of the
-Pro504
Ser mutant Hex A and Hex B isozymes produced
in CHO cells permanently transfected with the wild type
- and/or
mutant
-cDNAs. The mutant
-subunit produced isozymes with
decreased heat stability (Table II) and increased retention in the ER,
60 ± 10% as compared with 15 ± 5% for the wild type
(Figs. 5 and 6). The latter data indicate that the mutation reduces the
-containing Hex isozyme content in lysosomes by 30-60%. These data
are also consistent with the effects of other mutations producing the
chronic phenotype, i.e.
-Gly269
Ser (15,
45)
-Tyr180
His (16),
-Arg505
Gln
(43), and
-Ala543
Thr (42).
Dlott et al. (38) characterized the HEXB mutation
associated with clinically asymptomatic individuals whose biochemical phenotype of low levels of residual Hex A and undetectable levels of
Hex B, i.e. Hex A+/Hex B, had previously been designated
as "Hexosaminidase Paris" (46). In the same report, they
characterized another mutation associated with subacute Sandhoff
disease, which was also characterized by the Hex A+/Hex B
biochemistry. Both of these patients were also heterozygous for the
16kb allele; thus, their biochemical phenotype was due to their
second allele (26). In both cases, the second allele produced a partial
splicing defect in the HEXB gene encoding an elongated
-polypeptide, i.e. a duplication of bp
16 to +2 of
IVS-13
exon 14 (asymptomatic) and g-26a IVS-12 (subacute) (38). It
was also shown that the residual activity present in these patients'
samples was from a small amount of properly spliced
-mRNA
encoding the wild type protein. Activity measurements indicated that
the asymptomatic individuals had twice as much residual Hex A activity
as the subacute patients, 10 and 5% respectively (38).
In this and other reports (26, 27, 47), we have included the cell line
from the above subacute patient (g-26a IVS-12; 16kb) in our
analyses. In our hands, the residual Hex A activity in this line is
2-3% of normal, using artificial substrates (Table I) (27, 47). This
would suggest that Hex A levels of 4-6% of normal should prevent
GM2 storage and disease. This estimate is close to that set
as the "critical threshold" by Sandhoff and colleagues (5, 37).
Given this critical threshold and our previous Hex A activity data, it
has been difficult to explain why our two patients present with chronic
GM2 gangliosidosis. Two possibilities were considered:
first, that the
mutation is somehow affecting the
active site,
lowering its activity toward MUGS and GM2, e.g.
a new type of B1-variant; second, the mutant
-subunit is affecting
the ability of Hex A to bind the GM2
activator-GM2 ganglioside complex.
We now report the reexamination of residual Hex A* activities using the
-specific MUGS substrate (Table I) and the evaluation of both
-
and
-CRM levels in patients' cells using an anti-Hex A antiserum
(Fig. 3 and 4). These analyses confirmed our previous data,
particularly that the levels of MUGS activity from Hex A* are 9-23%
of normal, and the
-CRM present in the cell line from the
aforementioned subacute patient is much less than half that present in
cells from our patient (Fig. 3). Thus, this substitution does not
appear to specifically affect the
active site, e.g. through some induced conformational change. However, to fully eliminate
this possibility, we determined the Km of Hex A* for
both the MUG and MUGS substrates. These were found to be normal (Table
II).
Finally, we assessed the ability of the Hex A* produced in transfected
CHO cells and semipurified from one of our patient's fibroblasts to
hydrolyze its natural substrate, the GM2
activator-GM2 ganglioside complex (Fig. 7, Table II). These
data demonstrate that the -Pro504
Ser mutation
reduces the ability of the Hex A* to hydrolyze ganglioside in the
presence of human activator by 3-fold (Table II). If this 3-fold
reduction in the specific activity of Hex A* toward GM2
ganglioside, but not MUG or MUGS, is factored into our residual Hex A
activity measurement (Table I), the patients' Hex A* activity is
reduced to 3-9% of normal. This is very close to the critical
threshold values we discussed above and is consistent with the chronic
phenotype observed in the patients.
Recently, we (48) and others (49) have reported the characterization of
-
fusion proteins. Although some of our conclusions differed,
both studies concluded that the C terminus of the
-polypeptide is
important for the correct binding of the activator-ganglioside complex.
The characterization of this novel, naturally occurring mutation
strengthens these conclusions and identifies the region surrounding
Pro504 as the area in the C terminus most likely to be
responsible for this function.
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ACKNOWLEDGEMENT |
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We thank A. Leung for excellent technical assistance and I. B. Warren for preparing the 50 normal DNA samples used to screen for the C1510T substitution.
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FOOTNOTES |
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* This work was supported by a Medical Research Council of Canada Grant MT-10435 (to D. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Research
Institute, The Hospital For Sick Children, 555 University Ave.,
Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-6161; Fax:
416-813-8700; E-mail: hex{at}sickkids.on.ca.
The abbreviations used are:
GM2, GM2 ganglioside,
GalNAc(1-4)-[NANA
(2-3)-]-Gal
(1-4)-Glc-ceramideHex,
-hexosaminidaseactivator, GM2 activator proteinMU, 4-methylumbelliferoneMUG, 4-methylumbelliferone-
-N-acetylglucosamineMUGS, 4-methylumbelliferone-
-N-acetylglucosamine-6-sulfateCRM, cross-reacting materialER, endoplasmic reticulumkb, kilobase
pair(s)bp, base pair(s)PCR, polymerase chain reactionCHO, Chinese
hamster ovaryGA2, asialo GM2
(gangliotriaosylceramide).
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REFERENCES |
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