From the Service de Génétique
Médicale, Hôpital Sainte-Justine and Département de
Pédiatrie, Faculté de Médicine, Université de
Montréal, Montréal, Québec H3T 1C5, Canada and the
¶ Department of Anatomy and Cell Biology, McGill
University, Montréal, Québec H3A 2B2, Canada
Received for publication, January 17, 2001, and in revised form, February 15, 2001
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ABSTRACT |
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Sialidosis is an autosomal recessive disease
caused by the genetic deficiency of lysosomal sialidase, which
catalyzes the catabolism of sialoglycoconjugates. The disease is
associated with progressive impaired vision, macular cherry-red spots,
and myoclonus (sialidosis type I) or with skeletal dysplasia,
Hurler-like phenotype, dysostosis multiplex, mental retardation, and
hepatosplenomegaly (sialidosis type II). We analyzed the effect of the
missense mutations G68V, S182G, G227R, F260Y, L270F, A298V, G328S, and
L363P, which are identified in the sialidosis type I and sialidosis
type II patients, on the activity, stability, and intracellular
distribution of sialidase. We found that three mutations, F260Y,
L270F, and A298V, which are clustered in the same region on the surface
of the sialidase molecule, dramatically reduced the enzyme activity and
caused a rapid intralysosomal degradation of the expressed protein. We
suggested that this region might be involved in sialidase binding with
lysosomal cathepsin A and/or Sialidosis (also called mucolipidosis I and cherry-red spot
myoclonus syndrome) is an autosomal recessive disease caused by the
genetic deficiency of lysosomal sialidase, also called neuraminidase (reviewed in Refs. 1-3). The disease is characterized by tissue accumulation and urinary excretion of sialylated oligosaccharides and
glycoproteins (1) and includes two main clinical variants with
different ages of onset and degrees of severity. Sialidosis type
I or nondysmorphic type is a late-onset mild form, characterized by
bilateral macular cherry-red spots, progressive impaired vision, and
myoclonus syndrome (4-8). Sialidosis type II or dysmorphic type is the
infantile-onset form, which is also associated with skeletal dysplasia,
Hurler-like phenotype, dysostosis multiplex, mental retardation, and
hepatosplenomegaly (9-12). A severe form of the disease manifests
itself prenatally and is associated with ascites and hydrops fetalis
(13-15). The age of onset and severity of the clinical manifestations
correlate with the amount of residual sialidase activity, suggesting
the existence of considerable genetic heterogeneity (1-3).
Although sialidosis was recognized as a deficiency of lysosomal
sialidase from the moment of its discovery (16), the molecular mechanism of this disorder was not characterized for the following two
decades because the identification and sequencing of sialidase had been
hampered by low tissue content and instability of the enzyme. Several
works have shown that sialidase is a part of a multienzyme complex
containing other lysosomal enzymes such as cathepsin A (protective
protein), Recently the gene coding for sialidase was cloned, and a series of
mutations in sialidosis patients was identified (27-31). In
particular, we have found two frameshift and eight missense mutations
in nine sialidosis patients of multiple ethnic origin (28, 31). To
understand the effect of these mutations on sialidase, we modeled the
tertiary structure of the enzyme and localized the identified amino
acid substitutions (31). Surprisingly, none of mutations directly
affected the deduced active site residues or were found in the central
core of the sialidase molecule, but all of them involved residues on
the surface of the enzyme. Therefore, in most cases it was unlikely
that these mutations would introduce electrostatic or steric clashes in
the protein core leading to general folding defects of sialidase and
its retention in the endoplasmic reticulum/Golgi compartment, as
was observed in most of the mutations affecting cathepsin A (32).
In this paper we show that three sialidase mutants that have amino acid
substitutions clustered in one region on the surface of the sialidase
molecule were correctly processed and sorted but were not associated
with the complex and were rapidly degraded in the lysosome. These
results permitted us to conclude that the surface region containing
these mutations may be involved in the sialidase binding interface with
the lysosomal multienzyme complex and that sialidase deficiency in
sialidosis patients may be secondary to the disruption of the lysosomal
multienzyme complex.
Expression of Sialidase Mutants in COS-7
Cells--
Site-directed mutagenesis was performed using a
TransformerTM site-directed mutagenesis kit
(CLONTECH), the previously described pCMV-SIAL
expression vector, mutagenic primers corresponding to mutant sialidase
sequences, and a selection primer used to eliminate a unique
ScaI restriction site in the vector according to supplier's protocols. All primers were enzymatically phosphorylated, and for each
mutant the corresponding mutagenic primer and the selection primer were
annealed to a heat-denatured pCMV-SIAL plasmid. After elongation by T4
DNA polymerase, ligation, and primary digestion with ScaI
restriction enzyme to linearize all nonmutated DNA, the plasmid pool
was used to transform the mutS strain of BMH71-18 bacteria.
Plasmid DNA obtained from the pool of ampicillin-resistant transformants was subjected to a second ScaI digestion and
transformed into Escherichia coli DH5
COS-7 cells seeded in T-25 flasks or 60-mm round dishes were
co-transfected with pCMV-SIAL and pCMV-CathA expression vectors (31)
using LipofectAMINE Plus reagent (Life Technologies Inc.) in accordance
with the manufacturer's protocol. 48 h after transfection, sialidase and control N-acetyl- Metabolic Labeling--
48 h after transfection with wild-type
or mutant sialidase cDNA, COS-7 cells grown to confluency in 60-mm
dishes were washed twice with Hank's balanced salt solution, incubated
for 2 h in cysteine and methionine-free Dulbecco's modified
eagle's medium (Life Technologies, Inc.) supplemented with
L-glutamine and sodium pyruvate, then incubated again for
40 min in 5 ml of the same medium supplemented with a mixture of
[35S]Cys and [35S]Met
(Tran35S-label, ICN Pharmaceuticals Inc.; 0.1 mCi/ml
medium). The radioactive medium was then removed, and the cells
were washed twice with Hank's balanced salt solution and chased at
37 °C in Eagle's minimal essential medium containing 20%
(v/v) fetal calf serum with and without protease inhibitor leupeptin (5 µg/ml).
At the time indicated in the figures, the cells were placed on ice,
washed twice with ice-cold PBS, then lysed for 30 min in 1 ml of
radioimmunoprecipitation assay buffer containing 50 mM
Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) Nonidet P-40,
0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 5 µg/ml leupeptin, and 0.1 mM Immunoprecipitation, Electrophoresis, and Quantitation of
Sialidase--
1.0 ml of lysate was incubated for 4 h with
preimmune serum at a final dilution of 1:20. Then the pellet obtained
from 300 µl of Pansorbin cells (Calbiochem) was added, and the
resulting suspension was incubated for 2 h at 4 °C, followed by
centrifugation for 10 min at 13,000 × g. Supernatants
were incubated overnight with the anti-sialidase antibodies at a 1:100
final dilution, then for 2 h at 4 °C with the pellet from 100 µl of Pansorbin cells and precipitated as above. The pellet was
washed three times with 1 ml of radioimmunoprecipitation buffer. The
antigens were eluted from the pellet by the addition of 100 µl of a
buffer containing 0.1 M Tris-HCl (pH 6.8), 4% (w/v) SDS,
20% (v/v) glycerol, 0.2 M dithiothreitol, and 0.02% (w/v)
bromphenol blue. The proteins were denatured by boiling for 5 min, and
50 µl of each sample were subjected to SDS-polyacrylamide gel
electrophoresis according to Laemmli (38). The molecular weights were
determined with 14C-labeled protein markers (Amersham
Pharmacia Biotech). The gels were fixed in acetic acid/isopropyl
alcohol/water (10:50:40), soaked for 30 min in AmplifyTM solution
(Amersham Pharmacia Biotech), vacuum-dried at 60 °C, and analyzed by
quantitative fluorometry on a PhosphorImager SI analysis screen
(Molecular Dynamics) using the software supplied by the manufacturer.
Immunofluorescent Microscopy--
48 h after transfection with
wild-type or mutant sialidase, COS-7 cells were treated for 40 min with
75 nM LysoTracker Red DND-99 (Molecular Probes, Eugene, OR)
dye, washed twice with ice-cold PBS, and fixed with 3%
paraformaldehyde in PBS for 40 min. Cells were permeabilized by
incubating with 0.3% Triton X-100, washed twice with PBS, and stained
with rabbit polyclonal anti-sialidase antibodies and fluorescein
isothiocyanate-conjugated monoclonal antibodies against rabbit IgG.
Alternatively, cells were double-stained with rabbit polyclonal
anti-sialidase antibodies and monoclonal antibodies against lysosomal
membrane marker LAMP-2 (Washington Biotechnology Inc.,
Baltimore, MD). Slides were studied on a Zeiss LSM410 inverted confocal
microscope (Carl Zeiss Inc., Thornwood, NY).
Density Gradient Centrifugation of Cell Extracts--
COS-7
cells grown to confluency in T-25 flasks and harvested 48 h after
transfection with wild-type or mutant sialidase were solubilized in 0.2 ml of 0.15 M sodium acetate buffer, pH 5.2, containing 0.5 mg of BSA/ml and 1% (w/v) ZwitterionicTM detergent 3-12
(Calbiochem) as described (19) and centrifuged at 13,000 × g for 15 min. The supernatants were applied on the top of
the density gradient of 30% metrizamide (OptiPrep; Nycomed Amersham)
preformed by a 2-h ultracentrifugation at 45,000 rpm in a Beckman SW-55
Ti swinging bucket rotor. After application of the sample, the
centrifugation was continued for an additional 17 h at the same
speed. Immediately after centrifugation, each tube was divided
into 10 fractions using a Beckman tube slicer kit. Each fraction
was assayed for activities of sialidase, Modeling of Sialidase Tertiary Structure--
The modeling was
performed using the structures of homologous sialidases from
Micromonospora viridifaciens (Ref. 39; Protein Data Bank
file 1eur.pdb), Salmonella typhimurium (Ref. 40; Protein
Data Bank file 2sil.pdb), and Vibrio cholerae (Ref. 41;
Protein Data Bank file 1kit.pdb) as templates. These structures were
superimposed with ProSup King's Beech Biosoftware Solutions to
determine structurally conserved regions. The sequence of human
sialidase was manually aligned with the sequences of structurally
conserved regions. The modeling was then carried out with Modeler 4 software (Andrej Sali, The Rockefeller University, New York).
Expression and Intracellular Targeting of Sialidase
Mutants--
The effect of sialidase mutations on enzyme biogenesis
was studied by the transient expression of the mutant cDNA.
Mutations were generated by site-directed mutagenesis in the pCMV-SIAL
vector previously used for the expression of sialidase (28). Short restriction cassettes containing the mutations were then inserted into
the parental pCMV-SIAL vector replacing the corresponding fragments of
wild-type sialidase cDNA. The inserts and junction regions of the
resulting constructs were verified by sequencing to ensure the correct
introduction of mutations. Mutant or wild-type sialidase was
co-expressed with human cathepsin A, which is necessary for the
expression of sialidase activity. 48 h after transfection, the
cell lysates were assayed for sialidase, cathepsin A, and control
The expression results are shown in Table
I. All transfected cells had similar
cathepsin A activity, suggesting the same transfection efficiency for
all cells. Four of the expressed mutants, G68V, G227R, A298V, and
L363P, had very low (<10% of normal) sialidase activity. The activity
of F260Y and L270F mutants was between 10 and 20% of normal, and that
of S182G and G328S mutants was between 20 and 40% of normal.
Additional experiments showed that F260Y, A298V, and L270F mutants were
also significantly less stable than the wild-type sialidase. The
half-life of their enzymatic activity in cellular lysates at 37 °C
was about 30 min as compared with the 2-h half-life of the wild-type
enzyme.
Using immunolabeling, we studied the intracellular distribution of the
sialidase mutants expressed in COS-7 cells. To identify the lysosomal
late endosomal compartment, the COS-7 cells were treated for 40 min with 75 nM LysoTracker Red DND-99 dye prior to fixation
and immunostaining with anti-sialidase antibodies. Alternatively the
cells were double-stained with anti-sialidase antibodies and monoclonal
antibodies against human LAMP-2. For the wild-type sialidase we
have observed the complete
co-localization of anti-sialidase immunostaining with lysosomal markers
LysoTracker Red (Fig. 1) or LAMP-2 (not shown). The G68V, S182G,
F260Y, L270F, A298V, and G328S mutants showed similar localization,
suggesting that the mutant protein is able to reach the lysosomes.
Although partial co-localization of anti-sialidase and LysoTracker
staining was also detectable in the cells transfected with the G227R
and L363P mutants, the majority of the anti-sialidase antibodies
labeled distinct cellular areas, suggesting that in this case the
mutant protein is mostly retained in prelysosomal compartments. This finding is consistent with the results of structural modeling of
sialidase mutants that suggested general folding defects and retention in the endoplasmic reticulum/Golgi compartment for both G227R
and L363P substitutes (31).
Metabolic Labeling of Sialidase Mutants--
The results of
sialidase activity assay in COS-7 cells expressing sialidase mutants
have shown that some of them, i.e. L270F, A298V, and F260Y
mutants, had significantly lower stability in cellular homogenates than
the wild-type enzyme. To measure the stability of sialidase mutants in
the cell, we performed pulse-chase experiments. The 46-48-kDa
polypeptides similar to those previously observed by both
immunoprecipitation and Western blotting (20) were precipitated by
anti-sialidase antibodies from homogenates of cells transfected with
wild-type or mutant sialidase cDNA and pulsed for 40 min (Fig.
2). The intensities of both bands
decreased proportionally with the time of chase. By 4 h of chase,
normal wild-type sialidase was reduced to ~50% of total. In
contrast, the degradation rate of F260Y, L270F, and A298V mutants was
remarkably increased so that for all these cells 46-48-kDa sialidase
bands were already nearly undetectable after 4 h of chase. The
same degradation rate was observed with and without leupeptin, a potent inhibitor of lysosomal serine and thiol proteases, suggesting that they
are not involved in the degradation of mutant sialidase.
Association of Sialidase Mutants with the Lysosomal Multienzyme
Complex--
The ability of sialidase mutants to associate with the
lysosomal multienzyme complex was studied by the density gradient
ultracentrifugation of the cell extracts (Fig.
3). In the extracts of COS-7 cells co-transfected with wild-type sialidase and cathepsin A, all sialidase activity was associated with the peak that sedimented before
thyroglobulin (Mr = 669,000). This peak, which
also contained almost all the cathepsin A and the majority of
endogenous Analysis of molecular defects in the sialidase gene in sialidosis
patients shows that the spectrum of mutations is very different from
that in cathepsin A and -galactosidase in the multienzyme
lysosomal complex required for the expression of sialidase activity.
Transgenic expression of mutants followed by density gradient
centrifugation of cellular extracts confirmed this hypothesis and
showed that sialidase deficiency in some sialidosis patients results
from disruption of the lysosomal multienzyme complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase, and
N-acetylgalactosamine-6-sulfate sulfatase (17-19). Because
the functional activity of sialidase completely depends on the
integrity of its association with cathepsin A, it was hypothesized that
cathepsin A supports catalytically active conformation of this enzyme
(18). In addition, the complex protects sialidase and
-galactosidase
against rapid proteolysis (17, 20, 21) and may also be important
for proper sorting and processing of their precursors (22-25). In the
autosomal recessive disease galactosialidosis, a primary genetic defect
of cathepsin A (17, 21) results in disruption of the complex and
causes the combined deficiency of
-galactosidase and
sialidase activities. The clinical features and a composition of
storage products in galactosialidosis resemble those in
sialidosis (8, 9, 26).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Positive clones
were selected after a final ScaI restriction analysis, and
the entire sialidase cDNA was sequenced. Up to 80% of
transformants contained the desired mutation. DNA fragments of between
300 and 600 base pairs containing the introduced mutations were
obtained from the mutant pCMV-SIAL plasmids by double digestion with
either BstEII/NaeI,
NaeI/KpnI, or KpnI/EcoRV
enzymes and subcloned into the parental pCMV-SIAL plasmid. The final
constructs were verified by sequencing.
-glucosaminidase
activities were assayed in cellular homogenates using the corresponding
fluorogenic 4-methylumbelliferyl glycoside substrates as described
(33-35). The cathepsin A activity was determined with
benzyloxycarbonyl-Phe-Leu and
3-(2-furyl)acryloyl-Phe-Leu substrates (36). One unit of enzyme activity is defined as the conversion of 1 µmol of
substrate/min. Proteins were assayed according to Bradford (37) with
BSA1 (Sigma) as the standard.
To measure the stability of the expressed sialidase, the cellular
homogenate was incubated at 37 °C for 0.5, 1, 2, and 3 h before
the assay of sialidase activity.
-toluenesulfonyl fluoride
(phenylmethylsulfonyl fluoride). The lysate was collected and
centrifuged at 13,000 × g for 10 min to remove the
cell debris.
-galactosidase, and
cathepsin A as well as for the presence of human sialidase and
cathepsin A protein by Western blot as previously described (20). The
activity of endogenous N-acetyl-
-hexosaminidase in fractions was used as an internal control. The molecular masses of
proteins were approximated using the following
Mr standards (Amersham Pharmacia Biotech):
thyroglobulin (Mr = 669,000), catalase (Mr = 232,000), and BSA
(Mr = 69,000). Thyroglobulin and BSA were covalently labeled with fluorescein isothiocyanate to facilitate their
detection in fractions containing proteins from a COS-7 cell.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hexosaminidase activities.
Enzymatic and biochemical properties of sialidase mutants expressed in
COS-7 cells
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Fig. 1.
Immunohistochemical localization of sialidase
mutants expressed in COS-7 cells. COS-7 cells were probed with 75 nM lysosomal marker LysoTracker Red DND-99 for 30 min at
37 °C 48 h after transfection with cathepsin A and wild-type
(WT) or mutant sialidase cDNAs as indicated and stained
with rabbit polyclonal anti-sialidase antibodies and fluorescein
isothiocyanate-conjugated secondary antibodies. Slides were studied on
a Zeiss LSM410 inverted confocal microscope. Panels show
co-localization of anti-sialidase antibodies (green) and
LysoTracker marker (red). Magnification × 600.
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Fig. 2.
Metabolic labeling of sialidase.
Nontransfected COS-7 cells (Control) and cells
co-transfected with cathepsin A and wild-type sialidase (WT)
or mutant sialidase cDNA as indicated on the figure were
metabolically labeled with a mixture of [35S]Cys and
[35S]Met for 40 min and chased at 37 °C for 0, 1, and
4 h in Eagle's minimal essential medium supplemented with 20%
(v/v) fetal calf serum. The sialidase (SIAL) was
immunoprecipitated from cell lysates with rabbit anti-sialidase
antibodies and resolved on SDS-polyacrylamide gel
electrophoresis.
-galactosidase activities, probably represented the
lysosomal multienzyme complex (Fig. 3A). A similar
sedimentation profile was observed in the extracts of cells transfected
with sialidase G328S or S182G mutants. Although about 3-fold less
sialidase activity was detected in the collected fractions as compared
with that of the wild-type control, all activity was associated with
the high molecular weight fraction. The distribution of sialidase and
cathepsin A proteins detected by Western blot (Fig. 3B)
followed that of the enzyme activity. In contrast, in the cells
transfected with G227R, F260Y, L270F, and A298V mutants, the high
molecular weight form of sialidase was not detected. Both sialidase
protein and the trace levels of sialidase activity were found in the
peak that sedimented together with the low molecular weight marker, BSA
(Mr = 69,000), suggesting that the mutant enzyme
does not associate with the multienzyme complex. Moreover, although a
significant amount of sialidase cross-reacting protein was detected in
these fractions for F260Y, L270F, and A298V transfected cells, the
sialidase activity was ~50-100-fold reduced as compared with
wild-type enzyme, which is consistent with the inactivation of
sialidase after the dissociation from the complex.
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Fig. 3.
Density gradient centrifugation of cell
extracts. COS-7 cells were co-transfected with cathepsin A and
wild-type sialidase (WT) or mutant sialidase cDNA
as indicated on the figure. 48 h after transfection, cellular
extracts were analyzed by density gradient centrifugation as described
under "Experimental Procedures." A, the 0.5 ml-fractions
were collected and assayed for sialidase activity ( , left
ordinate), cathepsin A activity (
, right ordinate),
and
-galactosidase activity (
, right ordinate).
N-Acetyl-
-glucosaminidase activity in fractions (not
shown) was used as an internal control. Each curve
represents the average of several independent experiments. The
positions of the sedimentation peaks of the Mr
standards described under "Experimental Procedures" are shown by
arrows. B, the indicated fractions with the peak
sialidase and cathepsin A activities were analyzed for the presence of
sialidase and cathepsin A protein by Western blotting as described. The
protein bands are indicated on the right side of the
blots: Cath30 and Cath20, 30- and 20-kDa
polypeptide chains of cathepsin A; SIAL, sialidase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase, which underlies clinically
similar disorders galactosialidosis and GM1-gangliosidosis, respectively. Most of the sialidosis patients studied so far, 21 of 27, had amino acid substitutions and not frameshift or splicing defects2 (27, 28, 30, 31,
42). The localization of the missense mutations on the sialidase
structural model (Fig. 4) suggested that
only few of them (shown in blue in Fig. 4) affect active site residues (Y370C) or may interfere with their correct positions (L91R with the active site residue Arg78, P80L with
Arg97, duplication of His399 and
Tyr400 with Glu394, P316S with
Arg280, and P335Q with Arg341). In addition,
the L363P mutation is situated on a
-strand adjacent to that
containing the active site residue Tyr370. The
Leu363 residue is probably necessary to anchor this
-strand to the one containing Tyr370 so that the L363P
mutation can potentially also have an effect on the active site.
However, in contrast to cathepsin A mutations in galactosialidosis
patients, which mostly affect the enzyme central core and cause
unfolding of the protein (32), the majority of sialidase mutations
involves residues on the surface of the enzyme and is not supposed to
result in significant structural change. Moreover, the distribution of
mutations on the sialidase surface is uneven. The region that contains
the majority of mutations resulting in complete or almost complete
inactivation of the enzyme and causing severe sialidosis type II
phenotype is easily detectable (shown in red in Fig. 4). In
particular this region contains mutations G227R, F260Y, L270F, and
A298V (28, 31), R294S, L231H, and G218A (30), W240R2, and
V217M and G243R (42).
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Fig. 4.
Schematic diagram of the sialidase model,
showing the location of mutations identified in sialidosis
patients. Mutations localized in putative sialidase-cathepsin A
binding sites are shown in red, mutations in the active site
residues or those that may affect the positions of the active site
residues are in blue, and mutations that do not cause
obvious structural changes are in green.
We expressed eight sialidase mutants, four of which contained amino acid substitution in the defined surface patch (G227R, F260Y, L270F, and A298V) and four in the other areas of the sialidase molecule (G68V, S182G, G328S, and L363P) in COS-7 cells and studied trafficking, activity, and stability of the produced protein. We found that in two cases (G227R and L363P) the majority of the mutant protein was not sorted to the lysosomes, suggesting that these mutations can cause general folding defects and retention of the mutant in the pre-lysosomal compartments. All other expressed sialidase mutants were targeted to lysosomes and correctly processed.
Subsequent experiments revealed that the sialidase mutants F260Y,
L270F, and A298V containing amino acid substitutions in the surface
patch of the fifth -sheet have similar properties. First, they had
very low or absent sialidase activity. Second, stability of sialidase
mutants in cellular homogenates or their half-life in the cell as
estimated by metabolic labeling was significantly lower than that of
the wild-type enzyme. In addition, previous analysis of COS-7 cells
transfected with F260Y, L270F, and A298V mutants by Western blot (31)
demonstrated the presence of 37-, 26-, and 24-kDa fragments of
sialidase protein similar to those observed in COS-7 cells transfected
with wild-type sialidase cDNA in the absence of human cathepsin A. The same pattern of sialidase degradation products was also observed in
the cells of a galactosialidosis patient that lacked functional
cathepsin A (20). Metabolic labeling studies (20) also demonstrated the
dramatically reduced half-life of wild-type sialidase expressed in
galactosialidosis cells (30 min versus 2.7 h in normal
cells) similar to that observed in this work. Together these data
suggest that F260Y, L270F, and A298V mutants are not protected by
cathepsin A, although the same high amount of functional human
cathepsin A was expressed by COS-7 cells in all cases. Indeed, in the
extracts of cells transfected with F260Y, L270F, and A298V mutants we
could not detect a high molecular weight complex of sialidase with
cathepsin A. Instead we observed that sialidase protein sedimented
during the density gradient centrifugation together with low molecular
weight marker BSA. Therefore in the case of F260Y, L270F, and A298V
mutations, the deficit of sialidase activity resulted from the
disruption of normal protein-protein interactions in the lysosome.
Further studies should show if this mechanism is unique for sialidase or extends to other lysosomal enzymes.
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ACKNOWLEDGEMENTS |
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We thank Dr. Hitoshi Sakuraba, who generously provided unpublished information. We also thank Dr. Mila Ashmarina for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by operating grants from Canadian Institutes of Health Research (FRN-15079), Fonds de La Recherche en Santé du Québec, and Vaincre les Maladies Lysosomales Foundation (France) to (A.V.P.).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.
§ Present address: The Scripps Research Institute, La Jolla, CA 92037.
To whom correspondence should be addressed: Service de
génétique médicale Hôpital Sainte-Justine, 3175 Côte Ste-Catherine, Montréal, Quebec H3T 1C5, Canada. Tel.:
514-345-4931/2736; Fax: 514-345-4801; E-mail:
alex@justine.umontreal.ca.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M100460200
2 H. Sakuraba, private communication.
3 H. Sakuraba, private communication.
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ABBREVIATIONS |
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The abbreviations used are: BSA, bovine serum albumin; PBS, phosphate-buffered saline.
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REFERENCES |
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