Protein Misfolding as an Underlying Molecular Defect in Mucopolysaccharidosis III Type C

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DOI: 10.1371/journal.pone.0007434 · Source: PubMed
Abstract
Mucopolysaccharidosis type IIIC or Sanfilippo syndrome type C (MPS IIIC, MIM #252930) is an autosomal recessive disorder caused by deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT, EC 2.3.1.78), which catalyses transmembrane acetylation of the terminal glucosamine residues of heparan sulfate prior to their hydrolysis by α-N-acetylglucosaminidase. Lysosomal storage of undegraded heparan sulfate in the cells of affected patients leads to neuronal death causing neurodegeneration and is accompanied by mild visceral and skeletal abnormalities, including coarse facies and joint stiffness. Surprisingly, the majority of MPS IIIC patients carrying missense mutations are as severely affected as those with splicing errors, frame shifts or nonsense mutations resulting in the complete absence of HGSNAT protein. In order to understand the effects of the missense mutations in HGSNAT on its enzymatic activity and biogenesis, we have expressed 21 mutant proteins in cultured human fibroblasts and COS-7 cells and studied their folding, targeting and activity. We found that 17 of the 21 missense mutations in HGSNAT caused misfolding of the enzyme, which is abnormally glycosylated and not targeted to the lysosome, but retained in the endoplasmic reticulum. The other 4 mutants represented rare polymorphisms which had no effect on the activity, processing and targeting of the enzyme. Treatment of patient cells with a competitive HGSNAT inhibitor, glucosamine, partially rescued several of the expressed mutants. Altogether our data provide an explanation for the severity of MPS IIIC and suggest that search for pharmaceutical chaperones can in the future result in therapeutic options for this disease.
Protein Misfolding as an Underlying Molecular Defect in
Mucopolysaccharidosis III Type C
Matthew Feldhammer
1,2.
, Ste
´
phanie Durand
1.
, Alexey V. Pshezhetsky
1,2,3,4
*
1 Department of Medical Genetics, CHU Sainte-Justine University of Montreal, Montreal, Canada, 2 Department of Biochemistry, University of Montreal, Montreal, Canada,
3 Department of Pediatrics, University of Montreal , Montreal, Canada, 4 Department of Anatomy and Cell Biology, Faculty of Medicine, McGill University, Montreal, Canada
Abstract
Mucopolysaccharidosis type IIIC or Sanfilippo syndrome type C (MPS IIIC, MIM #252930) is an autosomal recessive disorder
caused by deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA: a-glucosaminide N-acetyltransferase
(HGSNAT, EC 2.3.1.78), which catalyses transmembrane acetylation of the terminal glucosamine residues of heparan sulfate
prior to their hydrolysis by a-N-acetylglucosaminidase. Lysosomal storage of undegraded heparan sulfate in the cells of
affected patients leads to neuronal death causing neurodegeneration and is accompanied by mild visceral and skeletal
abnormalities, including coarse facies and joint stiffness. Surprisingly, the majority of MPS IIIC patients carrying missense
mutations are as severely affected as those with splicing errors, frame shifts or nonsense mutations resulting in the
complete absence of HGSNAT protein. In order to understand the effects of the missense mutations in HGSNAT on its
enzymatic activity and biogenesis, we have expressed 21 mutant proteins in cultured human fibroblasts and COS-7 cells and
studied their folding, targeting and activity. We found that 17 of the 21 missense mutations in HGSNAT caused misfolding of
the enzyme, which is abnormally glycosylated and not targeted to the lysosome, but retained in the endoplasmic reticulum.
The other 4 mutants represented rare polymorphisms which had no effect on the activity, processing and targeting of the
enzyme. Treatment of patient cells with a competitive HGSNAT inhibitor, glucosamine, partially rescued several of the
expressed mutants. Altogether our data provide an explanation for the severity of MPS IIIC and suggest that search for
pharmaceutical chaperones can in the future result in therapeutic options for this disease.
Citation: Feldhammer M, Durand S, Psh ezhetsky AV (2009) Protein Misfolding as an Underlying Molecular Defect in Mucopolysaccharidosis III Type C. PLoS
ONE 4(10): e7434. doi:10.1371/journal.pone.0007434
Editor: Raphael Schiffmann, National Institutes of Health, United States of America
Received June 17, 2009; Accepted September 17, 2009; Published October 13, 2009
Copyright: ß 2009 Feldhammer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by research grants from March of Dimes Foundation, Sanfilippo Childrens Research Foundation and Canadian Institutes for
Health Research (MOP84430) to A.V.P. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: alexei.pchejetski@umontreal.ca
. These authors contributed equally to this work.
Introduction
Mucopolysaccharidosis III (also called Sanfilippo syndrome) is
an autosomal recessive disease caused by lysosomal accumulation
of heparan sulfate [reviewed in 1] and includes four allelic
subtypes caused by the genetic deficiencies of heparan N-sulfatase
(MPS III type A; MIM #252900), a-N-acetylglucosaminidase
(MPS III type B; MIM #252920), heparan sulfate acetyl-CoA: a-
glucosaminide N-acetyltransferase or HGSNAT (MPS III type C;
MIM #252930), and N-acetylglucosamine 6-sulfatase (MPS III
type D; MIM #252940). The majority of MPS IIIC patients have
severe clinical manifestations with onset in infancy or early
childhood. They rapidly develop progressive and severe neuro-
logical deterioration causing hyperactivity, sleep disorders, loss of
speech accompanied by behavioral abnormalities, neuropsychiat-
ric problems, mental retardation, hearing loss, and visceral
manifestations, such as mild hepatomegaly, mild dysostosis
multiplex, mild coarse facies, and hypertrichosis [2,3]. A majority
of patients experience severe mental retardation and die before
adulthood but some survive to the fourth decade with progressive
dementia and retinitis pigmentosa [1,3]. In the very rare MPS
IIIC patients with the onset of symptoms in adulthood the disease
progression was similar in severity and time course to the forms
with onset in childhood [4]. The birth prevalence of MPS IIIC in
Australia, Portugal and the Netherlands was estimated at 0.07,
0.12 and 0.21 per 100,000, respectively [5,6,7].
Although from the moment of discovery MPS IIIC was
recognized as a deficiency of an enzyme that transfers an acetyl
group from cytoplasmically derived acetyl-CoA to terminal N-
glucosamine residues of heparan sulfate within the lysosomes [8–
10], the molecular defects causing the disease have not been
characterized for almost three decades because the identification
and cloning of HGSNAT has been hampered by low tissue
content and instability of the enzyme. Recently our group and
others cloned the gene coding for HGSNAT [11,12] which paved
the way for characterization of the molecular defects in MPS IIIC
patients. To date, 54 HGSNAT sequence variants have been
identified including 13 splice-site mutations, 11 insertions and
deletions causing frame shifts and premature termination of
translation, 8 nonsense, 18 missense mutations and 4 polymor-
phisms [reviewed in 13, http://chromium.liacs.nl/LOVD2/
home.php?select_db = HGSNAT], While the HGSNAT tran-
scripts with abnormal splicing, frame shifts and premature stop
codons are rapidly degraded via the nonsense-mediated mRNA
decay pathway [11], the phenotypic pathogenicity of missense
mutations was impossible to predict. Here, in order to understand
the biochemical effects of the missense mutations we have
expressed mutant proteins in cultured human fibroblasts and
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COS-7 cells and found that all mutations result in misfolding of
the enzyme. As a consequence, it is abnormally glycosylated, and
is not targeted to the lysosome, but retained in the endoplasmic
reticulum (ER).
Results
Expression, processing and enzymatic activity of HGSNAT
mutants
The effect of HGSNAT mutations on the enzyme biogenesis
and catalytic activity was studied by the transient expression of the
mutant cDNA. Mutations were generated by site directed
mutagenesis in the pCTAP-HGSNAT construct that expresses
human HGSNAT with a C-terminal tandem affinity purification
(TAP) tag consisting of a high affinity streptavidin-binding peptide
(SBP) and a calmodulin-binding peptide (CBP) to allow purifica-
tion of the recombinant protein using successively-applied
streptavidin-resin and calmodulin-resin affinity purification steps
or its detection with anti-CBP antibodies [14,15]. In preliminary
experiments we have ensured that a human wild-type HGSNAT
carrying a TAP tag on its C-terminus had a lysosomal localization
and showed catalytic activity similar to that of the untagged
enzyme (not shown). The sequences of the constructs were verified
to ensure the correct introduction of mutations. In total, 21
constructs bearing nucleotide changes identified in patients were
transfected in COS-7 cells and assayed for N-acetyltransferase
activity (Figure 1). All tested cells showed similar endogenous
lysosomal b-hexosaminidase activity (not shown) but drastically
different levels of the N-acetyltransferase activity. The four
polymorphisms (P237Q, V481L, K523Q and A615T) displayed
activity similar to that of the wild-type enzyme, whereas the
activity of the rest of the mutants was below the detection level.
Kinetics studies were further conducted using partially purified
enzyme to determine if the P237Q and V481L mutants showing
full enzymatic activity would have a different affinity for the
substrate, however their K
m
and V
max
values were similar to those
of the wild-type (Figure S1).
The expression of HGSNAT mutants was studied by Western
blot analysis of cellular homogenates using anti-CBP antibodies
(Figure 1B). All mutants were expressed at a level approximately
similar to that of the wild-type HGSNAT, but showed a difference
in their molecular mass. The four polymorphisms (P237Q, V481L,
K523Q and A615T) showed a molecular mass of ,77 kDa,
similar to that of the wild-type enzyme, whereas all missense
mutants (C76F, L137P, G262R, N273K, P283L, R344C, R344H,
W403C, G424S, E471K, M482K, A489E, S518F, S539C, S541L,
D562V, P571L) had a smaller molecular mass of ,67 kDa. Since
HGSNAT is predicted to have 5 potential N-linked glycosylation
sites each potentially contributing ,2 kDa to the size of the
mature enzyme [11], this molecular mass difference was consistent
with the hypothesis that inactive mutants lacked proper glycosyl-
ation. To confirm this, the wild-type enzyme, 2 polymorphic
variants and 2 inactive mutants were treated with endoglycosidase
H which cleaves immature mannose-rich oligosaccharide chains
added to glycoproteins in the ER [16] Analysis by Western blot
(Figure 2) showed that upon deglycosylation the molecular mass of
the wild-type enzyme and polymorphic mutants was reduced to
67 kDa, whereas the inactive mutants displayed only a subtle shift
in their position on the gel, indicating that they already lacked
most of the glycans. The homogenates were also treated with the
peptide: N-Glycosidase F (PNGase F) which fully removes all
sugars from mature glycoproteins (Figure 2B). In addition to
Figure 1. Enzymatic activity and expression of HGSNAT mutants. COS-7 cells were harvested 42 h after transfection with HGSNAT-TAP
plasmids bearing missense mutations. Cell homogenates were (A) assayed for N-acetyltransferase activity and (B) analyzed by Western blot using anti-
CBP antibody as described in Materials and Methods. A. N-acetyltransferase activity is shown as a fraction of the activity measured in the cells
transfected with the wild-type HGSNAT-TAP plasmid. Values represent means 6 S.D. of three independent experiments. B. Blot shows a
representative image of triplicate experiments.
doi:10.1371/journal.pone.0007434.g001
HGSNAT Misfolding in MPS IIIC
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performing enzymatic deglycosylation of cell homogenates we
treated the COS-7 cells transiently expressing the wild-type enzyme,
a polymorphic and an inactive mutant with tunicamycin, an
inhibitor of N-acetylglucosamine transferase that blocks glycosyla-
tion of newly-synthesized proteins (Figure 2C). Results of both
experiments were consistent with those obtained with Endoglyco-
sidase H. Upon treatment with tunicamycin or PNGase F the size of
the wild type HGSNAT and the enzyme containing the
polymorphism was reduced by approximately ,10 kDa whereas
the inactive mutant displayed only a minor change in electropho-
retic mobility. Western blot analysis also revealed that homogenates
of cells transfected with both wild-type HGSNAT and polymorphic
mutants, but not of untransfected cells or cells transfected with
inactive mutants contained an immunoreactive protein with a
molecular mass of ,48 kDa. Since this form of HGSNAT contains
the C-terminal CBP cross-reacting with the antibodies we estimated
that it corresponds to the HGSNAT fragment missing ,300 amino
acids on the N-terminus. It seems that the appearance of this form is
associated with the proper glycosylation and lysosomal targeting of
HGSNAT, but further studies are necessary to conclude whether it
represents a product of intralysosomal maturation (with the N-
terminal chain not being detected by our antibodies) or a
degradation product. On the other hand, the 77-kDa form of
HGSNAT separated by FPLC anion-exchange chromatography
showed full enzymatic activity (Figure 3).
Subcellular localization of HGSNAT mutants
The effects of missense mutations on the subcellular localization
of the enzyme were studied by confocal immunofluorescence
microscopy. Immortalized human skin fibroblasts from a normal
control were transfected with constructs expressing each of the
missense mutations. The cells were allowed to express the mutant
and polymorphic enzymes for 42 hours and were then fixed by
paraformaldehyde. To identify the lysosomal-late endosomal
compartment, prior to fixation the cells were treated with
LysoTracker Red DND-99 dye. After fixation the cells were
permeabilized with Triton X-100 and probed with anti-CBP
antibodies to localize the HGSNAT and with antibodies against
the ER marker calnexin and lysosomal marker LAMP-2.
Distinct punctate staining characteristic of lysosomal targeting
of the protein was evident for the recombinant wild-type enzyme
and all active mutants. Accordingly, both wild-type recombinant
HGSNAT and polymorphic mutants almost completely co-
localized with the lysosomal markers LysoTracker Red or
LAMP-2 (representative data are shown in Figure 4). In contrast,
the inactive mutants exhibited a diffuse pattern throughout the cell
and were co-localized not with the lysosomal markers but with the
ER marker calnexin (representative data are shown in Figure 4,
see Figure S2 for the data on all mutants). Partial ER localization
(typically 2–5% of the total, not shown) was also observed for the
wild-type recombinant and active mutants and most likely
represented a pool of the enzyme processed in the ER on its
way to the lysosomes.
Glucosamine-mediated refolding of inactive HGSNAT
mutants
Since all the data were consistent with general folding defects
and retention in the ER compartment of the HGSNAT mutants
(C76F, L137P, G262R, N273K, P283L, R344C, R344H, W403C,
G424S, E471K, M482K, A489E, S518F, S539C, S541L, D562V,
P571L) we further tested whether competitive inhibitors of the
enzyme that mimic the substrate binding in the active site would
help to fold the enzyme in the ER so it can be properly modified
and exported to the lysosome. We first tested several potential
inhibitors of HGSNAT that were not expected to be highly toxic
for the cultured cells and found that one of them, D-(+)-
Figure 2. Deglycosylation of HGSNAT by endoglycosidase H,
PNGase F and tunicamycin treatment. COS-7 cells expressing
either the wild-type HGSNAT or the protein containing C76F, P237Q,
G262R or V481L variants were harvested 42 h post-transfection and
their homogenates were treated overnight with endoglycosidase H (A)
or PNGase F (B). (C) COS-7 cells expressing wild-type HGSNAT and C76F
or P237Q variants were cultured for 48 h in the presence or absence of
1
mg/ml of tunicamycin added to the culture medium 5 h after the
transfection. The treated and control homogenates were analyzed by
Western blot using anti-CBP antibodies as described in Materials and
Methods.
doi:10.1371/journal.pone.0007434.g002
Figure 3. Analysis of the wild-type recombinant HGSNAT by
anion-exchange FPLC. COS-7 c ells were harvested 42 h after
transfection with the wild-type HGSNAT plasmid, solubilized in a buffer
containing 0.1% NP-40, applied to an ion-exhange Mono Q HR 5/5
column and eluted by 0-0.5 M NaCl gradient as described in Materials
and Methods. Graph shows N-acetyltransferase activity (nmol/hr ml) in
the collected fractions. Dashed line represents the NaCl gradient. An
aliquot from each fraction was analyzed by Western blot using anti-CBP
antibodies (inset).
doi:10.1371/journal.pone.0007434.g003
HGSNAT Misfolding in MPS IIIC
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glucosamine hydrochloride, was a competitive inhibitor of the
enzyme with a K
I
of 0.28 mM close to the K
M
value for the 4MU-
bGlcN substrate (Figure S3). To establish the optimal concentra-
tion of the inhibitor in the culture medium and the length of the
treatment, we have used the immortalized skin fibroblasts of
previously reported MPS IIIC patient homozygous for the N273K
mutation that causes misfolding of the enzyme (Figure 1 and
Figure 4), since they were readily available in our lab [17].
Immortalized fibroblasts from a patient homozygous for a splice
site mutation (c.1726+1G.A) [11] and normal immortalized
Figure 4. Localization of HGSNAT mutants expressed in cultured human skin fibroblasts by immunofluorescence microscopy. The
cells transfected with wild-type or mutant HGSNAT-TAP constructs as indicated were fixed and stained with either mouse monoclonal anti-LAMP-2
antibodies, Lysotracker Red DND-99 or mouse monoclonal anti-calnexin antibodies (red) and rabbit polyclonal anti-CBP antibodies (green) as
indicated. Slides were studied on a Zeiss LSM510 inverted confocal microscope. Magnification 630x. Panels show representative images illustrating
co-localization of anti-CBP antibodies (green) and lysosomal and ER markers (red) for the wild-type HGSNAT, active enzyme containing P237Q
polymorphism and inactive L137P and P571L mutants. From 10 to 15 cells all showing similar localization patterns were studied for each variant. See
Figure S2 for the data on other mutants.
doi:10.1371/journal.pone.0007434.g004
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fibroblasts were used as controls. We found that the treatment of
fibroblasts from a patient homozygous for the N273K mutation
with 14 mM glucosamine results in the progressive increase of N-
acetyltransferase activity in cell homogenates which reaches 3 fold
induction after 5 days of treatment (Figure 5A). The N-
acetyltransferase activity in the normal cells and in the cells of
the MPS IIIC patient homozygous for the splice site mutation
c.1726+1G.A as well as the control b-hexosaminidase activity in
all cell lines were not increased upon the treatment (not shown)
suggesting that the observed induction of N-acetyltransferase
activity was caused by rescuing the N273K mutant but not by the
general induction of lysosomal enzymes. When the cells of the
patient affected with the N273K mutation were treated for 5 days
with increasing concentrations of glucosamine (Figure 5B) we
found that the effect was proportional to the concentration of the
inhibitor. At the glucosamine concentration of 14 mM (,50x K
I
)
the N-acetyltransferase activity in the treated cells was 0.3 nmol/h
mg which corresponds to ,7% of the average activity in normal
control fibroblasts, but further increase of glucosamine concen-
tration resulted in inhibition of cell growth. To understand
whether similar effects could be also observed for other missense
mutants the available primary cultures of skin fibroblasts from 9
MPS IIIC patients carrying missense HGSNAT mutations or a
missense mutation in combination with a splice site or nonsense
mutation were treated with glucosamine and assayed for N-
acetyltransferase activity. Nine missense mutations were studied
altogether (Figure 5C; patient genotypes are listed in the figure
legend). Although the maximal effect for different cell lines was
achieved at different concentrations of glucosamine and at
different treatment time (not shown), for 8 of 9 cell lines a
statistically significant increase in the activity was observed
(Figure 5C), suggesting that some of the mutants were partially
stabilized by the glucosamine, correctly processed and targeted to
the lysosomal compartment.
Discussion
The current work provides an explanation for the severe
phenotype of MPS IIIC patients carrying missense mutations in
the HGSNAT gene. Altogether, we studied the activity and
biogenesis of HGSNAT mutants having 21 amino acid substitu-
tions previously identified in MPS IIIC families and affecting 8 of
the 11 transmembrane segments of the enzyme as well as its
luminal and cytosolic domains (Figure 6). The missense mutations
studied in this paper represent all of the currently identified
missense HGSNAT mutations. The polymorphisms (P237Q,
V481L, K523Q and A615T, shown in green in Figure 6) identified
in MPS IIIC only in cis either with a splice site mutation or with a
missense mutation [11,13] were previously reported by us to result
in catalytically active protein [13]. They were included in the
current study because it was unclear whether they still could affect
kinetic parameters or targeting of HGSNAT and therefore
represent a clinically valuable phenotype. All variants were
expressed as TAP-tagged proteins since no Western blotting or
cellular immunostaining could be conducted using the patient
fibroblasts due to a lack of specific antibodies against human
HGSNAT. Our results show that the HGSNAT variants P237Q,
V481L, K523Q and A615T are all correctly processed targeted to
the lysosome and display full enzymatic activity. We conclude
therefore that these four mutations represent rare polymorphisms
in the HGSNAT gene and do not have clinical significance thus
confirming our previous hypothesis [13].
Seventeen mutations (C76F, L137P, G262R, N273K, P283L,
R344C, R344H, W403C, G424S, E471K, M482K, A489E,
Figure 5. Partial refolding of HGSNAT mutants by glucosamine.
A. Fifty percent confluent immortalized skin fibroblasts from a MPS IIIC
patient homozygous for N273K mutation [17] were cultured in the
presence or absence of 14 mM D-(+)-glucosamine hydrochloride.
Medium was replaced every day and at the indicated time intervals
cells were harvested and assayed for N-acetyltransferase activity. N-
acetyltransferase activity is shown as a fraction of that measured in non-
treated cells after 24 h of culturing. Data show mean values and
standard error of 2 independent experiments. ** Significantly different
(p,0.01) from non-treated cells according to repeated measurements
ANOVA. B. Same cells were cultured in the presence of increasing
glucosamine concentrations (0–14 mM) for 5 days, harvested and
assayed for N-acetyltransferase activity or b-hexosaminidase activity.
Data show mean values a nd s tandar d error of 2 independent
experiments. C. Primary skin fibroblasts of the MPS IIIC patients
carrying the missense HGSNAT mutations: L137P/S518F (P1), P283L/
R344C (P3), S518F/S518F (P4), N273K/N273K (P6), R344C/R344C (P7),
S518F/S518F (P8) and E471K/D562V (P9) or a missense mutation in
combination with a splice site (S541L/c.234+1G.A; P2) or nonsense
(R344H/R384X, P5) mutation were cultured for 2 (P1, P2, P5, P6, P8, P9)
or 3 (P3, P4, P7) days in the absence (open bars) or presence (filled bars)
of 7 mM (P3-P9) or 14 mM (P1, P2) glucosamine, harvested and assayed
for N-acetyltransferase activity. Data show mean values and standard
error of 2 independent experiments. The residual N-acetyltransferase
activity detectable in untreated cells most likely represents background
chemical or enzymatic reactions occurring with the substrate in the
presence of cell homogenates. Significantly (*, p,0.05; **, p,0.01; ***,
p,0.001) different from non-treated cells according to non-parametric
t-test.
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S518F, S539C, S541L, D562V and P571L, shown in red in
Figure 6) result in production of misfolded HGSNAT protein that
is abnormally glycosylated and not targeted to the lysosome. Seven
of these mutations (G262R, P283L, W403C, M482K, A489E,
S518F and P571L) are predicted to reside within the highly
hydrophobic transmembrane domains of the protein. Four of
them (G262R, W403C, M482K, A489E) introduce hydrophilic or
charged residues inside the transmembrane domains which usually
have a dramatic effect on the folding of the protein [reviewed in
18]. Three other changes (P283L, S518F and P571L) result in
replacement of hydrophilic residues for hydrophobic ones that also
could destabilize the transmembrane helix.
Six mutations (R344C, R344H, E471K, S539C, S541L and
D562V) are found adjacent to the predicted transmembrane
domains either on the cytoplasmic (D562V) or on the lumenal
(R344C, R344H, E471K, S539C, and S541L) side and 4
mutations (C76F, L137P, N273K, and G424S) reside inside the
hydrophilic lumenal domains of the enzyme. In most cases these
mutations are predicted to have a drastic effect on protein folding
since they involve replacements with amino acids significantly
different in hydrophobicity (C76F, D562V, S541L), charge
(R344C/H, N273K, E471K) or size (C76F, L137P, G424S).
Thus, enzyme folding defects due to missense mutations, together
with nonsense-mediated mRNA decay seem to be the major
molecular mechanisms underlying MPS IIIC.
For at least 5 of the above changes (N273K, R344C, R344H,
S518F and S541L) the active conformation can be stabilized by
the competitive inhibitor of HGSNAT glucosamine resulting in
part of the enzyme pool being properly processed and targeted to
the lysosomes. L137P and P283L mutants may also be stabilized
by the glucosamine treatment, however this could not be verified
experimentally because in the available patient cell lines they were
present together with the responsive mutations S518F and R344C,
respectively. Only one cell line carrying E471K and D562V
mutations did not show a significant increase in N-acetyltransfer-
ase activity in response to glucosamine. Further structural studies
are needed to fully understand the difference in the effect of
glucosamine on these mutants.
Although the spectrum of mutations in MPS IIIC patients shows
substantial heterogeneity, some of the missense mutations have a
high frequency within the patient population. Importantly, the two
mutations, R344C and S518F, responsive to glucosamine-mediated
refolding account for 22.0% and 29.3%, respectively, of the alleles
among the probands of Dutch origin [19]. The S518F mutation has
also been identified in a patient from Germany, while the R344C
change was found in families from France, UK, Germany and
Singapore. The responsive mutation R344H was found in 4 families
from Eastern and Northern Europe (2 from Poland, one from
Czech Republic and one from Finland) and the responsive mutation
S541L was reported in 4 families from France, Ireland, Poland and
Portugal. In general, the vast majority of patients is affected with at
least one missense mutation interfering with the proper folding of
the enzyme that could be partially rescued by the treatment of the
cells with the competitive inhibitor of HGSNAT, glucosamine. We
believe this makes MPS IIIC a good candidate for enzyme
enhancement therapy [reviewed in 20,21], where active site-specific
inhibitors are used as pharmacological chaperones to modify the
conformation of the mutant lysosomal enzymes usually retained and
degraded in the ER in order to increase the level of the residual
activity to a point sufficient to reverse the clinical phenotype.
Together with inhibitors of heparan sulfate synthesis, pharmaco-
logical chaperones could potentially reduce storage of this polymer
in the central nervous system to levels sufficient to stop neuronal
death and reverse inflammation.
Figure 6. Distribution of missense mutations in HGSNAT protein. Visual representation of HGSNAT membrane topology was created using
the TMRPres2D software [26]. The deduced amino acid sequence of HGSNAT predicts 11 transmembrane domains and five potential N-glycosylation
sites oriented towards the lysosomal lumen (shown in blue). Mutations that result in production of misfolded proteins are shown in red.
Polymorphisms are shown in green. Figure was adapted from our previous work [13].
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Materials and Methods
The current study was conducted with ethics approval from the
review board of CHU Ste-Justine, University of Montreal.
Generation of expression constructs and site-directed
mutagenesis
The wild-type HGSNAT-TAP plasmid was obtained by
subcloning the HGSNAT 1992 bp coding sequence into pCTAP
vector (Stratagene). Briefly, a 39 part of human pCMV-Script
construct [11] missing the stop codon was amplified by PCR using
primers 76-Cla-F 59-TTG CTC TTA TAC TCA TGG TCT TTG
TCA-39 and TM76-R4 59-ATA TGT CGA CGA GCC ATC
CGA TTT TCC-39 and then used to replace Cla I - Sal I segment
of the same construct prior to subcloning into pCTAP using Hind
III Sal I sites. Missense mutants were constructed using
QuikChange Lightning kit (Stratagene) with HGSNAT-TAP as a
matrix (see Table S1 for primers). All primers were designed using
QuikChange Primer Design Program (http://www.stratagene.
com/qcprimerdesign). For all constructs the coding sequence
included an extra 84 base pairs on their 59 end (encoding 28 amino
acids) as the sequence first described for HGSNAT gene [11], but
for clarity the nomenclature used reflects a unified numbering
system based on the sequence of GenBank entries NM_152419.2
and NG_009552.1 as in Fan et al. [12], Ruijter et al. [19], Fedele et
al. [22] and Feldhammer et al. [13].
Cell culture and transfection
Skin fibroblast lines of MPS IIIC patients, obtained with a written
informed consent [11], and COS-7 cells (ATCC) were cultured in
Eagles’s minimal essential medium supplemented with 10% (v/v) fetal
calf serum (Wisent). Transfections were carried out using polyethyle-
neimine 25,000 (PEI, Polysciences inc.) or Lipofectamine LTX
(Invitrogen). PEI was dissolved in water at 1 mg/ml and pH was
adjusted to 6.8 with HCl. For transfection, a mixture of 2
mgDNAand
8
mg PEI in 0.4 ml of serum free medium was incubated for 15 min at
room temperature and added to ,70% confluent cells growing in a
10 cm dish. The transfection media was replaced with growth media
18 h later. Lipofectamine LTX was used as described in the
manufacturer’s protocol. Patients (N273K and c.1726+1G.A) and
control fibroblasts immortalized by transfection with retroviral vectors
expressing the type 16 human papilloma virus E7 gene and the
catalytic component of human telomerase were described before [17].
Glucosamine-mediated refolding
Patient and control skin fibroblasts were cultured as described
above in growth media supplemented with various concentrations
of D-(+)-glucosamine hydrochloride (Sigma G4875). Medium was
replaced every day and at specified time cells were harvested and
assayed for N-acetyltransferase activity and b-hexosaminidase
activity as described below.
Enzyme assays
N-acetyltransferase enzymatic activity was measured using
fluorogenic substrate, 4-methylumbelliferyl b-D-glucosaminide
(4MU-bGlcN, Moscerdam, Rotterdam, The Netherlands) as
previously described by He et al. [23]. The reaction mixture
containing 5
ml of cell homogenate, 5 ml of 6 mM acetyl-CoA and 5
ml of 3 mM 4MU-bGlcN in McIlvain buffer (100 mM sodium
citrate, 200 mM sodium phosphate, pH 5.7) was incubated at 37uC
for 3–18 h. The reaction was terminated by adding 1.98 ml of
0.5 M Na
2
CO
3
/NaHCO
3
, pH 10.7, and fluorescence was mea-
sured and used to calculate the specific activity. Lysosomal b-
hexosaminidase activity was measured as previously described [24].
Protein concentration was measured according to the method of
Bradford [25] using a commercially available reagent (BioRad).
Deglycosylation of HGSNAT
To remove N-linked glycans from HGSNAT, cell homogenates
were treated with recombinant endoglycosidase H, or peptide: N-
Glycosidase F (Endo H; PNGase F New England Biolabs). Briefly,
the mix consisted of 10
mg of cell homogenate in 25 mM sodium
phosphate buffer, pH 7.5, to which 1
ml (500 U) of concentrated
Endo H or PNGase F was added before incubating at 37uC
overnight and analysis by Western blot.
To inhibit glycosylation of newly-synthesized HGSNAT COS-7
cells transfected with plasmids coding for the wild-type enzyme
and the C76F or P237Q variants were treated with 1
mg/ml of
tunicamycin (Sigma) added 5 h after transfection and allowed to
express the recombinant protein for 48 h in the presence of drug.
Media and drug were changed after 24 h and the homogenates
were analyzed by Western blot as described below.
Western blotting
Cell homogenates were sonicated and boiled in LDS sample buffer
(Invitrogen) in the presence of 25 mM DTT. Proteins were resolved by
SDS-polyacrylamide gel electrophoresis using NuPAGE 4–12% Bis-
Tris gels (Invitrogen) and electrotransferred to PVDF membrane.
Detection of TAP-tagged N-acetyltransferase protein was performed
using anti-calmodulin binding peptide epitope tag (CBP) rabbit
antibodies (Immunology Consultants Laboratory, dilution 1:30,000)
and the Amersham ECL Western Blotting Detection Reagents (GE
Healthcare) in accordance with the manufacturer’s protocol.
Analysis of the wild-type recombinant HGSNAT by anion-
exchange FPLC
COS-7 cells were harvested 42 h after transfection with the
wild-type HGSNAT plasmid and suspended in lysis buffer
(10 mM Tris-HCl, pH 7.5, 0.1% NP-40, 1 mM PMSF and Sigma
P8340 protease inhibitor cocktail at 10
ml per 1 ml of cell
suspension). The homogenate was sonicated, gently shaked at 4uC
for 2 h and centrifuged at 13,000 rpm for 30 min. One ml of the
supernatant containing 8 mg of total protein was applied to an
ion-exhange Mono Q HR 5/5 column equilibrated with 10 mM
Tris buffer, pH 8.2. The column was washed with 4 ml of the
same buffer and then eluted using a 20 mL gradient of NaCl (0–
0.5 M) at a flow rate of 0.5 ml/min. One ml fractions were
collected and assayed for N-acetyltransferase activity. Thirty
ml
aliquots from each fraction were analyzed by SDS-PAGE and
Western blot using anti-CBP antibodies as described above.
Confocal immunofluorescence microscopy
Immortalized control human skin fibroblasts were transfected
with HGSNAT-TAP or plasmids coding for the HGSNAT
mutants using Lipofectamine LTX (Invitrogen) as described in
the manufacturer’s protocol. Forty-two hours post-transfection
cells were incubated for 1 hour with 1
mM Lysotracker Red DND-
99 (Invitrogen) and then washed with ice-cold PBS. Cells were
fixed with 4% paraformaldehyde, 4% sucrose in PBS for 5 min,
and then rinsed 3 times with PBS. Cells were permeabilized by
0.25% Triton X-100 for 10 min and blocked for 1 h in 3% horse
serum and 0.1% Triton X-100. Cells were either co-stained with
rabbit anti-CBP (Immunology Consultants Laboratory; 1:400) and
mouse monoclonal anti-calnexin (Millipore; 1:250) antibodies in
3% horse serum, with anti-CBP antibodies and mouse monoclonal
antibodies against human LAMP-2 (Developmental Studies
Hybridoma Bank; 1:150), or with anti-CBP antibodies and
HGSNAT Misfolding in MPS IIIC
PLoS ONE | www.plosone.org 7 October 2009 | Volume 4 | Issue 10 | e7434
Lysotracker Red DND-99. Cells were then counterstained with
Oregon Green 488-conjugated anti-rabbit IgG antibodies or
Texas-Red-conjugated goat anti-mouse antibodies (Molecular
Probes; 1:1000). Slides were studied on a Zeiss LSM510 inverted
confocal microscope (Zeiss). Images were processed using the LSM
image browser software (Zeiss) and Photoshop (Adobe).
Supporting Information
Figure S1 Lineweaver-Burk plot of substrate dependance for
partially purified HGSNAT wild-type and P237Q and V481L
mutants. COS-7 cells were harvested 42 hrs after transfection with
the wild-type or mutant HGSNAT plasmids and suspended in lysis
buffer (40 mM Tris-HCl, 300 mM KCl, pH 7.5, 0.1% NP-40, 1 mM
PMSF and Sigma P8340 protease inhibitor cocktail at 10
mlper1 mlof
cell suspension). The homogenate was sonicated, gently shaked at 4uC
for 2 h and centrifuged at 13,000 rpm for 30 min. The supernatant
was first passed through an avidin-agarose column (Sigma A9207) then
affinity purification of TAP-tagged HGSNAT was performed using
streptavidin resin (Stratagene) according to the manufacturer’s
protocol. N-acetyltransferase activity was assayed as described in
Material and Methods using 0.05 to 2.0 mM 4MU-bGlcN and 18 h
incubation time. KM and VMAX values for all 3 enzymes were similar
within the statistical error.
Found at: doi:10.1371/journal.pone.0007434.s001 (0.07 MB PPT)
Figure S2 Localization of HGSNAT mutants expressed in
cultured human skin fibroblasts by immunofluorescence micros-
copy. The cells transfected with wild-type or mutant HGSNAT-
TAP constructs as indicated were fixed and stained with either
mouse monoclonal anti-LAMP-2 antibodies, Lysotracker Red
DND-99 or mouse monoclonal anti-calnexin antibodies (red) and
rabbit polyclonal anti-CBP antibodies (green) as indicated. Slides
were studied on a Zeiss LSM510 inverted confocal microscope.
Magnification 630x. Panels show representative images showing
co-localization of anti-CBP antibodies (green) and lysosomal and
ER markers (red) for the active enzyme containing polymorphisms
and all inactive mutants.
Found at: doi:10.1371/journal.pone.0007434.s002 (5.80 MB PPT)
Figure S3 Dixon plot showing the inhibition of HGSNAT by
glucosamine. COS-7 cells were harvested 42 hrs after transfection
with the wild-type HGSNAT plasmid and N-acetyltransferase
activity was measured in the homogenates for 3 h at 37uC in the
presence of 2 mM AcCoA, 0.0375 to 1.5 mM 4MU-bGlcN and 0
to 2 mM D-(+)-glucosamine hydrochloride.
Found at: doi:10.1371/journal.pone.0007434.s003 (0.06 MB PPT)
Table S1 Primers for site-directed mutagenesis of HGSNAT-
TAP plasmid.
Found at: doi:10.1371/journal.pone.0007434.s004 (0.04 MB
DOC)
Acknowledgments
The authors thank the patients and their families for participating in our
study. We also thank Maryssa Canuel for helpful advice, Mila Ashmarina
for critical reading and Eva Lacroix for help in preparation of the
manuscript.
Author Contributions
Conceived and designed the experiments: MF SD AVP. Performed the
experiments: MF SD. Analyzed the data: MF SD AVP. Wrote the paper:
MF SD AVP.
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HGSNAT Misfolding in MPS IIIC
PLoS ONE | www.plosone.org 8 October 2009 | Volume 4 | Issue 10 | e7434
    • "Despite the identification of several MPSIIIC-causing mutations, it has proven difficult to establish a clear genotype–phenotype correlation, except e.g. for mutations p.G262R and p.S539C from two sisters that were associated with an attenuated phenotype (Berger-Plantinga et al., 2004; Ruijter et al., 2008). In any case, the vast majority of patients are affected by at least one missense mutation in or adjacent to transmembrane domains of HGSNAT, interfering with the proper folding of the enzyme (Feldhammer et al., 2009a; Feldhammer et al., 2009b). "
    [Show abstract] [Hide abstract] ABSTRACT: Mucopolysaccharidosis type IIIC (MPSIIIC) is a severe lysosomal storage disease caused by deficiency in activity of the transmembrane enzyme Heparan acetylCoA:α-Glucosaminide N-Acetyltransferase (HGSNAT) that catalyses the N-acetylation of α-glucosamine residues of heparan sulfate. Enzyme deficiency causes abnormal substrate accumulation in lysosomes, leading to progressive and severe neurodegeneration, somatic pathology and early death. There is no cure for MPSIIIC, and development of new therapies is challenging due to the unfeasibility of cross-correction. We generated a new mouse model of MPSIIIC by targeted disruption of the Hgsnat gene. Successful targeting left LacZ expression under control of the Hgsnat promoter, allowing the study of sites of endogenous expression, which was particularly important in CNS, but was also detectable in peripheral organs. Signs of CNS storage pathology, including glycosaminoglycan accumulation, lysosomal distension, lysosomal dysfunction and neuroinflammation were detected in 2-month-old animals and progressed with age. Glycosaminoglycan accumulation and ultrastructural changes were also observed in most somatic organs, but lysosomal pathology seemed most severe in liver. Furthermore, HGSNAT-deficient mice had altered locomotor and exploratory activity and shortened lifespan. Hence, this animal model recapitulates human MPSIIIC and provides a useful tool for the study of disease physiopathology and the development of new therapeutic approaches.
    Full-text · Article · Aug 2016
    • "Having this in mind, the search for therapeutic alternatives has been initiated in the recent years. Studies on gene therapy aimed at establishing an endogenous source of functional enzyme, as well as a variety of mutation-specific solutions including the use of chaperones [17][18][19], stop-codon readthrough drugs [20][21][22]and/or splicing correction oligonucleotides [23,24] are being published by different teams, some of them with promising results. Recently, the first pharmacological chaperone to be proven effective in ameliorating LSD clinical symptoms has just been approved by the European Medicines Agency (EMA) for Fabry disease management: migalastat [25,26]. "
    [Show abstract] [Hide abstract] ABSTRACT: Lysosomal storage diseases (LSDs) are a group of rare, life-threatening genetic disorders, usually caused by a dysfunction in one of the many enzymes responsible for intralysosomal digestion. Even though no cure is available for any LSD, a few treatment strategies do exist. Traditionally, efforts have been mainly targeting the functional loss of the enzyme, by injection of a recombinant formulation, in a process called enzyme replacement therapy (ERT), with no impact on neuropathology. This ineffectiveness, together with its high cost and lifelong dependence is amongst the main reasons why additional therapeutic approaches are being (and have to be) investigated: chaperone therapy; gene enhancement; gene therapy; and, alternatively, substrate reduction therapy (SRT), whose aim is to prevent storage not by correcting the original enzymatic defect but, instead, by decreasing the levels of biosynthesis of the accumulating substrate(s). Here we review the concept of substrate reduction, highlighting the major breakthroughs in the field and discussing the future of SRT, not only as a monotherapy but also, especially, as complementary approach for LSDs.
    Full-text · Article · Jul 2016
    • "The reaction mixture was incubated for 3 h at 37 C, and after the reaction termination step, the assay followed the procedure for measuring HGSNAT activity in fibroblasts. Enzymatic activity of HGSNAT against MU-b GlcN substrate was measured as previously described (Feldhammer et al. 2009b). "
    [Show abstract] [Hide abstract] ABSTRACT: Heparan sulfate acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT) catalyzes the transmembrane acetylation of heparan sulfate in lysosomes required for its further catabolism. Inherited deficiency of HGSNAT in humans results in lysosomal storage of heparan sulfate and causes severe neurodegenerative disease, mucopolysaccharidosis III type C (MPS IIIC). MPS IIIC patients can potentially benefit from a therapeutic approach based on active site-specific inhibitors of HGSNAT used as pharmacological chaperons to modify the folding of the mutant protein in the patient's cells. This research however was hampered by the absence of the assay suitable for high-throughput screening of drug libraries for HGSNAT inhibitors. The existing method utilizing 4-methylumbelliferyl-β-D-glucosaminide (MU-βGlcN) requires the sequential action of two enzymes, HGSNAT and β-hexosaminidase, whereas the radioactive assay with [C(14)]-AcCoA is complicated and expensive. We describe a novel direct method to assay HGSNAT enzymatic activity using fluorescent BODIPY-glucosamine as a substrate. The specificity of the assay was tested using cultured fibroblasts of MPS IIIC patients, which showed a profound deficiency of HGSNAT activity as compared to normal controls as well as to MPS IIIA and D patients known to have normal HGSNAT activity. Known competitive HGSNAT inhibitor, glucosamine, had similar inhibition constants for MU-βGlcN and BODIPY-glucosamine acetylation reactions. Altogether our data show that novel HGSNAT assay is specific and potentially applicable for the biochemical diagnosis of MPS IIIC and high-throughput screening for HGSNAT inhibitors.
    Full-text · Article · Oct 2015
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