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J. Biol. Chem., Vol. 277, Issue 46, 44079-44084, November 15, 2002
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From the Department of Nutritional Sciences, University of
Missouri, Columbia, Missouri 65211
Received for publication, August 26, 2002
Copper is an essential co-factor for several key
metabolic processes. This requirement in humans is underscored by
Menkes disease, an X-linked copper deficiency disorder caused by
mutations in the copper transporting P-type ATPase, MNK. MNK is located in the trans-Golgi network where it transports copper to
secreted cuproenzymes. Increases in copper concentration stimulate the trafficking of MNK to the plasma membrane where it effluxes copper. In
this study, a Menkes disease mutation, G1019D, located in the large
cytoplasmic loop of MNK, was characterized in transfected cultured
cells. In copper-limiting conditions the G1019D mutant protein was
retained in the endoplasmic reticulum. However, this mislocalization
was corrected by the addition of copper to cells via a process that was
dependent upon the copper binding sites at the N-terminal region of
MNK. Reduced growth temperature and the chemical chaperone, glycerol,
were found to correct the mislocalization of the G1019D mutant,
suggesting this mutation interferes with protein folding in the
secretory pathway. These findings identify G1019D as the first
conditional mutation associated with Menkes disease and demonstrate
correction of the mislocalized protein by copper supplementation. Our
findings provide a molecular framework for understanding how mutations
that affect the proper folding of the MNK transporter in Menkes
patients may be responsive to parenteral copper therapy.
Copper is essential for the growth and development of all
organisms. Several key metabolic processes are
copper-dependent, including oxidative respiration,
neurotransmitter synthesis, connective tissue formation, free radical
detoxification, iron homeostasis, and pigmentation (1). This copper
dependence for humans is most dramatically illustrated in Menkes
disease, an X-linked recessive copper deficiency disorder that is
generally lethal in early childhood (2). Menkes disease is caused by
mutations in a transmembrane copper transporting P-type ATPase, MNK (or
ATP7A), which is expressed in virtually all non-hepatic tissues (3-5).
Menkes disease is characterized by reduced copper transport from
intestinal enterocytes into the bloodstream. This reduced supply of
dietary copper is further compounded by reduced copper transport across
the blood-brain barrier to the central nervous system. Consequently,
Menkes patients exhibit a range of symptoms attributable to
deficiencies in copper-dependent metabolism, including
connective tissue defects, neurological degeneration, mental
retardation, seizures, osteoporosis, and hypopigmentation (2).
Studies using cultured cells suggest that MNK is located in the
trans-Golgi network (TGN),1
where it transports copper to copper-dependent enzymes
synthesized within secretory compartments (6-8). Elevated copper
concentrations stimulate the trafficking of MNK to the plasma membrane
where it is involved in copper efflux (6, 9). The copper efflux function of MNK is likely to be essential for supplying the blood with
dietary copper, as well as the central nervous system. Menkes patients
are treated by parenteral copper injections, which in the most
successful cases reduces neurological defects and prolongs life
expectancy (2). However, responses to treatment are variable and likely
to depend on the age at which treatment commences, and the extent to
which mutations impair copper transport or trafficking functions of the
Menkes protein. Indeed, the more positive outcomes are thought to
involve patients whose mutations permit the expression of some
functional MNK protein (10).
Here we characterize the effect of a Menkes disease missense mutation,
G1019D, on the intracellular location and trafficking of MNK. The
G1019D mutation was found to impair normal secretion and maturation of
MNK in the early secretory pathway. Surprisingly, these defects were
exacerbated by copper depletion of the growth media and corrected by
copper supplementation. This copper-dependent rescue of
mislocalized G1019D mutant protein occurred in a
dose-dependent and metal-specific manner and was dependent
on the N-terminal copper binding sites. Conditions such as reduced
temperature or glycerol supplementation, which are known to correct
misfolding of other membrane proteins, also prevented mislocalization
of the G1019D mutant protein. Our study reports the first conditional Menkes disease mutation that affects secretion and maturation of the
transporter and demonstrates correction of this defect by copper
supplementation. These observations provide new insights into
understanding how parenteral copper treatment of Menkes patients, whose
mutations affect protein folding, may yield positive outcomes.
Reagents and Cell Culture--
All chemicals were purchased from
Sigma. The immortalized Menkes fibroblast cell line, Me32a, was used to
generate cell lines expressing wild type MNK (WtMNK) and the G1019D
mutant. Me32a cells lack detectable MNK protein due to a frameshift
mutation, as described previously (11). Cells were cultured in minimal essential medium (Invitrogen) supplemented with 10% fetal bovine serum, penicillin/streptomycin in a 5% CO2, 37 °C
incubator. In some experiments, copper chloride or the copper chelator,
bathocuproine disulfonic acid was added to the culture medium,
as indicated in the figure legends. In some experiments various
concentrations of tunicamycin or 10% glycerol were added to the growth
media for 16 h.
Transfections--
The Transformer mutagenesis kit
(Clontech) was used to engineer the G1019D mutation
into an EcoRV subclone of the MNK cDNA and
verified by DNA sequencing. This mutant EcoRV fragment was then cloned into the MNK cDNA expression plasmid (12) by replacing the corresponding wild type fragment. The G1019D mutant fragment was
also cloned into the previously generated construct in which all six
Cys-X-X-Cys copper binding sites in the
N-terminal region were mutated to Ser-X-X-Ser
(13). Plasmid constructs bearing the wild type MNK or the G1019D mutant
cDNAs were transfected into Me32a cells using LipofectAMINE 2000 (Invitrogen), and G418 selection was used to isolate stably expressing
cell lines, as previously described (14). In the experiments shown
below in Figs. 6 and 7, Me32a cells were transiently transfected with
the indicated plasmids using LipofectAMINE 2000.
Immunofluorescence Microscopy and
Immunoblots--
Immunofluorescence microscopy was performed as
previously described (14), using affinity-purified MNK antibodies or
anti-PDI antibodies (Stressgen Biotech), and detected using Alexa594
anti-sheep or Alexa488 anti-mouse secondary antibodies (Molecular
Probes). Protein lysates were prepared as previously described and
fractionated using either 6% SDS-PAGE for MNK immunoblots or 8%
SDS-PAGE for all other immunoblots (8). Immunoblotting experiments were performed using either anti-MNK antibodies or anti-tubulin antibodies (Sigma), as indicated in the figure legends using an enhanced chemiluminescence detection kit (Roche Molecular Biochemicals).
The G1019D mutation was previously identified in a patient with
classic Menkes disease (15). Glycine 1019 is located in the largest
cytoplasmic loop of the ATPase, near the aspartic acid 1044 that is the
conserved phosphorylation site of all P-type ATPases (Fig.
1A). Although there has been
no specific role assigned to G1019, the non-conservative replacement of
a small neutral glycine with a large charged residue such as aspartic
acid, was predicted to reduce copper transport activity (15). Indeed, a
previous study of the recombinant G1019D MNK protein in the yeast,
Saccharomyces cerevisiae, demonstrated that this mutation severely reduced but did not eliminate copper transport, as determined by a complementation growth assay (16). In this study, we investigated the consequences of the G1019D mutation on the cell biology of the MNK
copper transporter. In vitro mutagenesis was used to
generate the G1019D mutation in a plasmid bearing the MNK cDNA.
Both wild type MNK (WtMNK) and the G1019D mutant protein were stably
expressed in the immortalized fibroblast cell line, Me32a. This cell
line was derived from a Menkes patient and does not express endogenous MNK protein (11). Three cell lines expressing the G1019D protein were
isolated by selection in G418 and used for immunofluorescence microscopy and immunoblotting experiments. Immunofluorescence microscopy showed that the WtMNK protein was located in the perinuclear region of cells (Fig. 1B), consistent with its location in
the TGN (6, 7). There was no signal detected in Me32a cells stably
transfected with the empty vector (data not shown). Although the G1019D
protein appeared concentrated in the perinuclear region (Fig.
1B, arrowhead), there was also staining
associated with reticular structures extending into cytoplasmic
regions. This same distribution was observed in three independent
G1019D cell lines (data not shown). This distribution suggested partial
location of G1019D protein in both the perinuclear Golgi complex and
the endoplasmic reticulum (ER).
Immunoblotting analysis detected the expected 180-kDa MNK protein in
cells expressing the WtMNK protein (Fig. 1B). However, the
G1019D protein was resolved as two bands that corresponded to the
wild type size, and a protein with an apparent molecular mass of
175 kDa (Fig. 1B). MNK has been previously shown to be a
glycosylated protein (7). The existence of this lower G1019D band,
together with the immunofluorescence data suggesting partial localization in the ER, indicated that the G1019D mutation may impair
the glycosylation of MNK resulting in this faster migrating species. To
explore this further, we treated cells with the glycosylation inhibitor, tunicamycin. This resulted in the generation of the faster
migrating non-glycosylated form of the WtMNK protein, which was similar
to the lower form of the G1019D protein in untreated cells.
Significantly, the G1019D protein was more sensitive to tunicamycin
treatment compared with wild type MNK and caused G1019D to migrate as a
single lower band. These data suggest that the double band of G1019D
protein in untreated cells arises from incomplete or defective
glycosylation and were consistent with the immunofluorescence data
suggesting partial retention of the G1019D protein in the ER.
We characterized the G1019D mutant protein further by exploring whether
varying the copper availability affected the localization of the
protein. To mimic the copper deficiency that occurs in various tissues
of Menkes disease patients, we limited the copper availability by
culturing the cell lines in the presence of the copper chelator,
bathocuproine disulfonate (BCS). Copper depletion had no apparent
effect on the WtMNK protein, which appeared perinuclear in both
untreated (Fig. 2A) and
BCS-treated cells (Fig. 2C). As seen earlier in Fig. 1, in
untreated G1019D-expressing cells, G1019D protein was observed in the
perinuclear region as well as a diffuse reticular distribution (Fig.
2B). Surprisingly, BCS treatment caused a striking change in
the location of the G1019D protein to a predominant reticular
distribution, with a notable absence of perinuclear signal (Fig.
2D). This BCS effect on the location of G1019D was due to
copper depletion, because it was not observed when BCS was added in
combination with copper (Fig. 2F). Indeed, the addition of
BCS and copper had the opposite effect by shifting the G1019D protein
to a predominantly perinuclear location (Fig. 2F). This
localization was indistinguishable from the location of the WtMNK
protein, which remained perinuclear in cells treated either with BCS
alone or BCS plus copper (Fig. 2, C and E). We
further increased the media copper levels to an excess concentration of
100 µM Cu, which is known to bring about copper-induced
trafficking of MNK to the plasma membrane, as shown for the WtMNK
protein (Fig. 2G). Notably, the G1019D mutant protein also
relocalized to the plasma membrane in copper-treated cells (Fig.
2H), suggesting the G1019D mutation did not inhibit
copper-induced trafficking from the TGN.
A Conditional Mutation Affecting Localization of the Menkes
Disease Copper ATPase
SUPPRESSION BY COPPER SUPPLEMENTATION*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (39K):
[in a new window]
Fig. 1.
Mislocalization and reduced glycosylation of
the G1019D mutant protein. A, schematic illustration of
the MNK copper ATPase showing the location of the G1019D mutation in
the large cytoplasmic loop that contains the conserved phosphorylated
aspartic acid D1044. Filled ovals indicate the six
N-terminal copper-bindings sites. B, left panels
demonstrate indirect immunofluorescence detection of WtMNK and G1019D
proteins in stably transfected cell lines using affinity-purified MNK
antibodies, as described under "Experimental Procedures." The
arrowhead indicates partial staining of G1019D in the
perinuclear region. Immunoblot detection of WtMNK and G1019D protein is
shown in the right panel in stably transfected cells using
affinity-purified MNK antibodies. The G1019D protein appears as a
doublet band corresponding to the normal size and a smaller product.
C, inhibition of glycosylation using tunicamycin causes
WtMNK to migrate as a double band of similar sizes as the G1019D
doublet in untreated cells. WtMNK- and G1019D-expressing cells were
incubated in the presence of increasing concentrations of tunicamycin
for 16 h prior to immunoblotting analysis as described under
"Experimental Procedures."
View larger version (66K):
[in a new window]
Fig. 2.
The mislocalization of the G1019D protein is
copper-responsive. Cells were cultured for 48 h in media
supplemented with the copper chelator, BCS, and/or copper, to assess
the effect of varying copper availability on the subcellular
distribution on the G1019D protein using immunofluorescence microscopy.
The distribution of WtMNK (A) and G1019D (B) is
shown after growing cells in basal media (~0.5 µM Cu).
When copper availability was limited by adding 500 µM BCS
to the culture medium, WtMNK remained perinuclear (C),
however, G1019D was mislocalized in a reticular distribution
(D). After supplementing 500 µM BCS plus 100 µM copper to cells, both WtMNK (E), and G1019D
(F) were predominantly perinuclear, demonstrating correction
of the G1019D mislocalization by copper. The expected redistribution of
WtMNK to the plasma membrane was observed when 100 µM
copper was added to the media (G), and this was also
observed for the G1019D protein (H). Images were viewed
using a ×60 oil objective after staining fixed cells with
affinity-purified MNK antibodies (1:1500) followed by a fluorescent
Alexa488-labeled secondary antibody (1:1000).
The effect of copper depletion on the G1019D protein was further
explored by testing whether the BCS-induced changes in subcellular location were also accompanied by changes in its electrophoretic mobility. In untreated cells, the G1019D protein appeared as the double
band (Fig. 3A, lane
1), however, in BCS-treated cells, the lower band was the
predominant form of G1019D and there was a marked diminution of the
normal sized upper band (Fig. 3A, lane 2).
Significantly, the combined addition of BCS and copper to G1019D-expressing cells reversed the BCS effect by causing a marked increase in levels of the upper band with a concomitant decrease in
levels of the lower band (Fig. 3A, lane 3). None
of the treatments affected the mobility of the WtMNK
protein (Fig. 3A, lanes 4-6). These
observations, together with the immunofluorescence data in Fig. 2,
suggested that copper limitation reduces secretion and glycosylation of
the G1019D mutant protein in early secretory compartments, which can be
rescued by copper supplementation.
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We then investigated the concentrations of copper required to increase the levels of the higher molecular weight G1019D product. The shift toward the higher form of the G1019D protein was observed when G1019D-expressing cells were cultured in 5 µM copper (Fig. 3B, lane 2) and was saturable with 30 µM copper (Fig. 3B, lane 4). To test metal specificity, we investigated whether exposure of cells to high levels of zinc, silver, or cadmium could enrich levels of the higher form of the G1019D protein (Fig. 3B, lanes 6-8). The metal concentrations tested were ~50% of the levels that retarded cell growth (data not shown). None of these heavy metals changed the ratio of upper and lower bands from those observed in untreated G1019D-expressing cells. These data suggest that the copper-dependent maturation of the G1019D protein is dose-dependent and -specific for this metal ion.
In Fig. 2D, a reticular distribution reminiscent of the ER
was observed for the G1019D protein when cells were depleted of copper
by BCS treatment. However, to more rigorously test this localization,
we tested if the BCS-induced distribution of the G1019D protein
co-localized with an ER marker, protein disulfide isomerase (PDI). Fig.
4 shows that the reticular localization of the G1019D protein in BCS-treated cells (Fig. 4D)
co-localized with PDI (Fig. 4E), as indicated by the
extensive yellow labeling in the merged image (Fig.
4F). There was no apparent co-localization of the WtMNK and
PDI in BCS-treated cells (Fig. 4, A-C). These data
indicated that the G1019D mutant was retained in the ER under in
copper-limiting conditions.
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Some disease-causing mutations in other membrane proteins have been
shown to cause retention in the ER due to protein misfolding, and some
of these misfolded proteins can be corrected by reducing the culturing
temperature or by treating cells with the chemical chaperone glycerol
(17-21). Therefore, we tested whether glycerol or reduced temperature
could prevent the BCS-induced retention of the G1019D protein in the ER
(Fig. 5B). Glycerol could
indeed suppress the BCS-induced retention of G1019D protein in the ER, as evident by strong perinuclear staining of G1019D when BCS and 10%
glycerol was added to the culture media (Fig. 5D). The
BCS-induced reticular distribution of G1019D was also found to be
temperature-sensitive, because it was suppressed by growing cells with
BCS at a reduced temperature of 30 °C (Fig. 5F). None of
these treatments altered the perinuclear location of WtMNK (Fig. 5,
A, C, and E). These data further
support the notion that the G1019D mutation causes temperature-sensitive misfolding of the MNK protein in the endoplasmic reticulum, which can be pharmacologically corrected using copper or
glycerol.
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Our studies indicate that increasing copper availability allows the
G1019D protein to migrate through the ER and correctly localize to the
TGN. One possible manner by which this occurs is via copper binding to
the N-terminal copper binding sites of the mutant G1019D protein, which
may stabilize the protein and permit glycosylation and secretion to
post-ER compartments. To test this hypothesis, we engineered a G1019D
mutant in which all six Cys-X-X-Cys copper
binding sites were mutated to Ser-X-X-Ser; refered to as
mutant Metal Binding
Sites, mMBS1-6/G1019D. Me32a cells were transiently
transfected with plasmids for WtMNK, G1019D, mMBS1-6 (mutant metal
binding sites alone), and mMBS1-6/G1019D and then cultured in either
basal medium or elevated copper. The localization of these proteins was
then investigated by immunofluorescence microscopy. As expected, WtMNK
was perinuclear in untreated cells (Fig.
6A) and dispersed to the
plasma membrane in elevated copper conditions (Fig. 6B). As
shown earlier, the G1019D protein was both perinuclear and reticular in
untreated cells (Fig. 6C) and dispersed to the plasma
membrane by elevated copper (Fig. 6D). The mutation of the
copper binding sites alone did not affect the normal perinuclear
location in untreated cells (Fig. 6E), however, these sites
were essential for copper-induced trafficking to the plasma membrane
under elevated copper conditions (Fig. 6F), as shown in
previous studies (13). Significantly, when all six copper binding sites
were mutated in combination with the G1019D mutation, the resulting
mMBS1-6/G1019D mutant was completely located in a reticular
distribution (Fig. 6G), and this resembled the ER retention
of G1019D in copper-deficient conditions shown earlier (Fig.
2F). It was notable that increasing the copper concentration of the media failed to change the reticular distribution of the mMBS1-6/G1019D mutant (Fig. 6H). These data suggest that
the secretion of the G1019D mutant protein to post-ER compartments
requires the N-terminal copper binding sites.
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We then determined whether the copper binding sites were required for
the enrichment of the higher molecular weight G1019D protein in cells
exposed to elevated copper. Me32a cells were transiently transfected
with expression plasmids encoding WtMNK, G1019D, mMBS1-6, or
mMBS1-6/G1019D mutants and then exposed for 48 h to either BCS or
elevated copper. In BCS-treated cells, both G1019D and mMBS1-6/G1019D
proteins were resolved as single bands that were smaller than both the
WtMNK and mMBS1-6 proteins (Fig. 7).
Significantly, elevated copper treatment of cells rescued the G1019D
mutant protein by enriching expression of the correctly sized protein
(Fig. 7, lane 6), however, copper treatment did not change
the apparent size of the smaller mMBS1-6/G1019D protein (Fig. 7,
lane 8). These data, together with the immunofluorescence findings in Fig. 6, suggest that copper binding to the N-terminal copper binding sites is required for proper secretion and glycosylation of the G1019D mutant protein.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The G1019D mutant protein was previously shown to have reduced copper transport activity in a complementation assay in the yeast, S. cerevisiae (16). Here we expand on these previous studies by using a mammalian cell model to explore the effect of this mutation on the cell biology of MNK. The recombinant G1019D protein in cultured cells was partially localized in the ER and the TGN, and this was associated with the existence of two electrophoretic mobilities for the protein. Inhibition of glycosylation using tunicamycin resulted in the appearance of only a single lower G1019D band. This suggested that the faster mobility of the lower G1019D band was the result of defective glycosylation, rather than truncation, degradation, or premature termination of the G1019D protein. Our findings suggest that the mislocalization of the G1019D protein probably contributed to Menkes disease pathology by limiting the transport of dietary copper from enterocytes into circulation and, subsequently, the transport of copper into the appropriate secretory compartments for cuproenzymes.
An important finding of our study was that the extent of the mislocalization of the G1019D protein was dependent on copper levels in the cell growth media. Extensive mislocalization in the ER was observed when copper availability was limited by chelation using BCS, and this was associated with the presence of only the lower molecular weight protein. Adding copper in combination with the BCS restored normal perinuclear localization of the G1019D mutant and increased the amount of the normal sized product. This copper-dependent correction was dose-dependent and copper-specific. Collectively, these data suggest that copper limitation impaired glycosylation and secretion of the G1019D mutant protein in early secretory compartments and that these defects could be rescued by copper supplementation. The copper-dependent correction of the G1019D mutant required the N-terminal copper binding sites, because the G1019D mutant in which these sites were mutated was retained in the ER and failed to become glycosylated when exposed to elevated copper. Because mutation of the six copper binding sites alone did not result in ER mislocalization or impaired glycosylation of MNK, these sites are not critical for secretion through the ER per se. Our data suggest that, in the context of a destabilized protein such as G1019D, the binding of copper to these sites may serve to promote a conformation that is able to bypass the ER quality control mechanisms. To date the N-terminal copper binding sites of MNK have been shown to have roles in the acquisition of copper under copper limiting conditions (16, 22) and in the copper-regulated trafficking of MNK from the TGN (13, 23). Our findings provide evidence that these sites are capable of copper binding early in the biosynthesis of MNK and that this process can be essential for secretion and maturation of mutant MNK proteins, such as G1019D.
The export of newly synthesized proteins from the ER to the Golgi is regulated by a quality control mechanism that ensures that only properly folded and assembled proteins exit the ER and misfolded proteins are retained and ultimately degraded. Defective processing of the cystic fibrosis transmembrane regulator protein was originally recognized as an important cause of cystic fibrosis (20) and has been documented in several proteins involved in other congenital diseases (17, 24). The most notable of these is Wilson disease, which is caused by mutations in a copper ATPase, WND, that shows a high degree of sequence similarity to MNK and is also is localized in the TGN (25). Wilson disease is caused by a toxic accumulation of hepatic copper due to defective biliary excretion of copper via the WND ATPase. A common Wilson disease mutation, H1069Q, causes retention of the WND protein in the ER (18), and ER retention has been subsequently identified for several other Wilson disease mutations (26). In several diseases involving misfolded proteins, glycerol treatment or reduced temperature facilitates the proper folding and secretion of the affected mutant protein in cultured cells. The Wilson disease H1069Q mutation is corrected by reduced temperature, and, similarly, we found both reduced temperature and glycerol could suppress mislocalization of the G1019D protein under copper-depleted conditions. It is possible that the large cytoplasmic loop of MNK and WND copper ATPases, which contains the respective G1019 and H1069 amino acids, is a region important in the folding and secretion of these transporters. It is notable that this cytoplasmic loop of WND has been recently shown to interact with the N-terminal region in a copper-dependent manner in vitro (27). Our data showing a dependence of the N-terminal copper bindings for the secretion of the G1019D mutant highlights the possibility that such an interaction in WND and MNK proteins may influence the processing and secretion of these copper transporters in vivo.
It is notable that certain misfolded proteins, such as the vassopressin receptor, P-glycoprotein, and apolipoprotein A, which are retained in the ER, can be rescued by exposing cells to their respective ligands (28-30). It is thought that the binding of the ligands to these proteins early in the secretory pathway confers a more stable conformation of the mutant proteins, allowing evasion of the ER quality control mechanisms. Moreover, in some diseases, such as nephrogenic diabetes insipidus, which involves mutations in the vassopressin receptor, the same ligand with protein-stabilizing ability is used to treat patients (29, 31). The demonstration that copper could correct the mislocalization of the Menkes G1019D mutant is clearly analogous to the ligand-dependent rescue of these other mutant membrane proteins. Moreover, our findings offer novel insights at the molecular level to explain how current treatments for Menkes disease, which involve parenteral administration of copper, may yield positive outcomes in certain individuals whose mutations affect the processing and secretion of MNK.
Although we have no specific information on the copper status of the
G1019D Menkes patient, there is typically a marked decrease in
circulating serum copper levels due to reduced intestinal absorption. A
more severe copper deficiency in the brain occurs due to the predicted
role of MNK in the transport of copper across the blood-brain barrier.
Our findings allow us to speculate that reduced circulating copper
levels in the G1019D Menkes patient may have impaired the maturation of
the G1019D protein in various tissues, especially in the brain.
Moreover, our finding that copper could suppress this mislocalization
in cultured cells suggests in principle that a course of treatment
involving copper injections may have promoted normal localization of
the G1019D protein and sufficient copper transport activity for a
positive clinical response. Clearly, future studies with transgenic
mice and cultured cells that express various Menkes disease alleles,
such as G1019D, will be a useful strategy for understanding the affects
of copper treatments on the function of mutant MNK proteins at the
cellular and physiological levels.
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ACKNOWLEDGEMENTS |
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We thank Drs. David Eide and Elizabeth Rogers for critical evaluation of the manuscript and Dr. Julian Mercer for MNK antibodies and the mutant metal binding site plasmid.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health (DK 59893) and the University of Missouri Research Board (RB 01-115).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: Dept. of Nutritional
Sciences, 217 Gwynn Hall, University of Missouri-Columbia, Columbia, MO
65211. Tel.: 573-882-9685; Fax: 573-882-0185; E-mail: PetrisM@missouri.edu.
Published, JBC Papers in Press, September 6, 2002, DOI 10.1074/jbc.M208737200
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ABBREVIATIONS |
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The abbreviations used are: TGN, trans-Golgi network; WtMNK, wild type MNK; ER, endoplasmic reticulum; BCS, bathocuproine disulfonate; PDI, protein-disulfide isomerase; WND, Wilson disease protein.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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| 1. | Linder, M. C., Wooten, L., Cerveza, P., Cotton, S., Shulze, R., and Lomeli, N. (1998) Am. J. Clin. Nutr. 67, 965S-971S[Abstract] |
| 2. | Kaler, S. G. (1998) Am. J. Clin. Nutr. 67, 1029S-1034S[Abstract] |
| 3. | Mercer, J. F. B., Livingston, J., Hall, B. K., Paynter, J. A., Begy, C., Chandrasekharappa, S., Lockhart, P., Grimes, A., Bhave, M., Siemenack, D., and Glover, T. W. (1993) Nat. Genet. 3, 20-25[CrossRef][Medline] [Order article via Infotrieve] |
| 4. | Vulpe, C., Levinson, B., Whitney, S., Packman, S., and Gitschier, J. (1993) Nat. Genet. 3, 7-13[CrossRef][Medline] [Order article via Infotrieve] |
| 5. | Chelly, J., Tumer, Z., Tonnerson, T., Petterson, A., Ishikawa-Brush, Y., Tommerup, N., Horn, N., and Monaco, A. P. (1993) Nat. Genet. 3, 14-19[CrossRef][Medline] [Order article via Infotrieve] |
| 6. | Petris, M. J., Mercer, J. F. B., Culvenor, J. G., Lockhart, P., Gleeson, P. A., and Camakaris, J. (1996) EMBO J. 15, 6084-6095[Medline] [Order article via Infotrieve] |
| 7. |
Yamaguchi, Y.,
Heiny, M. E.,
Suzuki, M.,
and Gitlin, J. D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14030-14035 |
| 8. |
Petris, M. J.,
Strausak, D.,
and Mercer, J. F. B.
(2000)
Hum. Mol. Genet.
9,
2845-2851 |
| 9. |
Camakaris, J.,
Petris, M.,
Bailey, L.,
Shen, P.,
Lockhart, P.,
Glover, T. W.,
Barcroft, C. L.,
Patton, J.,
and Mercer, J. F. B.
(1995)
Hum. Mol. Genet.
4,
2117-2123 |
| 10. | Kaler, S. G., Das, S., Levinson, B., Goldstein, D. S., Holmes, C. S., Patronas, N. J., Packman, S., and Gahl, W. A. (1996) Biochem. Mol. Med. 57, 37-46[CrossRef][Medline] [Order article via Infotrieve] |
| 11. |
La Fontaine, S.,
Firth, S. D.,
Camakaris, J.,
Engelzou, A.,
Theophilos, M. B.,
Petris, M. J.,
Lockhart, P.,
Greenough, M.,
Brooks, H.,
Reddel, R. R.,
and Mercer, J. F. B.
(1998)
J. Biol. Chem.
273,
31375-31380 |
| 12. |
Petris, M. J.,
and Mercer, J. F. B.
(1999)
Hum. Mol. Genet.
8,
2107-2175 |
| 13. |
Strausak, D., La,
Fontaine, S.,
Hill, J.,
Firth, S. D.,
Lockhart, P. J.,
and Mercer, J. F. B.
(1999)
J. Biol. Chem.
274,
11170-11177 |
| 14. |
Petris, M. J.,
Camakaris, J.,
Greenough, M., La,
Fontaine, S.,
and Mercer, J. F. B.
(1998)
Hum. Mol. Genet.
7,
2063-2071 |
| 15. | Tümer, Z., Lund, C., Tolshave, J., Vural, B., Tonnesen, T., and Horn, N. (1997) Am. J. Hum. Genet. 60, 63-71[Medline] [Order article via Infotrieve] |
| 16. |
Payne, A. S.,
and Gitlin, J. D.
(1998)
J. Biol. Chem.
273,
3765-3770 |
| 17. |
Halaban, R.,
Svedine, S.,
Cheng, E.,
Smicun, Y.,
Aron, R.,
and Hebert, D. N.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5889-5894 |
| 18. |
Payne, A. S.,
Kelly, E. J.,
and Gitlin, J. D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
10854-10859 |
| 19. |
Sato, S.,
Ward, C. L.,
Krouse, M. E.,
Wine, J. J.,
and Kopito, R. R.
(1996)
J. Biol. Chem.
271,
635-638 |
| 20. | Cheng, S. H., Gregory, R. J., Marshall, J., Paul, S., Souza, D. W., White, G. A., O'Riordan, C. R., and Smith, A. E. (1990) Cell 63, 827-834[CrossRef][Medline] [Order article via Infotrieve] |
| 21. |
Loo, T. W.,
and Clarke, D. M.
(1994)
J. Biol. Chem.
269,
7243-7248 |
| 22. |
Voskoboinik, I.,
Mar, J.,
Strausak, D.,
and Camakaris, J.
(2001)
J. Biol. Chem.
276,
28620-28627 |
| 23. |
Goodyer, I. D.,
Jones, E. E.,
Monaco, A. P.,
and Francis, M. J.
(1999)
Hum. Mol. Genet.
8,
1473-1478 |
| 24. |
Partridge, C. J.,
Beech, D. J.,
and Sivaprasadarao, A.
(2001)
J. Biol. Chem.
276,
35947-35952 |
| 25. |
Hung, I. H.,
Suzuki, M.,
Yamaguchi, Y.,
Yuan, D. S.,
Klausner, R. D.,
and Gitlin, J. D.
(1997)
J. Biol. Chem.
272,
21461-21466 |
| 26. |
Forbes, J. R.,
and Cox, D. W.
(2000)
Hum. Mol. Genet.
9,
1927-1935 |
| 27. |
Tsivkovskii, R.,
MacArthur, B. C.,
and Lutsenko, S.
(2001)
J. Biol. Chem.
276,
2234-2242 |
| 28. |
Wang, J.,
and White, A. L.
(1999)
J. Biol. Chem.
274,
12883-12889 |
| 29. | Morello, J. P., Salahpour, A., Laperriere, A., Bernier, V., Arthus, M. F., Lonergan, M., Petaja-Repo, U., Angers, S., Morin, D., Bichet, D. G., and Bouvier, M. (2000) J. Clin. Invest. 105, 887-895[Medline] [Order article via Infotrieve] |
| 30. |
Loo, T. W.,
and Clarke, D. M.
(1997)
J. Biol. Chem.
272,
709-712 |
| 31. | Serradeil-Le Gal, C. (2001) Cardiovasc. Drug Rev. 19, 201-214[Medline] [Order article via Infotrieve] |
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