THEMES
Genetic Disorders of Membrane Transport
IV. Wilson's disease and
Menkes disease*
Mark
Schaefer and
Jonathan D.
Gitlin
Edward Mallinckrodt Department of Pediatrics, Washington
University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
Copper is an essential
transition metal that permits the facile transfer of electrons in a
series of critical biochemical pathways. Menkes disease and Wilson's
disease are inherited disorders of copper metabolism resulting from the
absence or dysfunction of homologous copper-transporting ATPases that
reside in the trans-Golgi network of all cells. Despite striking
differences in the clinical presentation of these two diseases, the
respective ATPases function in precisely the same manner within the
cell and the unique clinical features of each disease are entirely the
result of the tissue-specific expression of each protein. Elucidation
of the basic defect in these rare genetic disorders has provided a
valuable heuristic paradigm for understanding the mechanisms of
cellular copper homeostasis.
Wilson's disease; Menkes disease; adenosine
triphosphatase
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INTRODUCTION |
MENKES DISEASE IS AN X-linked disorder of copper
metabolism resulting in growth failure and severe neurodegenerative
disease in early childhood. The Menkes disease gene was physically
mapped by analysis of a balanced translocation in an affected female infant, and subsequent cloning of the gene revealed a predicted protein
with marked similarity to a cation transporting P-type ATPase essential
for copper homeostasis in Enterococcus
hirae. The Menkes disease ATPase
transports copper across the placenta, the gastrointestinal tract, and
the blood-brain barrier, and the clinical features of this disease
arise from deficient activity of essential cuproenzymes. Such features
include abnormal hair, absence of pigmentation, laxity of the skin and
joints, bony dysplasia, and cerebellar degeneration. Allelic
heterogeneity accounts for milder forms of the disease with minimal or
absent neurological symptoms (1).
Wilson's disease is an autosomal recessive disorder of copper
metabolism resulting in hepatic cirrhosis and neuronal degeneration. Based on homology to the Menkes disease gene, the Wilson's disease locus was cloned and shown to encode a novel member of this same family
of copper-transporting ATPases with 55% amino acid identity to the
Menkes ATPase. The Wilson's disease protein is expressed in the liver
and transports copper into the hepatocyte secretory pathway for
subsequent incorporation into ceruloplasmin and excretion into bile.
Affected individuals present with signs and symptoms arising from
impaired biliary copper excretion. Because this route represents the
only physiological mechanism of copper excretion, excessive
accumulation of copper in the hepatocyte cytoplasm eventually results
in cellular necrosis with leakage of copper into the plasma and
deposition of copper in extrahepatic tissues, including the limbus of
the cornea and the basal ganglia of the brain (1).
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STRUCTURE OF COPPER TRANSPORTERS |
Sequence comparison and hydropathy plot analysis of the derived amino
acid sequence of the Menkes and Wilson's ATPases reveal a polytopic
membrane protein predicted to transport copper across biological
membranes in an ATP-dependent manner (Fig.
1). Homologous proteins have now been
identified in a wide range of prokaryotic and eukaryotic species and
shown to play an analogous role in copper transport in these organisms.
Conserved amino acid motifs in such proteins include the MXCXXC
copper-binding sequences in the amino terminus, an IGTEA phosphatase
domain, a conserved DKTGT sequence that includes the invariant aspartyl
residue, which is reversibly phosphorylated in the process of energy
transduction, and a GDGVND ATP binding domain (Fig. 1). In addition to
these functionally defined regions, a highly conserved SEHPL sequence is present within the large cytoplasmic loop containing the ATP binding
domain. The histidine residue in this motif is conserved in all known
copper-transporting P-type ATPases and is the site of the most common
mutation (H1069Q) in patients with
Wilson's disease (21).

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Fig. 1.
Proposed topological model of the human Wilson's disease P-type
ATPase. The directed movement of copper from the cytoplasm into the
secretory compartment is shown. Conserved amino acid motifs are
indicated in circles. * Histidine residue 1069, which is the site
of common disease mutation. Modified from Payne and Gitlin (14) with
permission.
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Biosynthetic studies of the Wilson's and Menkes proteins indicate that
each ATPase is synthesized as a single-chain polypeptide, localized to
the trans-Golgi network of the cell (3, 8, 17, 20, 22). In this
location, these ATPases transport copper into the secretory pathway of
the cell for incorporation into specific cuproenzymes and export from
the cell. Support for this model has come from studies in
Saccharomyces
cerevisiae deficient in the homologous
copper-transporting ATPase CCC2 (23, 24). Expression of the Wilson's
or Menkes proteins in ccc2
yeast
restores copper incorporation into the ceruloplasmin homologue FET3,
providing direct evidence of copper transport by these ATPases into the secretory pathway of the cell (8, 14).
Mutation of a conserved histidine to glutamine
(H1086Q) in the Menkes protein,
homologous to the common H1069Q
mutation in Wilson's disease, abrogates copper transport by this
protein in ccc2
yeast (14). The
finding that the most commonly occurring mutation in Wilson's disease
similarly compromises function of the Menkes protein provides evidence
of a commonality of transporter function. Studies demonstrating that
the human Wilson's protein can rescue the phenotype of Menkes disease
protein-deficient cells also provide compelling evidence that these
ATPases work through common biochemical mechanisms (15).
These data support the concept that the differences in clinical
presentation of these diseases are the result of tissue-specific
differences in expression and raise the possibility that interventions
to induce expression of the Wilson's protein early in development in
cells normally expressing the Menkes protein might provide a novel
therapeutic approach in this otherwise fatal disease.
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MECHANISMS OF CELLULAR COPPER HOMEOSTASIS |
Although the Menkes and Wilson's ATPases are localized to the
trans-Golgi network under steady-state conditions, an increase in the
copper concentration results in trafficking of these proteins to a
cytoplasmic vesicular compartment visible by electron microscopy (8,
10, 17). In the polarized hepatocytes of the liver these vesicles are
localized adjacent to the canalicular membrane (20). As copper is
transported into this compartment, the intracellular copper
concentration falls and these proteins are recycled back to the
trans-Golgi network while copper is exported from the cell. This
copper-dependent trafficking of the Menkes and Wilson's proteins is
rapid, occurring within minutes of exposure to increased copper, and
represents a novel posttranslational mechanism allowing for restoration
of cellular copper homeostasis (8, 17, 20).
The mechanisms determining the intracellular location of these ATPases
are not well understood. In a patient with a mild form of Menkes
disease, in which mutation of the splice donor site of exon 10 results
in an in-frame deletion of the third and fourth transmembrane domains
of the Menkes protein, immunofluorescence studies indicate that this
transmembrane region is essential for the trans-Golgi network
localization (4, 19). Studies of the
H1069Q mutant Wilson's protein
suggest that this conserved histidine residue is also essential for
localization to the trans-Golgi network and copper-dependent recycling
(15). Recent studies (16) utilizing site-directed mutagenesis of the
Menkes protein have revealed that a dileucine motif in the carboxy
terminus is necessary for this protein to respond to intracellular
copper, suggesting that trafficking signals common to the cytoplasmic domain of many intracellular cargo proteins may be utilized by these
ATPases in the physiological response to copper.
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THE COPPER CHAPERONE HAH1 |
A series of genetic studies in
Saccharomyces
cerevisiae have revealed that the
delivery of copper to specific proteins within the cell is mediated by
a group of intracellular proteins termed copper chaperones. ATX1
encodes a small cytosolic copper-binding protein in
Saccharomyces originally identified as
a multicopy suppressor of sod1
mutants (11). This protein delivers copper to CCC2 for subsequent
transport into the secretory pathway and incorporation into the
multicopper oxidase FET3, which is required for high-affinity iron
uptake (12). The identification of a homologous protein in humans
termed HAH1 has defined a role for this chaperone in mammalian cells
and reveals a remarkable evolutionary conservation of the mechanisms of
copper trafficking and compartmentalization (9).
HAH1 contains a single copy of the amino acid sequence MTCGGC in the
amino terminus. A homologous sequence is present in the amino terminus
of the Menkes and Wilson's proteins and has been shown to bind copper
in vitro and in vivo (2, 13). Solution structure analysis of a single
domain containing this motif from the Menkes protein and
structure-function studies on ATX1 and HAH1 reveal a novel linear
bicoordinate copper ligand capable of binding and rapidly transferring
this metal (6, 7, 18) (Fig. 2). Consistent
with this, in vitro and in vivo studies have revealed the essential
role of these cysteine residues in HAH1 in copper trafficking to the
secretory pathway (7). The homologous MXCXXC motifs on the yeast copper
transporter CCC2 are the site of interaction with ATX1 and play an
essential role in copper transfer between these proteins (18).

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Fig. 2.
Structural model of the human copper chaperone HAH1. Linear
bicoordinate copper binding site is illustrated with conserved cysteine
ligands. From Hung et al. (7) with permission. N-ter and C-ter, amino
and carboxy terminals, respectively.
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FUTURE DIRECTIONS |
The last several years have witnessed significant progress in our
understanding of the molecular mechanisms of cellular copper transport.
As the balance of copper in the body is determined entirely by
gastrointestinal absorption and biliary excretion, understanding these
mechanisms is of direct relevance to gastrointestinal physiology. Most
interestingly, recent data from yeast suggest that the function of a
CLC chloride channel homologue is essential for copper transport by
CCC2 into a post-Golgi vacuolar compartment (5). These findings suggest
that the physiological principles derived for the movement of other
cations across biological membranes may also prove relevant for the
function of the copper-transporting ATPases and imply that the spectrum
of diseases involving abnormal copper homeostasis may extend to
abnormalities in associated anion transport proteins. Elucidation of
the molecular basis of the genetic disorders of copper transport has
thus provided useful direction for the future of investigation in this field.
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FOOTNOTES |
*
Fourth in a series of invited articles on Genetic
Disorders of Membrane Transport.
Address for reprint requests: J. D. Gitlin, St. Louis Children's
Hospital, One Children's Place, St. Louis, MO 63110.
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REFERENCES |
1.
Culotta, V. C., and J. D. Gitlin. The
disorders of copper transport. In: The Metabolic and
Molecular Basis of Inherited Disease, edited by
C. R. Scriver, A. L. Beaudet, W. S. Sly,
and D. Valle. New York: McGraw-Hill. In press.
2.
DiDonato, M.,
S. Narindrasorasak,
J. R. Forbes,
D. W. Cox,
and
B. Sarkar.
Expression, purification, and metal binding properties of the N-terminal domain from the Wilson disease putative copper-transporting ATPase (ATP7B).
J. Biol. Chem.
272:
33279-33282,
1997[Abstract/Free Full Text].
3.
Dierick, H. A.,
A. N. Adam,
J. F. Escara-Wilke,
and
T. W. Glover.
Immunocytochemical localization of the Menkes copper transport protein (ATP7A) to the trans-Golgi network.
Hum. Mol. Genet.
6:
409-416,
1997[Abstract/Free Full Text].
4.
Francis, M. J.,
E. E. Jones,
E. R. Levy,
S. Ponnambalam,
J. Chelly,
and
A. P. Monaco.
A Golgi localization signal identified in the Menkes recombinant protein.
Hum. Mol. Genet.
7:
1245-1252,
1998[Abstract/Free Full Text].
5.
Gaxiola, R. A.,
D. S. Yuan,
R. D. Klausner,
and
G. R. Fink.
The yeast CLC chloride channel functions in cation homeostasis.
Proc. Natl. Acad. Sci. USA
95:
4046-4050,
1998[Abstract/Free Full Text].
6.
Gitschier, J.,
B. Moffat,
D. Reilly,
W. I. Wood,
and
W. J. Fairbrother.
Solution structure of the fourth metal-binding domain from the Menkes copper-transporting ATPase.
Nat. Struct. Biol.
5:
47-54,
1998[Medline].
7.
Hung, I. H.,
R. L. Casareno,
G. Labesse,
F. S. Mathews,
and
J. D. Gitlin.
HAH1 is a copper-binding protein with distinct amino acid residues mediating copper homeostasis and antioxidant defense.
J. Biol. Chem.
273:
1749-1754,
1998[Abstract/Free Full Text].
8.
Hung, I. H.,
M. Suzuki,
Y. Yamaguchi,
D. S. Yuan,
R. D. Klausner,
and
J. D. Gitlin.
Biochemical characterization of the Wilson disease protein and functional expression in the yeast Saccharomyces cerevisiae.
J. Biol. Chem.
272:
21461-21466,
1997[Abstract/Free Full Text].
9.
Klomp, L. W. J.,
S.-J. Lin,
D. S. Yuan,
R. D. Klausner,
V. C. Culotta,
and
J. D. Gitlin.
Identification and functional expression of HAH1, a novel human gene involved in copper homeostasis.
J. Biol. Chem.
272:
9221-9226,
1997[Abstract/Free Full Text].
10.
LaFontaine, S.,
S. D. Firth,
P. J. Lockhart,
H. Brooks,
R. G. Parton,
J. Camakaris,
and
J. F. B. Mercer.
Functional analysis and intracellular localization of the human Menkes protein (MNK) stably expressed from cDNA construct in Chinese hamster ovary cells (CHO-K1).
Hum. Mol. Genet.
7:
1293-1300,
1998[Abstract/Free Full Text].
11.
Lin, S. J.,
and
V. C. Culotta.
The ATX1 gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity.
Proc. Natl. Acad. Sci. USA
92:
3784-3788,
1995[Abstract/Free Full Text].
12.
Lin, S.-J.,
R. A. Pufahl,
A. Dancis,
T. V. O'Halloran,
and
V. C. Culotta.
A role for the Saccharomyces cerevisiae ATX1 gene in copper trafficking and iron transport.
J. Biol. Chem.
272:
9215-9220,
1997[Abstract/Free Full Text].
13.
Lutsenko, S.,
K. Petrukhin,
M. J. Cooper,
C. T. Gilliam,
and
J. H. Kaplan.
N-terminal domains of human copper-transporting adenosine triphosphatases (the Wilson's and Menkes disease proteins) bind copper selectively in vivo and in vitro with stoichiometry of one copper per metal-binding repeat.
J. Biol. Chem.
272:
18939-18944,
1997[Abstract/Free Full Text].
14.
Payne, A. S.,
and
J. D. Gitlin.
Functional expression of the Menkes disease protein reveals common biochemical mechanisms among the copper-transporting P-type ATPases.
J. Biol. Chem.
273:
3765-3770,
1998[Abstract/Free Full Text].
15.
Payne, A. S.,
E. J. Kelly,
and
J. D. Gitlin.
Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation.
Proc. Natl. Acad. Sci. USA
95:
10854-10859,
1998[Abstract/Free Full Text].
16.
Petris, M. J.,
J. Camakaris,
M. Greenough,
S. LaFontaine,
and
J. F. B. Mercer.
A C-terminal di-leucine is required for localization of the Menkes protein in the trans-Golgi network.
Hum. Mol. Genet.
7:
2063-2071,
1998[Abstract/Free Full Text].
17.
Petris, M. J.,
J. F. B. Mercer,
J. G. Culvenor,
P. Lockhart,
P. A. Gleeson,
and
J. Camakaris.
Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking.
EMBO J.
15:
6084-6095,
1996[Abstract].
18.
Pufahl, R. A.,
C. P. Singer,
K. L. Peariso,
S.-J. Lin,
P. Schmidt,
V. C. Culotta,
J. E. Penner-Hahn,
and
T. V. O'Halloran.
Metal ion chaperone function of the soluble Cu(I) receptor, Atx1.
Science
278:
853-856,
1997[Abstract/Free Full Text].
19.
Qi, M.,
and
P. H. Byers.
Constitutive skipping of alternatively spliced exon 10 in the ATP7A gene abolishes Golgi localization of the Menkes protein and produces the occipital horn syndrome.
Hum. Mol. Genet.
7:
465-469,
1998[Abstract/Free Full Text].
20.
Schaefer, M., R. G. Hopkins, M. L. Failla,
and J. D. Gitlin. Hepatocyte-specific localization,
copper dependent trafficking and developmental expression of the Wilson
disease protein in the liver. Am.
J.
Physiol. In press.
21.
Thomas, G. R.,
J. R. Forbes,
E. A. Roberts,
J. M. Walshe,
and
D. W. Cox.
The Wilson disease gene: spectrum of mutations and their consequences.
Nat. Genet.
9:
210-217,
1995[Medline].
22.
Yamaguchi, Y.,
M. E. Heiny,
M. Suzuki,
and
J. D. Gitlin.
Biochemical characterization and intracellular localization of the Menkes disease protein.
Proc. Natl. Acad. Sci. USA
93:
14030-14035,
1996[Abstract/Free Full Text].
23.
Yuan, D. S.,
A. Dancis,
and
R. D. Klausner.
Restriction of copper export in Saccharomyces cerevisiae to a late Golgi or post-Golgi compartment in the secretory pathway.
J. Biol. Chem.
272:
25787-25793,
1997[Abstract/Free Full Text].
24.
Yuan, D. S.,
R. Stearman,
A. Dancis,
T. Dunn,
T. Beeler,
and
R. D. Klausner.
The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake.
Proc. Natl. Acad. Sci. USA
92:
2632-2636,
1995[Abstract].
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