loss of atp-dependent transport activity in pseudoxanthoma elasticum-associated mutants of ...

37 Pages·2002·0.28 MB·English

JBC Papers in Press. Published on March 5, 2002 as Manuscript M110918200
Loss-of-function mutants of ABCC6/MRP6 in PXE
LOSS OF ATP-DEPENDENT TRANSPORT ACTIVITY
IN PSEUDOXANTHOMA ELASTICUM-ASSOCIATED
MUTANTS OF HUMAN ABCC6 (MRP6)
Attila Iliás1, Zsolt Urbán2, Thomas L. Seidl2, Olivier Le Saux2,
Emese Sinkó3, Charles D. Boyd2, Balázs Sarkadi3 and András
Váradi1
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1 Institute of Enzymology, Hungarian Academy of Sciences, 1113 nlo
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Budapest, Hungary d fro
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2 Laboratory of Matrix Pathobiology, The Pacific Biomedical h
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Research Center, University of Hawaii, Honolulu, Hawaii 96822, w
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USA c
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3 National Medical Center, Institute of Haematology and b/
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Immunology, Research Group of the Hungarian Academy of st o
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Sciences, 1113 Budapest, Hungary o
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Corresponding author: Dr. András Váradi , 2
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Institute of Enzymology, Hungarian Academy of Sciences,
Karolina ut 29, 1113 Budapest, Hungary
Tel: [phone]; Fax: [phone]; email: [email protected]
Running title: Loss-of-function mutants of ABCC6/MRP6 in PXE
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Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Loss-of-function mutants of ABCC6/MRP6 in PXE
SUMMARY
Mutations in the ABCC6 (MRP6) gene cause pseudoxanthoma
elasticum (PXE), a rare heritable disorder resulting in the
calcification of elastic fibers. In the present study a cDNA
encoding a full-length normal variant of ABCC6 was amplified
from a human kidney cDNA library and the protein was
expressed in Sf9 insect cells. In isolated membranes ATP binding
as well as ATP-dependent active transport by ABCC6 were
demonstrated. We found that glutathione conjugates, including
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leukotriene-C4 (LTC4) and N-ethylmaleimide S-glutathione (NEM- nloa
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GS), were actively transported by human ABCC6. Organic anions d fro
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(probenecid, benzbromarone, indomethacin), known to interfere h
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with glutathione conjugate transport of human ABCC1 and w
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ABCC2, inhibited the ABCC6-mediated NEM-GS transport in a c
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specific manner, indicating that ABCC6 has a unique substrate- b/
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specificity. We have also expressed three missense mutant forms st o
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of ABCC6, which have recently been shown to cause PXE. MgATP- o
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binding was normal in these proteins; ATP-dependent NEM-GS or e
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LTC transport, however, was abolished. Our data indicate that , 2
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human ABCC6 is a primary active transporter for organic anions.
In the three ABCC6 mutant forms examined, the loss of transport
activity suggests that these mutations result in a PXE phenotype
through a direct influence on the transport activity of this ABC
transporter.
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Loss-of-function mutants of ABCC6/MRP6 in PXE
INTRODUCTION
Pseudoxanthoma elasticum (PXE) is a heritable disorder,
affecting several different elastic tissues, including the skin, the
elastic Bruch’s membrane of the eye and the arterial system.
Calcification of elastic fibers within the extracellular matrix in
these tissues contributes towards an inelastic and lax dermal
phenotype, angioid streaks in the retina and subsequent vision
loss and a variety of vascular abnormalities, including
gastrointestinal bleeding and premature atherosclerosis. It has
been demonstrated recently and very unexpectedly that
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mutations in a gene coding for an ABC transporter protein, o
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ABCC6 (MRP6) are responsible for the development of PXE, that is a
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traditionally thought of as a connective tissue disease [1 - 4]. fro
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ABC proteins have been found in each genome studied, and the ttp
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most recent annotation of the human genome sequence revealed w
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48 ABC-protein genes, which were grouped into seven sub-classes .org
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[see: http://nutrigene.4t.com/humanabc.htm]. The human ABCC g
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subfamily consists of twelve members, and most of these are t o
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identified as active membrane transporters of various organic ve
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anions (for recent reviews see: Refs. 5-8). Although the majority r 1
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of the proteins in the ABCC subfamily are active pumps, there are 1
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several exceptions. The cystic fibrosis transmembrane
conductance regulator, ABCC7 (CFTR) is a chloride channel, that
may also regulate other channel proteins [9], and the
sulfonylurea receptors, ABCC8 (SUR1) and ABCC9 (SUR2),
regulate the permeability of specific K+ channels [10]. Mutations
in the CFTR gene cause cystic fibrosis [11, 12], while mutations in
the SUR1 gene are causative in a rare genetic disorder, familial
persistent hyperinsulinemic hypoglycemia of infancy [10].
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Loss-of-function mutants of ABCC6/MRP6 in PXE
Inherited diseases have already been also shown to be caused by
the loss of function of active ABC transporters – e.g. missense
mutations in the ABCC2 gene cause conjugated
hyperbilirubinemia (Dubin-Johnson syndrome) [13, 14]. While a
spectrum of mutations within the ABCC6 gene is clearly
responsible for PXE, the functional relationship between altered
ABCC6 gene expression and the PXE phenotype is still unknown.
Recent studies have revealed that human and rat ABCC6 are
abundantly expressed in both liver and kidney [15 - 17], two
organs not thought to be involved in the development of PXE. It is
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not known therefore, whether the pathomechanism for PXE o
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involves aberrant ABCC6 activity directly within the affected a
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elastic tissues, as would be expected for a heritable connective fro
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tissue disorder, or whether PXE is more likely to be a metabolic ttp
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disease [18], involving abnormal hepatic and/or renal function of w
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ABCC6. .org
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If the major function of ABCC6 is primary active transport, a g
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critical step to understanding the pathomechanism of PXE would t o
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be the identification of a physiological metabolite(s) transported ve
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by ABCC6. To date, however, there have been no data reported in r 1
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this regard. One study on the rat ortholog of ABCC6 reported that 1
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an anionic cyclic pentapeptide was a relatively poor transported
substrate for the rat Mrp6/Abcc6 [17].
In the present paper, we describe the first studies of the
transport activity of human ABCC6. We have established that this
protein actively transports at least two anionic glutathione
conjugates, and this transport is abolished by three missense
mutations in ABCC6, that are known to cause PXE. These new
findings establish that aberrant transport is a primary
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Loss-of-function mutants of ABCC6/MRP6 in PXE
determinant in the PXE phenotype and should contribute
significantly to a detailed elucidation of how such functionally
null alleles of ABCC6 contribute toward abnormal elastic fiber
assembly in patients with PXE.
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Loss-of-function mutants of ABCC6/MRP6 in PXE
EXPERIMENTAL PROCEDURES
[3H]BQ-123 (36.0 Ci/mmol) was purchased from Amersham
Pharmacia Biotech (Buckinghamshire, UK). [3H]NEM-GS was
prepared from [3H]NEM (51.7 Ci/mmol; DuPont-New England
Nuclear, Boston, MA, USA) by mixing the radioactive reagent in
10 mM Tris-HCl (pH 7.0) with freshly dissolved reduced
glutathione in a 1:1.1 molar ratio. [α32P]8-azidoATP (20 Ci/mmol)
was obtained from ICN Pharmaceuticals, Inc. (Costa Mesa, CA,
USA). [3H]LTC (130 Ci/mmol) was purchased from DuPont-New
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England Nuclear (Boston, MA, USA).
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Generation of MRP6/ABCC6 cDNAs, expression in Sf9 insect cells o
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A human kidney Marathon-Ready cDNA library, which was a
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prepared from pooled RNA from 8 donors (7405-1, Clontech, Palo fro
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Alto, CA, USA) was used to amplify full-length MRP6/ABCC6 ttp
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cDNAs. As the initial amplification reaction generated multiple w
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products, the expected 4.5 kb DNA fragment was gel-isolated and .org
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reamplified. The amplified cDNA was cloned into a pBluescript SK g
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vector (Stratagene) and the DNA sequence of 6 clones was t on
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determined by sequencing both strands using gene specific e
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primers. r 1
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The sequences of all 6 clones unequivocally predict an Arg in 1
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position 64 of the amino acid sequence, while in one of published
sequences a Trp was found in this position [15]. Sequencing of
exon 2 in 50 normal individuals also detected only the 64R allele
[19]. Our sequence analysis predicts a Leu at position 377 in
ABCC6; the same amino acid is published in [15], while a Pro was
found in the other published sequence [16].
In order to study the effect of PXE mutations on MRP6/ABCC6
function, we have inserted three naturally occurring PXE
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Loss-of-function mutants of ABCC6/MRP6 in PXE
mutations, V1298F, G1302R and G1321S [20] into our cDNA by
using inverse PCR mutagenesis [21].
One of the missense mutations (G1302R) we studied was found in
a homozygous state [20] in one patient with sporadic PXE and in
heterozygote state in two unrelated patients with sporadic PXE.
The V1298F allele was present in a heterozygote state in two
patients with sporadic PXE, while the G1321S allele was present in
a single patient with a sporadic form of PXE [20]. None of these
variants were found in a panel of 200 alleles of unaffected
control individuals [20].
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Full length cDNA was inserted into a baculovirus transfer vector o
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(pAcUW21, Invitrogen, San Diego, CA, USA), and the sequence of a
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segment generated by PCR was checked by DNA sequencing. fro
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Sf9 cells were cultured and infected with the recombinant ttp
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baculoviruses as described [22]. w
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Membrane preparation and Western blotting .org
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Virus-infected Sf9 cells were harvested, cell membranes were g
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isolated and membrane protein concentrations were determined t o
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as described previously [23]. The Sf9 total membrane ve
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preparations contained both plasma membrane and intracellular r 1
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membrane material. 1
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A polyclonal antibody, named HB6, was raised in rabbits against
a synthetic peptide corresponding to aa 233-257 of the human
ABCC6 sequence (Sigma, Genosys, The Woodlands, TX, USA). The
location of this peptide segment within the human ABCC6 protein
is shown on Figure 1/a. Gel-electrophoresis and immunoblotting
was performed as described previously [23]. Protein-antibody
interaction was detected by using the enhanced
chemiluminenscence technique (ECL, Amersham). The
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Loss-of-function mutants of ABCC6/MRP6 in PXE
quantitative measure of expression level was determined by
densitometry of the immunoblots.
ATP-binding, nucleotide trapping and ATPase activity
measurements
Isolated membranes were incubated with 5 µM [α32P]-8-azido-
ATP, under non-hydrolytic conditions (at 0oC) for binding or at
37oC for nucleotide trapping. In the binding studies the
membranes were irradiated with UV light in the presence of the
labeled nucleotide, while in the case of nucleotide
occlusion/trapping measurements, the membranes were first
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washed in the presence of 10 mM ATP, and then UV-irradiated. o
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Thereafter the membrane proteins were separated by gel- a
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electrophoresis and electroblotted to PVDF membranes. 32P fro
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labeling was quantitated using a phospho-imager (Bio-Rad). The ttp
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identity of the 32P-azido-nucleotide labeled bands was confirmed w
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by immunostaining of the same blot, using the anti-ABCC6 .org
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polyclonal antibody (see above). ATPase activity was measured g
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by colorimetric detection of inorganic phosphate liberation, as t on
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previously described [23, 28]. e
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Vesicular transport measurements were performed using a r 1
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rapid filtration method as we have previously described [28, 29]. 1
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ATP-dependent tracer uptake was calculated by subtracting the
radioactivity on the filter obtained in the absence of MgATP (or
the presence of 4 mM MgAMP) from those obtained in the
presence of 4 mM MgATP. The figures represent the mean values
of at least three independent experiments. Transport values were
corrected for non-specific transport observed in the control, β-
galactosidase expressing membranes. The relative amount of
uptake-competent inside-out vesicles in different membrane
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Loss-of-function mutants of ABCC6/MRP6 in PXE
preparations was determined from the rate of endogenous ATP-
dependent 45Ca2+ uptake (see [28, 29]). The Michaelis-Menten
kinetic parameters of transport have been calculated by linear
regression using a software (Origin 5.0; Microcal Software Inc.,
Northhampton, MA).
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Loss-of-function mutants of ABCC6/MRP6 in PXE
RESULTS
Expression of wild-type and mutant ABCC6 and measurement of
specific ATP binding
In order to establish an experimental system to study the
functional properties of both normal and mutant forms of the
human ABCC6 transporter, we have amplified a normal variant of
the human ABCC6 cDNA and expressed the protein in Sf9 insect
cells. This approach was chosen, as several mammalian ABC
transporters have already been successfully expressed in insect
cells. In general, these expression systems have yielded high
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protein levels in a functionally active state [23, 24, 30, 31]. o
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Recently the expression of the functional form of the rat a
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Mrp6/Abcc6 in Sf9 cells has also been demonstrated [17]. fro
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Transcription of the ABCC6 gene occurs predominantly in the ttp
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liver and the kidney [15 - 17], therefore we used an amplification w
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strategy to obtain the full-length cDNA from a human kidney .org
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cDNA library. Using this cDNA, with a verified sequence, we have g
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generated recombinant baculoviruses containing the wild type t o
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human ABCC6 cDNA, as well as that of three ABCC6 mutant ve
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variants (V1298F, G1302R and G1321S). These amino acid r 1
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replacements are targeted to the C-proximal ABC-domain (see 1
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Figure 1/a), and have recently been identified as ABCC6 mutants
that will result in PXE [20].
Sf9 cells were infected with the recombinant baculovirus
supernatants, and virus clones producing the highest level of
ABCC6 protein expression were selected. By using these viruses, a
new protein band could be detected on the Coomassie-stained
PAGE of membrane preparations from recombinant virus-infected
Sf9 cells. This protein band corresponded to the expected
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