Expression and in vivo rescue of human ABCC6 disease-causing mutants in mouse liver.
ABSTRACT Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6 protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. We have established a complex experimental strategy to determine the structural and functional consequences of disease-causing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the biology of the fully differentiated human liver and could have future human therapeutic application.
- SourceAvailable from: Michael Freissmuth[Show abstract] [Hide abstract]
ABSTRACT: SLC6 family members and ABC transporters represent two extremes: SLC6 transporters are confined to the membrane proper and only expose small segments to the hydrophilic milieu. In ABC transporters the hydrophobic core is connected to a large intracellular (eponymous) ATP binding domain that is comprised of two discontiguous repeats. Accordingly, their folding problem is fundamentally different. This can be gauged from mutations that impair the folding of the encoded protein and give rise to clinically relevant disease phenotypes: in SLC6 transporters, these cluster at the protein-lipid interface on the membrane exposed surface. Mutations in ABC-transporters map to the interface between nucleotide binding domains and the coupling helices, which provide the connection to the hydrophobic core. Folding of these mutated ABC-transporters can be corrected with ligands/substrates that bind to the hydrophobic core. This highlights a pivotal role of the coupling helices in the folding trajectory. In contrast, insights into pharmacochaperoning of SLC6 transporters are limited to monoamine transporters - in particular the serotonin transporter (SERT) - because of their rich pharmacology. Only ligands that stabilize the inward facing conformation act as effective pharmacochaperones. This indicates that the folding trajectory of SERT proceeds via the inward facing conformation. Mutations that impair folding of SLC6 family members can be transmitted as dominant or recessive alleles. The dominant phenotype of the mutation can be rationalized, because SLC6 transporters are exported in oligomeric form from the endoplasmic reticulum (ER). Recessive transmission requires shielding of the unaffected gene product from the mutated transporter in the ER. This can be accounted for by a chaperone-COPII (coatomer protein II) exchange model, where proteinaceous ER-resident chaperones engage various intermediates prior to formation of the oligomeric state and subsequent export from the ER. It is likely that the action of pharmacochaperones is contingent on and modulated by these chaperones.Pharmacological Research 12/2013; · 4.35 Impact Factor
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ABSTRACT: Because vascular or cardiac mineralization is inversely correlated with morbidity and long-term survival, we investigated the role of ABCC6 in the calcification response to cardiac injury in mice. By using two models of infarction, nonischemic cryoinjury and the pathologically relevant coronary artery ligation, we confirmed a large propensity to acute cardiac mineralization in Abcc6−/− mice. Furthermore, when the expression of ABCC6 was reduced to approximately 38% of wild-type levels in Abcc6+/− mice, no calcium deposits in injured cardiac tissue were observed. In addition, we used a gene therapy approach to deliver a functional human ABCC6 via hydrodynamic tail vein injection to approximately 13% of mouse hepatocytes, significantly reducing the calcification response to cardiac cryoinjury. We observed that the level and distribution of known regulators of mineralization, such as osteopontin and matrix Gla protein, but not osteocalcin, were concomitant to the level of hepatic expression of human and mouse ABCC6. We notably found that undercarboxylated matrix Gla protein precisely colocalized within areas of mineralization, whereas osteopontin was more diffusely distributed in the area of injury, suggesting a prominent association for matrix Gla protein and osteopontin in ABCC6-related dystrophic cardiac calcification. This study showed that the expression of ABCC6 in liver is an important determinant of calcification in cardiac tissues in response to injuries and is associated with changes in the expression patterns of regulators of mineralization.American Journal Of Pathology 01/2014; 184(1):159-70. · 4.60 Impact Factor
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ABSTRACT: Mutations in the ABCC6 ABC-transporter are causative of pseudoxanthoma elasticum (PXE). The loss of functional ABCC6 protein in the basolateral membrane of the kidney and liver is putatively associated with altered secretion of a circulatory factor. As a result, systemic changes in elastic tissues are caused by progressive mineralization and degradation of elastic fibers. Premature arteriosclerosis, loss of skin and vascular tone, and a progressive loss of vision result from this ectopic mineralization. However, the identity of the circulatory factor and the specific role of ABCC6 in disease pathophysiology are not known. Though recessive loss-of-function alleles are associated with alterations in ABCC6 expression and function, the molecular pathologies associated with the majority of PXE-causing mutations are also not known. Sequence analysis of orthologous ABCC6 proteins indicates the C-terminal sequences are highly conserved and share high similarity to the PDZ sequences found in other ABCC subfamily members. Genetic testing of PXE patients suggests that at least one disease-causing mutation is located in a PDZ-like sequence at the extreme C-terminus of the ABCC6 protein. To evaluate the role of this C-terminal sequence in the biosynthesis and trafficking of ABCC6, a series of mutations were utilized to probe changes in ABCC6 biosynthesis, membrane stability and turnover. Removal of this PDZ-like sequence resulted in decreased steady-state ABCC6 levels, decreased cell surface expression and stability, and mislocalization of the ABCC6 protein in polarized cells. These data suggest that the conserved, PDZ-like sequence promotes the proper biosynthesis and trafficking of the ABCC6 protein.PLoS ONE 01/2014; 9(5):e97360. · 3.53 Impact Factor
Expression and In Vivo Rescue of Human ABCC6
Disease-Causing Mutants in Mouse Liver
Olivier Le Saux1, Krisztina Fu ¨lo ¨p2, Yukiko Yamaguchi1, Attila Ilia ´s2, Zala ´n Szabo ´2, Christopher N.
Brampton1, Viola Pomozi2, Krisztina Husza ´r2, Tama ´s Ara ´nyi2, Andra ´s Va ´radi2*
1Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii, United States of America, 2Institute of Enzymology,
Hungarian Academy of Sciences, Budapest, Hungary
Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease
models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6
protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes.
We have established a complex experimental strategy to determine the structural and functional consequences of disease-
causing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport
activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S
and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport
activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive
V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed
mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic
reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their
intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was
significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the
feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the
biology of the fully differentiated human liver and could have future human therapeutic application.
Citation: Le Saux O, Fu ¨lo ¨p K, Yamaguchi Y, Ilia ´s A, Szabo ´ Z, et al. (2011) Expression and In Vivo Rescue of Human ABCC6 Disease-Causing Mutants in Mouse
Liver. PLoS ONE 6(9): e24738. doi:10.1371/journal.pone.0024738
Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America
Received June 22, 2011; Accepted August 16, 2011; Published September 14, 2011
Copyright: ? 2011 Le Saux 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 the Hungarian research grants (by the National Research Foundation, OTKA, www.otka.hu) OTKA CK 80135, OTKA NK
81204, OTKA PD 79183 and by National Institute of Health (www.nih.gov) NIH R01AR055225 (subaward) to A.V.; NIH HL087289, Hawaii Community Foundation
(HCF, www.hawaiicommunityfoundation.org) 20080443 and American Heart Association (AHA, www.heart.org) 11GRNT5840005 grants to O.L.S. and by PXE
International Inc. (www.pxe.org). T.A. is a recipient of Bolyai Fellowship of the Hungarian Academy of Sciences (www.mta.hu). The NIH grants RR003061 and
RR016453 supported the Histology and Imaging Core Facility of the John A. Burns School of Medicine. 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: firstname.lastname@example.org
Commonly found in aging tissue, dystrophic calcification is
defined as the abnormal deposition of calcium salts in altered or
diseased tissues. It also occurs in pathologies such as diabetes,
hypercholesterolemia, chronic renal failure and certain genetic
conditions. Under pathological conditions, this abnormal miner-
alization can occur in response to metabolic, mechanical, infec-
tious, or inflammatory injuries and its etiology is heterogeneous
with overlapping yet distinct molecular mechanisms of initiation
and progression. Pseudoxanthoma elasticum (PXE, OMIM
26480) in human and dystrophic cardiac calcification (DCC) in
mice are similar pathologies both defined by dystrophic mineral-
ization of cardiovascular, ocular and dermal tissues. Both
conditions derive from loss-of-function mutations in the human
ABCC6 and mouse Abcc6 genes [1,2,3,4,5]. PXE is characterized
by dystrophic calcification primarily affecting elastic fibers in skin,
arteries and the Bruch’s membrane of the eye [6,7,8]. The
causality of mutations of the ABCC6 gene in PXE was demon-
strated in 2000 [2,3,5] and since then the clinico-genetic
characteristics of the disease have been established [7,8]. Interest-
ingly, heterozygous carriers of ABCC6 mutant alleles present an
increased susceptibility to cardiovascular diseases [9,10,11]. The
transcriptional regulation of the gene [6,12,13] and the biochem-
ical characteristics of the ABCC6 protein have been well defined
. ABCC6 functions as an organic anion efflux pump [14,15]
transporting an as yet unidentified substrate(s) from the liver to the
circulation. Because ABCC6 is predominantly found in liver and
kidney and with little or no expression in tissues affected by PXE
[8,16,17] this pathology appears to be systemic in nature. This
implies the presence of an abnormal circulating molecule(s) that
results from the failure of ABCC6 to export its substrate(s),
ultimately promoting calcification in peripheral tissues. We’ve
detected the presence of such circulating molecules in the serum of
adult PXE patients through their effects on elastic fibers deposited
in cultures  and others have reached similar conclusions using
a mouse model [19,20].
Since the first PXE-causing mutations were characterized
[2,3,5,21], the number of identified disease-causing variants has
exceeded 300 . Despite the large number of PXE-specific
PLoS ONE | www.plosone.org1 September 2011 | Volume 6 | Issue 9 | e24738
mutations that have been identified in ABCC6, no clear genotype-
phenotype correlation has emerged [7,21,22]. However, a signi-
ficant clustering of missense mutations was identified in domain-
domain interfaces predicted from a homology model of the human
ABCC6 protein . Amino acid substitutions in large plasma
membrane proteins such as ABCC6 generally result in decreased
activity, major conformation changes, low level of plasma
membrane targeting or a combination thereof. Therefore,
studying the consequences of naturally occurring disease-causing
missense mutations can provide important insights into the
relationship between protein structure and function, which may
later assist in the development of therapeutic applications. In
recent years, several studies have shown that sodium 4-
phenylbutyrate (4-PBA) can partially restore the cellular trafficking
of a mutated ABCC7/CFTR (DF508) and parts of its cellular
function were restored in both cultured cells and human cystic
fibrosis patients [24,25,26], though how much phenotype
improvement was achieved is less clear. Other proteins with
disease-causing mutations were also subjected to 4-PBA treatment
to improve their folding/trafficking like ABCA3 , LDL-
receptor , the bile salt export pump/ABCB11 [29,30,31],
ATP7B  and ATP 8B1  and ATP-Sensitive Potassium
Channel/ABCC8 . 4-PBA is a butyrate analogue approved for
clinical use in human with urea cycle disorders and thalassemia
[35,36,37]. It is thought to interfere with the Hsc70 protein in
endoplasmic reticulum (ER), allowing a proportion of misfolded
proteins to escape association with the chaperon thus improving
cellular trafficking [26,38]. To capitalize on these precedent
studies, we characterized the structural and functional conse-
quences of selected PXE-causing missense mutations on ABCC6
transport activity, protein stability and conformation using in vitro
assays. We also investigated for the first time, the in vivo stability
and cellular location of WT and mutated ABCC6 in fully
differentiated hepatocytes by transiently expressing the human
mutant proteins in the liver of C57BL/6J mice. We focused on
identifying those mutants with preserved transport activity and
intracellular mistargeting. Such mutants are candidates for
pharmacological rescue of their intracellular maturation. Indeed,
we determined the potential for recovery of plasma membrane
targeting of a transport-competent ABCC6-mutant in liver of
C57BL/6J mouse after 4-PBA treatments.
Materials and Methods
The ABCC6 model, previously generated by Fu ¨lo ¨p et al  was
analysed with the PyMOL Molecular Graphics System, Version
1.3, Schro ¨dinger, LLC.
Expression of ABCC6 variants in Sf9 insect cells, ATP binding, nucleotide
trapping and vesicular transport were performed as described in our
previous papers [14,39,40,41].
Expression of ABCC6 variants in MDCKII cells were achived by
retroviral gene delivery as described .
Liver-specific expression of ABCC6 variants in mice
The WT and mutant ABCC6 cDNA constructs were sub-cloned
into the pLIVE vector (Mirus Bio, Madison, WI) and expressed
under the control of a liver-specific promoter. Plasmid DNA
constructs were delivered by hydrodynamic tail vein injections
[43,44]. We used 3-month-old C57BL/6J wild type mice. The tail
vein injections were performed with a 27-gauge needle with a
volume of 1.5 to 2 ml of DNA in a solution of TransIT EEH
following the manufacturers instructions (Mirus Bio Madison, WI).
Mice were injected with 60 mg of a plasmid. At least 3 mice per
mutant were injected.
All mice were kept under routine laboratory conditions with
12 hours light-dark cycle with access ad libitum to water and
standard chow. Mice were euthanized by standard CO2
procedures 24 hrs after tail vein injections. This study was
approved by the Institutional Animal Care and Use Committee
of the University of Hawaii.
Immunoblotting and immunohistochemical staining of
mouse liver samples
Multiple liver lobes were quickly harvested, placed in Optimum
Cutting Temperature (OCT) compound and stored at 280uC.
Immunofluorescent staining of liver samples was performed using
5 mm-thick frozen sections. The rat monoclonal anti-ABCC6
M6II-31 antibody (sc-59618) was used to specifically detect the
human ABCC6. The rabbit polyclonal anti-Abcc6 antibody (S-20)
was used to identify the mouse Abcc6. These primary antibodies
were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz,
CA). The secondary antibodies were labeled with Alexafluor 488
and 568. We also used the rabbit polyclonal K-14 antibody,
previously described . A rabbit polyclonal antibody (Ab10286)
from Abcam (Cambridge, MA) was used to detect the mouse
Calnexin. The subcellular localization of the mouse Abcc6 and the
human ABCC6 proteins was determined by imaging using an
Axioscope 2 fluorescent microscope (Zeiss, Thornwood, NY). Fifty
to one hundred individual images of cells for each mutant from at
least 3 mice were collected and processed with Photoshop CS3
(Adobe, San Jose, CA) and then evaluated. Images were also
analyzed using the ImageJ64 software (NIH) using the Graphic
Dynamic Profiler tool.
4-PBA treatment of MDCKII cells and mouse
MDCKII cells were cultured as previously described  and in
the presence of 1 mM 4-PBA (Tocris Biosciences, Ellisville, MO).
Mice received 3 intraperitoneal injections of 4-PBA (100 mg/kg/
day) prior to performing hydrodynamic tail vein injections.
The aim of our study was to characterize the structural and
functional consequences of PXE-causing mutations in ABCC6
with a focus on the in vivo intracellular targeting of the human
mutant proteins in mouse liver. Our main motivation was to
identify disease-causing missense mutants with normal transport
activity but with aberrant intracellular targeting. Such mutants
were candidates for rescue of their intracellular maturation using
chemical chaperons. We previously found an unequal distribution
of ABCC6 missense mutations with high frequency on the ABC-
ABC contact or in the transmission interface . Therefore, we
chose five missense mutations in these regions, V1298F and
G1321S in the C-proxymal ABC domain and R1138Q, R1314W
and R1339C in the transmission interface. The location of the
mutated residues is illustrated on Figure 1, which depicts a model
of the membrane topology of ABCC6 as well as a homology model
of the protein. A N-terminal truncated mutant, del1–277ABCC6
(DABCC6) missing the TMD0and L0domains has also been
constructed as a control (Figure 1).
In vitro expression and functional characterization
The wild type (WT) protein, DABCC6 and the 5 PXE-
associated mutant ABCC6 proteins were expressed in Sf9 insect
cells to first establish whether the mutants could be overexpressed.
This was an important step to assess the overall stability of the
ABCC6 mutants. We used western blotting with the M6II-7
Rescue of Human ABCC6 Mutants in Mouse Liver
PLoS ONE | www.plosone.org2September 2011 | Volume 6 | Issue 9 | e24738
monoclonal antibody and found that V1298F, G1321S, R1138Q,
R1314W showed high levels of expression comparable to the WT
ABCC6 at the expected molecular mass of ,160 kDa (Figure 2A).
The DABCC6 mutant was also stably overexpressed and displayed
a molecular mass of ,130 kDa, as expected. Surprisingly, the
R1339C mutant could not be detected with the M6II-7 antibody.
Using the polyclonal HB6 antibody, which recognizes a different
region of ABCC6 (L0), we found that the R1339C variant was in
fact degraded (Figure 2A). As both fragments (75 kDa and 85 kDa)
reacted with the HB6 antibody recognizing an epitop in the N-
proxymal part of the protein (for the location of the epitop, see
Figure 1A), these were overlapping fragments indicating multiple
degradation sites in the protein. As we were able to produce
sufficient amounts of mutant ABCC6 proteins (except for
R1339C), we analyzed the transporter characteristics of the
mutants ABCC6. V1298F, G1321S, R1138Q, R1314W were
capable of binding labeled MgATP (Figure 2A), indicating that the
mutations did not alter significantly the conformation of the ATP-
binding site(s). To determine if the PXE-mutants could form
catalytic intermediate in which the gamma phosphate of ATP is
cleaved, we studied the occluded nucleotide intermediate state
(‘‘vanadate trapping’’) of the variants under catalytic conditions
(37uC). Mutants V1298F and G1321S presented impaired activity
while both R1138Q and R1314W mutants were capable of
forming occluded nucleotide transitory complex as efficiently as
the WT ABCC6 (Figure 2B). As we have previously demonstrated
that glutathione-conjugated N-ethylmaleimide (NEM-GS) and
Leukotriene C4 (LTC4) were both efficiently transported by
ABCC6 in vitro (13), we continued the characterization of the
mutants by measuring their transport activity with these two
substrates. As shown on Figure 2C, mutants R1138Q and
R1314W were able to actively transport both substrates at levels
comparable to that of the WT ABCC6. In contrast, V1298F and
G1321S exhibited little transport capacity as expected . To
complement these data, we also determined that the concentra-
tion-dependent LTC4 transport kinetics of R1138Q and R1314W
was similar to the WT ABCC6 (not shown).
Intracellular targeting of PXE-causing human ABCC6 in
the liver of living mice
We studied the consequences of disease-causing mutations of
ABCC6 in the liver of living mice, the organ where most of its
physiological function is performed. We first sub-cloned the
cDNAs of the 6 mutants and the WT protein into pLIVE vectors
under the control of a liver-specific promoter consisting of the
mouse albumin promoter and alpha fetoprotein enhancers. We
then transiently expressed these cDNAs into the liver of adult
normal mice using hydrodynamic tail vein injections (HTVI) .
Individual hepatocytes expressing the ABCC6 mutants were
detected on frozen sections by immunofluorescence with the
Figure 1. Membrane topology and homology model of the human ABCC6. A: The membrane topology was based on prediction previously
described . The locations of the various domains are indicated as horizontal arrows above the topology model. Various lines indicate the locations
of the epitopes for the polyclonal antibody HB6 and K-14 and monoclonal antibodies M6II-7, M6II-31 utilized in the present study and arrows point to
the position of the PXE missense mutations. B: The location of mutants in the three-dimensional homology model of the human ABCC6 protein 
are shown. The entire model is shown with the two ABC halves of the molecule distinguished by grey and pink colors. The left panel is a schematic
illustrating the domain swap characteristic of the ABC-protein superfamily. Protein segments of the ABC-domains forming the transmission interface
are space-filled and colored in blue. Protein segments of the intracellular loops forming the transmission interface are colored in red.
Rescue of Human ABCC6 Mutants in Mouse Liver
PLoS ONE | www.plosone.org3 September 2011 | Volume 6 | Issue 9 | e24738
monoclonal antibody M6II-31 specific to the human ABCC6
along with a polyclonal antibody specific to the mouse Abcc6.
Based on these immunostainings, we estimated that 5 to 10
percent of the mouse liver hepatocytes expressed detectable levels
of the human ABCC6 (Figure 3A). The WT ABCC6 was fully
integrated into the basolateral membrane of hepatocytes and co-
localized with the mouse Abcc6 protein (Figure 3B). The ABCC6
mutants were also successfully expressed in mouse liver including
R1339C despite the previously noted instability of this mutant in
Sf9 cells. The cellular localization of all 6 mutants was determined
by co-immunofluorescence staining (Figure 3C). The transport-
incompetent mutant V1298F was the only mutant that showed
a cellular localization identical to the human WT protein. In
contrast, mutants R1314W, G1321S, R1314W, R1339C and
DABCC6 were primarily located in the intracellular compartment
with little or no plasma membrane localization (Figure 3C). The
cellular targeting of R1138Q, however, showed an intermediary
distribution both in the plasma membrane and in the intracellular
space. To determine whether the immunofluorescent staining we
obtained reflected the cellular localization of the integral protein
for each ABCC6 variant, we performed immunostainings of the
same liver samples with the polyclonal antibody K-14 that
recognizes both the human ABCC6 and mouse Abcc6 proteins.
This antibody was raised against the C-terminal end of the rat
Abcc6  We found identical intracellular staining patterns for
each mutant (Figure 4), suggesting that the ABCC6 mutants were
expressed as full size proteins in mouse liver. Next, we determined
if mutants with residual transport activity and intracellular
accumulation (R1138Q and R1314W) were retained in the
endoplasmic reticulum. Mutant R1339C was also included into
this experiment (our Sf9 exprression and transport assay was not
suitable to determine its potential transport activity, see Figure 2A).
We have addressed this question because it is established that the
pharmacological rescue with 4-PBA can be successful in the case of
ER-retained protein species [26,38]. Figure 5 shows the results
obtained of co-localization between Calnexin, a marker for the ER
Figure 2. Biochemical characterization of the human WT and mutant ABCC6. A: Expression of ABCC6 variants in Sf9 insect cells as detected
by western blotting. The WT ABCC6 and R1339C mutant were detected with the HB6 antibody, whereas the expression of DABCC6, V1298F, G1321S,
R1138Q and R1314W was revealed with the M6II-7 antibody. B: Upper panel: specific MgATP-binding as revealed by photo-crosslinking of Sf9
membrane preparations with 8-N3-[a-32P] ATP under non-catalytic conditions (at 0uC).. b-gal: control membrane isolated from cells overexpressing b-
galactoside. Lower panel: detection of the trapped ADP-vanadate complex (occluded nucleotide intermediate) as revealed by photo-crosslinking of
the membrane preparation derived from Sf9 cells with 8-N3-[a-32P] ATP under catalytic conditions (at 37uC). b-gal: control membrane isolated from
cells overexpressing b-galactoside. The arrow indicates the position of the labelled proteins C: Relative transport activities of ABCC6 and PXE-causing
missense variants. Dark grey columns represent LTC4-transport activities whereas light grey columns indicate GS-NEM transport activities.
Rescue of Human ABCC6 Mutants in Mouse Liver
PLoS ONE | www.plosone.org4 September 2011 | Volume 6 | Issue 9 | e24738
and variants R1138Q, R1314W and R1339C. The immunoflu-
orescence images showed little of the WT protein resided in the
ER. Interestingly, mutants R1318Q and R1339C also showed
some co-localization with Calnexin while mutant R1314W was
found to be predominantly associated with the ER.
Table 1 summarizes the structural and functional consequences
of PXE-causing missense mutations in ABCC6 identified in this
Pharmacological rescue of ABCC6 mutants
As it was shown that 4-PBA can partially restore cellular
trafficking (thus the function) of the ABCC7 cystic fibrosis mutant
protein (DF508), we studied whether pre-treating mice with 4-PBA
before HTVI could restore at least partial cellular trafficking of
ABCC6 mutants that retained substantial transport activity. In the
group of mutants we studied, R1138Q and R1314W showed near
normal transport capacity despite incorrect cellular localization in
the mouse liver. We also used the WT ABCC6 as a positive
control as well as R1339C (though no information on its transport
activity was obtained). Mice received 3 daily injections of 4-PBA
before HTVI. We observed that the treatments did not alter the
expression or plasma membrane localization of the WT ABCC6
and had no effect on the intracellular localization of R1138Q
and R1339C. However, 4-PBA treated mice expressing R1314W
showed a clearly improved membrane localization of this mutant
as compared to untreated mice (Figure 6A and Supplementary
Figure 3. Intracellular localization of human ABCC6 variants expressed in vivo in mouse liver. The human and mouse ABCC6/Abcc6 were
detected on frozen sections by immunofluorescence using the M6II-31 monoclonal antibody (green) and the S-20 polyclonal antibody (red),
respectively, Panel A: A low magnification image shows the overall distribution of the human WT ABCC6 in a cross-section of single liver lobe. B: The
basolateral plasma membrane localization of the WT ABCC6 expressed in mouse liver hepatocytes was confirmed by immunofluorescent imaging.
The Z-stack cross-section images are also shown and arrows point to the basolateral membrane. C: Localization of the human WT and mutant ABCC6.
Individual channels and the merged images of the endogenous Abcc6 (red) and the human ABCC6 variants (green) are shown. The scale bar
represents 50 mm.
Rescue of Human ABCC6 Mutants in Mouse Liver
PLoS ONE | www.plosone.org5 September 2011 | Volume 6 | Issue 9 | e24738
Figure S1). This was confirmed by the lack of co-localization of
R1314W with Calnexin (Figure 6B). For confirmation, we
generated a MDCKII cell line overexpressing this mutant and
found that R1314W was located in the intracellular space as was
observed in the mouse liver. Treating these cells with 1 mM 4-
PBA corrected the targeting of the mutant to the plasma
membrane (Figure 6C).
These results not only showed that the structural consequence of
the R1314W mutation could at least in part, be corrected by 4-
PBA but also suggested that the incorrect intracellular trafficking
of this mutant was likely due to ER retention and suggested
We and others have accumulated a body of data that implicated
ABCC6 as a new modulator of ectopic calcification in both human
and mice [1,2,3,4,5]. This transmembrane protein is primarily
expressed in liver, kidneys and intestine and transports unknown
metabolite(s), which directly or indirectly control mineralization of
dermal, ocular and cardiovascular tissues . Mutations in the
human ABCC6 gene cause pseudoxanthoma elasticum, a recessive
disorder [2,3,5,22,47]. The vast majority of ABCC6 mutations are
missense [7,21,22,23] and several categories of missense substitu-
tions can be distinguished based on the functional consequence of
the amino acid replacement. Based on published data [26,38],
we have also anticipated that mutants with certain transport-
and intracellular characteristics can be subjected to rescue by
treatment with pharmacological compounds (called ‘‘chemical
chaperons’’). Therefore, the main objective of this study was to
characterize the structural and functional consequences of ABCC6
missense mutations in vitro as well as in an accurate in vivo model
and to identify mutants suitable for rescue. ABC transporters are
traditionally studied in cultures of MDCKII cells, but these are not
ideal for studying hepatic proteins. Indeed, MDCKII cells are
transformed cells derived from canine kidney tissues and do not
reproduce the biology of the liver where ABCC6 is primarily
expressed. A transgenic animal approach would be more accurate,
yet it is time-consuming and poorly suited to the analysis of
multiple protein variants. We have overcome these obstacles by
demonstrating the feasibility of using HTVI to study normal and
mutant forms of the human ABCC6 protein in the fully
differentiated liver of a living mouse. This method delivers DNA
to the liver very effectively [43,44,48] and ensures the selective
hepatic expression of the WT human protein in mouse liver with
an adequate basolateral targeting. We also analyzed an artificially
truncated mutant, DABCC6 as similar truncated ABCC1 and
ABCC2 proteins were found to be inactive and not integrated into
the plasma membrane [41,49]. The WT human protein co-
localized with the mouse Abcc6 (Figure 3A, B) and thus validated
our in vivo experimental model. Conversely, DABCC6 was
completely absent from the plasma membrane arguing for the
similar role of the TMD0and L0domains of ABCC6 with those of
ABCC1 and ABCC2 in intracellular trafficking. Interestingly, two
of the PXE-causing mutants we characterized, R1138Q and
R1314W showed a near normal transport activity in in vitro assays,
Figure 4. Intracellular localization of human ABCC6 variants expressed in vivo in mouse liver. The human ABCC6 variants were detected
by immunofluorescence on liver frozen sections using the M6II-31 monoclonal antibody (upper panels) and the K-14 polyclonal antibody (lower
panels). Subsequent frozen sections from the same blocks were used for the two different stainings. Note that K-14 polyclonal antibody recognizes
both the human and mouse ABCC6/Abcc6. Immunostaining with K-14, which recognized the extreme C-terminal segment of ABCC6/Abcc6, indicated
that full size proteins were expressed in mouse liver.
Rescue of Human ABCC6 Mutants in Mouse Liver
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Figure 5. Co-localization of the endoplasmic reticulum (ER) marker Calnexin and selected ABCC6 mutants. The liver cells expressing
the human ABCC6 variants (WT, R1339C, R1138Q and R1314W) were visualized by immunofluorescence on liver frozen sections using the M6II-31
monoclonal antibody (green) and a rabbit polyclonal antibody to the mouse ER marker Calnexin (red). Minimal co-localization (yellow) was seen
between Calnexin and the WT and R1138Q mutant, while some spotty co-localization was visible for the R1339C mutant, primarily around the nuclei
indicating partial ER retention or slower trafficking of this variant. The R1314W mutant showed the highest level of co-localization, which suggested
that a large fraction of this protein is unable to leave the ER and translocate to the plasma membrane. Scale is shown on the upper left image
Table 1. Function and intracellular localization of ABCC6 variants.
(% of WT)
localization in mouse
WT Stableyes yes100%
DABCC6 Stable n.d.n.d.
G1321SStable yes no
R1339C Unstable n.a. n.a.n.a.
n.d.: not determined.
n.a.: not applicable.
(ER): mostly retained with the endoplasmic reticulum.
*Plasma membrane and intracellular localization was determined independently by five of the co-authors using (+++++) for the plasma membrane and (2) for the
intracellular localization of the WT and (2) for the plasma membrane and+++++for the intracellular localization of DABCC6, respectively.
Rescue of Human ABCC6 Mutants in Mouse Liver
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whereas mutants V1298F and G1321S were transport-deficient.
We also observed that mutant R1339C could not be expressed in
insect cells as an intact protein, but was successfully produced as
full-length polypeptide in mouse liver following tail vein injection.
The five mutants we examined demonstrated various degrees of
intracellular accumulation in vivo ranging from WT behavior with
normal plasma membrane position (V1298) to intracellular
localization similar to DABCC6 (R1321S, R1339C or R1314W).
R1138Q, however, showed an intermediate behavior with partial
plasma membrane localization and a relatively abundant intra-
cellular presence (Figure 3, see also Table 1). Because we have
established that mutants G1321S, R1314W, R1339C and
R1138Q were likely retained in intracellular compartments as
non-degraded, full-size polypeptides (Figure 4), we used immuno-
fluorescent imaging to identify the potential intracellular com-
partments involved. We have found that mutants R1138Q and
R1339C showed some association with the ER suggesting partial
ER retention. R1314W was mostly retained in the ER (Figure 5)
indicating that the main consequence of pathologic amino acid
substitutions in the human ABCC6 protein is the alteration of
folding and/or trafficking. Though we have not specifically
investigated it in this study, we found no evidence of protein
accumulation in aggresomes or microtubule-organizing centers in
images of hepatocytes expressing the ABCC6 mutants [50,51,52].
However, this would certainly be worth investigating, perhaps
using MDCKII cells.
In summary, we have identified three possible consequences of
PXE-causing mutations: 1) Transport deficiency due to the
inability to use ATP (V1298F); 2) Altered folding and/or protein
stability leading to intracellular retention (R1314W, R1339C,
G1321S) in the ER or other cellular organelles, which could be
pharmacologically corrected; and 3) Reduced trafficking efficiency
with normal in vitro transport activity (R1138Q). We have
summarized these results in Table 1. Despite the variability in
the fate of these mutated proteins, all five mutations resulted in loss
of physiological function, which to some extent, provide an
explanation for the observed lack of phenotype–genotype
correlation in PXE [21,22]. In these experiments, we identified
two mutant candidates to test whether a chemical chaperon could
facilitate intracellular trafficking in mouse liver. Several studies
have shown the possibility of rescue of membrane targeting of
misfolded cystic fibrosis ABCC7/CFTR mutant (DF508) by
treating either cultured cells or human patients with 4-PBA
[24,25,26]. Similar results were obtained with disease-causing
mutants ABCB11 in MDCKII cells . Therefore, we explored
the effect of 4-PBA in mice transiently expressing transport-
competent ABCC6 mutants, R1138Q and R1314W as well as the
R1339C variant and the WT protein for control purposes
Figure 6. Pharmacological rescue of ABCC6 mutants. A: Mice were treated with 4-PBA prior to being subjected to HTVI with plasmids
expressing the WT ABCC6 and mutants R1339C, R1138Q and R1314W. Immunostaining on frozen sections of mouse liver were performed to reveal
both the mouse Abcc6 (red) and the human ABCC6 (green). The arrows show the improved plasma membrane localization of mutant R1314W as
compared to the untreated controls. Each image was derived from a different mouse. The scale bar represents 50 mm. B: Co-localization between the
endogenous ER marker Calnexin and mutant R1314W is indicated by yellow color. Treatment with 4-PBA facilitated the release of the latter mutant
from the ER as indicated by the reduced co-localization. C: Effect of 1 mM 4-PBA treatment on the intracellular localization of R1314W in MDCKII cells
as detected by immunofluorescence using the M6II-7 monoclonal antibody. The improved cellular localization of mutant R1314W is indicated by
Rescue of Human ABCC6 Mutants in Mouse Liver
PLoS ONE | www.plosone.org8 September 2011 | Volume 6 | Issue 9 | e24738
(Figure 6). The treatment had a remarkable effect on R1314W
reverting the apparent ER retention of this mutant protein back to
a near normal membrane localization in hepatocytes. A detailed
analysis of liver sections from 4-PBA treated mice expressing
R1314W is shown on Supplementary Figure S1. Treatment of
MDCKII cells expressing the R1314W mutant with PBA resulted
in a similar improvement (Figure 6C) thereby confirming the in vivo
In the present work we demonstrated the efficient association of
in vitro studies with in vivo experiments to evaluate the consequences
of missense ABCC6 mutations providing a unique insight into the
intracellular processing of this human ABC transporter in
physiological conditions similar to that of human primary
hepatocytes. We also found that our integrated approaches
constituted an appropriate base not only for testing pharmaco-
logical compounds with the ultimate aim of finding allele-specific
therapeutic solutions for PXE but could also be a model for the
systematic investigation of hepatic ABC transporters and other
liver-specific membrane proteins.
distribution of R1314W ABCC6 mutant. Mice were treated
with 4-PBA prior to being subjected to HTVI with plasmids
expressing ABCC6 R1314W. Immunostaining were performed on
liver frozen sections from mice exposed to 4-PBA (rows 3 and 4) or
from untreated animals (rows 1 and 2). Each row represents a
different animal. Histograms were generated by ImageJ64
software using the Graphic Dynamic Profiler tool. Arrows indicate
the lines of signal sampling.
Effect of 4-PBA treatment on the intracellular
The authors are thankful to Drs Sarkadi and Szaka ´cs for valuable
Conceived and designed the experiments: OLS TA AV. Performed the
experiments: OLS KF YY AI ZS CNB VP KH. Analyzed the data: OLS
KF ZS VP TA AV. Contributed reagents/materials/analysis tools: ZS.
Wrote the paper: OLS AV.
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