S -Decyl-glutathione nonspecifically stimulates the ATPase activityof the nucleotide-binding domains of the human multidrug resistance-associated protein, MRP1 ABCC1 Robbert H.. Remarkab
Trang 1S -Decyl-glutathione nonspecifically stimulates the ATPase activity
of the nucleotide-binding domains of the human multidrug
resistance-associated protein, MRP1 (ABCC1)
Robbert H Cool1, Marloes K Veenstra1, Wim van Klompenburg1, Rene´ I R Heyne1, Michael Mu¨ller2,*, Elisabeth G E de Vries3, Hendrik W van Veen1,†, and Wil N Konings1
1
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen,
Haren, the Netherlands;2Department of Gasteroenterology and Hepatology, University Hospital Groningen, the Netherlands;
3 Department of Medical Oncology, University Hospital Groningen, the Netherlands
The human multidrug resistance-associated protein (MRP1)
is an ATP-dependent efflux pump that transports anionic
conjugates, and hydrophobic compounds in a glutathione
dependent manner Similar to the other,
well-character-ized multidrug transporter P-gp, MRP1 comprises two
nucleotide-binding domains (NBDs) in addition to
transmembrane domains However, whereas the NBDs of
P-gp have been shown to be functionallyequivalent,
those of MRP1 differ significantly The isolated NBDs of
MRP1 have been characterized in Escherichia coli as
fusions with either the glutathione-S-transferase (GST) or
the maltose-binding domain (MBP) The nonfused NBD1
was obtained bycleavage of the fusion protein with
thrombin The GST-fused forms of NBD1 and NBD2
hydrolyzed ATP with an apparent Km of 340 lM and a
Vmax of 6.0 nmol PIÆmg)1Æmin)1, and a Km of 910 lM ATP and a Vmaxof 7.5 nmol PIÆmg)1Æmin)1, respectively Remarkably, S-decyl-glutathione, a conjugate specifically transported byMRP1 and MRP2, was able to stimulate the ATPase activities of the isolated NBDs more than 2-fold in a concentration-dependent manner However, the stimulation of the ATPase activitywas found to coincide with the formation of micelles by S-decyl-glutathione Equivalent stimulation of ATPase activitycould be obtained bysurfactants with similar critical micelle con-centrations
Keywords: ABC; MRP; multidrug resistance; ATPase; nucleotide-binding domain
Multidrug resistance of human tumour cells is an
impedi-ment to successful cancer treatimpedi-ment and is frequently
associated with the overexpression of certain members of
the ATP-binding cassette (ABC) transporter superfamily
such as the MDR1 P-glycoprotein (P-gp; ABCB1) and the
human multidrug resistance-associated protein (MRP1;
ABCC1) [1–3] As other ABC transporters, MRP1 is a
membrane-bound transport protein that mediates the
extrusion of its substrates at the expense of ATP Studies
on MRP1-expressing cells and membrane vesicles derived
thereof have demonstrated that MRP1 has a broad specificityfor glutathione S-conjugates, most notably cysteinyl leukotrienes, and for anionic conjugates of bile salts and steroid hormones [2,4] In addition, MRP1 is able
to extrude natural product drugs that are used in chemo-therapeutic strategies, such as daunorubicin and vincristine,
in cotransport with reduced glutathione [5–7]
MRP and P-gp proteins mayshare a mechanism by which ATP hydrolysis is coupled to drug transport [8,9] Indeed, the basal ATPase activityof P-gp and MRP proteins can be stimulated bysome of their transported substrates or allocrites [10–18] However, two aspects blur
a clear view on the coupling mechanism Firstly, whereas some allocrites do not stimulate, and other allocrites show onlya weak stimulation of the ATPase activity, modu-lators of transport activitythat are not transported can also affect ATPase activity Secondly, the concentration-dependencyof the allocrite-stimulated ATPase activity often is represented bya bell-shaped curve, and most allocrites even inhibit the ATPase activityat high concentrations [12,13,19,20] This can be explained by assuming the presence of one high-affinitystimulatory binding site, and one low-affinityinhibitorybinding site These sites maybe identical to the on and off sites, which are distinct allocrite-binding sites involved in transmem-brane transport [11]
A typical, but complicating, characteristic of multidrug transporters is their capabilityto efficientlyexpel struc-turallyunrelated compounds from the cell It was shown
Correspondence to R H Cool, Pharmaceutical Biology, Groningen
University, A Deusinglaan 1, 9713 AV Groningen, the Netherlands,
Fax: + 31 50 3633000, Tel.: + 31 50 3638154
E-mail: r.h.cool@farm.rug.nl
Abbreviations: ABC, ATP-binding cassette; GST,
glutathione-S-transferase; MBP, maltose-binding protein; MRP, multidrug
resistance-associated protein; NBD, nucleotide-binding domain.
Note: We prefer to use the term allocrite to describe transported
compounds as proposed previously[8] in order to distinguish these
compounds from the substrate, which bydefinition is ATP, and
nontransported modulators.
*Present address: Division of Nutrition, Metabolism and Genomics,
UniversityWageningen, the Netherlands.
Present address: Department of Pharmacology, University of
Cambridge, UK.
(Received 14 January2002, revised 8 May2002,
accepted 30 May2002)
Trang 2that different allocrites bind to different regions of the
transporter, as revealed bykinetic measurements and
mutational studies [21] Although these drug-binding sites
are still ill defined, there is ample evidence that most, if
not all, of the residues involved in allocrite binding and
transport are located within the transmembrane domains
in P-gp and MRP [22–30] Interestingly, however, two
studies point to the binding of an allocrite bythe
ATP-binding domain of an ABC transporter The
oleando-mycin transporter OleB from Streptomyces antibioticus
was suggested to have an allocrite-binding site located on
the binding domain [31], and KpsT, the
ATP-binding component of the Escherichia coli ABC
trans-porter KspTM involved in the transport of polysialic
acid, was shown to co-immunoprecipitate with polysialic
acid [32]
In order to reveal the characteristics of the individual
nucleotide-binding domains (NBDs) of MRP1, and in
search of a putative interaction of these NBDs with
allocrites, we have overproduced these domains as
gluta-thione-S-transferase or maltose binding domain fusion
proteins in E coli Both NBDs were found to be able to
hydrolyse ATP with comparable activity Remarkably, the
MRP1- and MRP2-specific allocrite S-decyl-glutathione
significantlystimulates the ATPase activityof these
do-mains, quite similar to its effect on the ATPase activityof
reconstituted MRP2 However, this stimulation was found
to relate to micelle formation bythis compound
M A T E R I A L S A N D M E T H O D S
Cloning of the nucleotide-binding domains of MRP
DNA coding for the first nucleotide-binding domain
(NBD1) of human MRP1 (residues Lys614–Lys959) was
amplified byPCR from plasmid pJ3W (kindlydonated by
P Borst, Netherlands Cancer Institute, Amsterdam) using
the forward primer 5¢-GGCGGGATCCGATATCA
AACGCCTGAGGATCTTTC-3¢ and the reverse primer
5¢-TACTAGCGGCGCCGTTAGAATTCCTTGACCTG
CCCTGTCTGCGC-3¢ (the introduced BamHI and EcoRI
sites, respectively, are underlined) To obtain a translational
fusion between glutathione S-transferase (GST) of
Schisto-soma japonicumand NBD1, the PCR product was cloned
as a BamHI–EcoRI fragment into pGEX4T-1
(Pharma-cia), giving pGST-NBD1 The fusion of the NBD1 with
the maltose-binding domain (MBP) was accomplished
through subcloning of the BamHI–SalI fragment from
pGST-NBD1 into the pMalc2 expression vector (New
England Biolabs)
DNA coding for NBD2 of MRP1 (residues Glu1294–
Val1531) was directlycloned as an EcoRI–NotI fragment in
pGEX4T-1 to create pGST-NBD2 A slightlylarger
fragment of NBD2 (residues Gly1291–Val1531) was
pro-duced byPCR (forward primer 5¢-CCCGGATCCGGC
CGAGTGGAGTTCCGGAAC-3¢ and reverse primer
5¢-GCCAGGTCGACTTATCACACCAAGCCGGCGT
CTTTGG-3¢) and subcloned into the expression vector
pMBPT [33] This vector, kindlyprovided byG Altenberg
(Universityof Texas Medical Branch, Galveston, USA),
comprises a factor Xa and a thrombin cleavage site between
the MBP and the fusion partner, so that the nonfused
protein can be obtained byincubation with either protease
Protein overproduction and purification Overproduction of the fusion proteins was performed principallyaccording to standard procedures, even though several deviations were tested A low incubation tempera-ture after induction of gene expression was essential to increase solubilityof NBD2-fusion proteins Escherichia coli strains DH5a and the protease-poorAD202 were both used for expression and did not result in large differences
In short, an overnight preculture in Luria–Bertani medium containing 50–100 lgÆmL)1 ampicillin and 0.2– 0.5% glucose was used to inoculate a larger volume of the same medium After incubation at 37C up to D660 0.6, 0.05–0.1 mMof isopropyl thio-b-D-galactoside (Boehringer Mannheim, Germany) was added NBD1 fusion protein-producing cultures were further incubated at 30C for 5 h, whereas NBD2-fusion protein producing cultures were cooled to 25 or 18C and incubated for an additional 5 or
16 h, respectively Cells were harvested by centrifugation and resuspended in buffer A (20–50 mMTris/HCl, pH 7.5, 50–100 mMNaCl, 5–10 mMdithiothreithol, 10% glycerol)
in the ratio of 3 mL of buffer per g of wet cells The resuspended cells were lyzed by ultra-sonication or by passage through a French pressure cell at 16 000 p.s.i., and centrifuged for 30–45 min at 40 000 g and 4C to remove cell debris The supernatant was used for purification according to the protocols of the supplier of the column material: glutathione–Sepharose 4B (Pharmacia Biotech) for the purification of GST-fusion proteins, or amylose resin (New England Biolabs) for the purification of MBP-fusion proteins Column material with bound proteins was washed extensivelywith buffer A, after which fusion proteins were eluted with buffer A plus 10 mM glutathione or maltose Protein containing fractions were determined bySDS/ PAGE and pooled fractions were concentrated using VivaspinTMcentrifugation vials (Vivascience Inc.)
To purifyNBD1 as a separate domain in the absence of GST, the specific thrombin cleavage site located between GST and NBD1 was employed A 20-mL column contain-ing glutathione–Sepharose 4B with GST–NBD1 was pre-pared as described above, after which 20 units of thrombin (Sigma) were added to the column After an incubation of
16 h at 4C, the nonfused NBD1 was eluted from the column with buffer A and concentrated using Vivaspin centrifugation vials (Vivascience Inc.)
Purified protein was rapidlyfrozen in liquid nitrogen and stored at)80 C Protein concentrations were determined bythe Bradford assayusing BSA as the standard
The following purified proteins were kindlyprovided by colleagues: the MBP fusion protein of the NBD of the lactococcal half-transporter LmrA (residues Glu328-Lys590) by M Pasmooij (Groningen University, the Netherlands); E coli MalK byH Landmesser (Humboldt University, Berlin, Germany); and GlcV, the ATP-binding subunit of a glucose uptake system from the thermoacido-philic Sulfolobus solfataricus, byS Albers (Groningen University, the Netherlands)
ATPase activity The ATPase activityof NBDs was measured bya colori-metric assay[34] Protein was incubated in buffer, supple-mented with ATP (from a 100-m stock of Na-ATP,
Trang 3brought to pH 7.5 with NaOH; Sigma), and allocrites or
inhibitors, as described in the legends Substrates and
inhibitors were added to the reaction mixtures or inorganic
phosphate standards prior to the addition of ATP The
transfer of the mixture from ice to a water bath of 30C
started the reaction At this temperature, the reaction was
linear over the 30 min-incubation interval Samples of
30 lL were added to wells of a 24-well plate precooled on
ice, where after 150 lL of colour reagent [0.034% (w/v)
malachite green base (Sigma), 1.05% (w/v) ammonium
molybdate and 0.1% (v/v) Triton X-100] was added After
5 min on ice, 75 lL of 34% (w/v) citric acid was added, and
the plate was incubated for 30 min at room temperature
Subsequently, the D650was measured in an ELISA plate
reader As a control, samples were incubated for 30 min on
ice and treated in the same way A calibration was
performed using 0–160 lMof inorganic phosphate
For clarity, in Table 1 ATPase activities are presented
relative to the activitymeasured at 2 mM Mg2+, and in
Figs 2,3,4 and 6 relative to the activitymeasured in absence
of surfactant The absolute ATPase activities of the proteins
in these experiments were, depending on protein
prepar-ation, in the range of 0.3–0.5 (pmol Pi)Æ(pmol
pro-tein))1Æmin)1 for the GST- and MBP-fused versions of
NBD, NBD2, and LmrA-NBD, and approximately
0.02 (pmol Pi)Æ(pmol protein))1Æmin)1 for the nonfused
version of NBD1
Micelle formation
Micelle formation was followed bymeasurement of the
fluorescence of 1,6-diphenyl-1,2,3-hexatriene (Fluka), using
excitation wavelength 355 and emission wavelength
428 nm 1,6-diphenyl-1,2,3-hexatriene was dissolved to
2.5 mM in dimethylsulfoxide, and used at a final
concen-tration of 5 lM 1,6-Diphenyl-1,2,3-hexatriene was
incu-bated with surfactants for at least 1 h at room
temperature, as time-dependent measurements
demonstra-ted that an equilibrium was reached after 30 min
Although micelle formation is usuallydetermined with
1,6-dihydro-1,2,3-hexatriene using fluorescence anisotropy
[35], standard fluorescence measurements give a
reason-ablygood indication of the critical micelle formation value
of a surfactant This was checked bydetermining the
critical micelle formation value of dodecylmaltoside, which
corresponded to the critical micelle formation value given
bythe manufacturer
R E S U L T S
Production and purification of the NBDs of MRP1 The amplified PCR products for the first and second NBD
of MRP1 were cloned into the pGEX-2T expression vector
in frame with GST domain The overexpression at 30C of the gene encoding the GST–NBD1 fusion protein under control of the isopropyl thio-b-D-galactoside-inducible tac promoter was evident as shown in Fig 1 For the overpro-duction of GST–NBD2 at 30C, most fusion protein was found in inclusion bodies However, when the protein overproduction was performed at lower temperature, most GST–NBD2 was present in the soluble fraction A yield of approximately15 mg GST–NBD1 and 3 mg GST–NBD2 per litre of culture was achieved The resultant proteins appear more than 95% pure as judged bySDS/PAGE analysis
Comparable results were obtained with the MBP-fused versions of NBD1 and NBD2 However, thrombin cleavage appeared less efficient for the MBP-fusion proteins as compared to the GST-fusion proteins
Basal ATPase activity
In order to studythe role of NBD1 and NBD2 in MRP1-mediated drug transport, the ATPase activityof the fusion proteins was determined Kinetic analysis revealed for GST-NBD1 an apparent Kmof 340 lM ATP and a Vmax
of 6.0 nmol PiÆmg)1min)1, and for GST-NBD2 an
Table 1 Dependence of the ATPase activity of GST–NBD1 and GST–
NBD2 on divalent cations ATPase activitywas measured as described
in Materials and methods in the presence of 2 m M ATP and 2 m M
divalent cations or EDTA For comparison, the ATPase activityof
each NBD are presented as percentages of its activityin the presence of
magnesium.
Fig 1 Overproduction and purification of MRP1 NBDs in Escherichia coli Fractions obtained for overproduction and purification of NBDs were analyzed by 10% SDS/polyacrylamide gel electrophoresis and stained bycolloidal Coomassie staining Lane 1–3, total bacterial proteins of cell cultures producing GST–NBD1: lane 1, overnight without IPTG; lane 2, before addition of isopropyl thio-b- D -galacto-side (t ¼ 0); lane 3, harvested cells after induction The arrow indicates the position of GST–NBD1 Lane 4–6, overloaded samples of (semi)-purified fractions: lane 4, GST–NBD1 (semi)-purified byglutathione-Seph-arose 4B affinitychromatography; lane 5, MBP–NBD2 purified by amylose/agarose; lane 6, NBD1 purified by glutathione-Sepharose 4B affinitychromatographyfollowed bythrombin cleavage The positions
of molecular mass markers (in kDa) are indicated.
Trang 4apparent Km of 910 lM ATP and a Vmax of 7.5 nmol
PiÆmg)1Æmin)1(data not shown) Similar Vmaxvalues (5–
10 nmol PiÆmg)1Æmin)1), but higher Kmvalues (1.5–1.8 mM)
were obtained recentlywith His-tagged NBDs of MRP1
[36] When compared to the full length MRP1, the obtained
Kmvalues are somewhat higher than the apparent Kmof
100 lMATP for MRP1-mediated leukotriene C4transport
[37], and than the apparent Kmof 104 lMfound for purified
MRP1 after reconstitution in proteoliposomes [38]
Signi-ficantlydifferent values were obtained with the purified
MRP1 in presence of phospholipids: Km¼ 3 mM and
Vmax¼ 460 nmol PiÆmg)1Æmin)1[14]
The MBP-fused versions of NBD1 and NBD2 have
similar ATPase activities as the GST-fused proteins In
contrast, the ATPase activityof the nonfused NBD1
(without GST- or MBP-moiety) was one order of
magni-tude lower than that of the fused protein As yet, we have no
evident explanation for this The nonfused version of NBD2
could not be isolated bythrombin cleavage of the GST–
NBD2 protein due to proteolytic degradation
GTP was hydrolyzed by both GST–NBDs (data not
shown), in agreement with the finding that reconstituted
MRP1 does not show nucleotide specificity[38] The
ATPase activityof GST–NBD1 and -NBD2 was dependent
on the presence of divalent cations The highest ATPase
activitywas obtained with magnesium ions but the
hydro-lysis of ATP was also observed in the presence of
manganese, cobalt and calcium ions (Table 1)
To further characterize the ATPase activityof both
GST-NBDs, the effect of ATPase inhibitors was tested
The cysteinyl-reactive N-ethylmaleimide did not
signifi-cantlyinhibit the ATPase activities at concentrations of
up to 2 mM However, the ATPase activityof GST–NBD1
and GST–NBD2 was inhibited more than 70% by
2 mM sodium azide and approximately40% by2 mM
ortho-vanadate In comparison, the ATPase activityof
reconstituted MRP1 was efficientlyinhibited
byortho-vanadate (IC50¼ 10 lM), less
efficientlybyN-ethylmalei-mide (IC50¼ 0.5 mM), and hardlybysodium azide
(IC50> 6 mM) [38] Surprisingly, the ATPase activities of
the His-tagged NBDs were inhibited by N-ethylmaleimide
[36]
In an attempt to obtain information about interdomain
interactions, the basal ATPase activities were measured at
different NBD concentrations No significant deviations
from a linear concentration-dependence were observed in
the tested concentration range up to 4 lMfor the separate
domains and up to 1 lMfor the mixture of the MBP-NBDs
Allocrite-mediated effects on the ATPase activity
The capabilitywas tested of a number of allocrites to
stimulate the ATPase activityof the NBDs of MRP1 The
following compounds (tested at the indicated
concentra-tions) had no significant effect on the basal ATPase activity:
vincristine (46 lM); vincristine (46 lM) plus reduced
gluta-thione (500 lM); reduced glutathione (500 lM); oxidized
glutathione (500 lM); probenecid (1 mM); sulfinpyranoside
(1 mM); N-ethyl-maleimide-glutathione (0.1–4 mM;
pre-pared as described previously; [18]); LTC4 (0.1–1.6 lM);
17b-estradiol 17-(b-D-glucuronide) (10–75 lM)
Strikingly, the ATPase activity of GST- and MBP-NBD1
was stimulated by S-decyl glutathione: approximately
twofold at 250 lM of S-decyl-glutathione (Fig 2) Up to
250 lM of S-decyl-glutathione, the stimulatory action increased, whereas the stimulation decreased at higher concentrations At 1 mMof S-decyl-glutathione, the meas-ured ATPase activityrepresented approximatelythe basal ATPase activity This type of behaviour was also observed for the stimulatoryeffect of S-decyl-glutathione on the ATPase activityof reconstituted MRP2 [17] The effect was
Fig 2 ATPase activity of GST–NBD1 and MBP–NBD1 is stimulated
by S-decyl-glutathione GST–NBD1 (upward diagonallyhatched bars)
or MBP–NBD1 (black bars) at 2.5 l M was incubated with 7 m M ATP and different concentrations of S-decyl-glutathione (A), decyl-malto-side (B) or decanol (C) in 50 m M Tris/HCl, pH 7.5, 50 m M NaCl, and
50 m M MgCl 2 , for 30 min at 37 C The ATPase activity was deter-mined as described in Materials and methods and calculated relative to the activitymeasured in absence of surfactant Error bars indicate standard deviations Each determination was performed at least twice.
Trang 5specific for S-decyl-glutathione as alkyl-glutathione
conju-gates of shorter chain length (ethyl-, propyl-, butyl-, hexyl-,
and octyl-glutathione) did not show a significant effect on
the ATPase activityat concentrations up to 1 mM As a
control, the effects of two molecules with a similar alkyl
chain were compared: decyl-maltoside and decanol
(Fig 2B,C) Decyl-maltoside did not stimulate the ATPase
activityat lower concentrations, but induced a
concentra-tion-dependent stimulation at concentrations above
500 lM, at least up to 1 mM(we did not measure at higher
concentrations) In contrast, decanol did not show any
stimulation, and caused a small, but significant and
concentration-dependent inhibition of the ATPase activity
The ATPase activityof MBP–NBD2 was stimulated in
the same manner as MBP–NBD1, indicating that in this
respect there is no difference between the two NBDs
Surprisingly, the MBP-fusion protein of the isolated NBD
of the bacterial multidrug transporter LmrA [39] also
showed a similar stimulation pattern (Fig 3) LmrA is the
bacterial homologue of P-gp, and is not expected to bind or
transport glutathione-conjugates In comparison, we have
measured the effect of S-decyl-glutathione on the ATPase
activityof MalK [40] and of GlcV, the ATP-binding subunit
of a glucose uptake system from the thermoacidophilic
Sulfolobus solfataricus [41] In both cases, the ATPase
activitywas slightlyinhibited in a near-linear
concentration-dependent manner over the concentration range 0–0.5 mM
of S-decyl-glutathione (data not shown) Thus, the ATPase
activityof these proteins is differentlyaffected when
compared to the NBDs of MRP and LmrA
In further analysis, the concentration of
S-decyl-glutathi-one, which optimallystimulates the ATPase activityof
MBP-NBD1 appeared to depend on the concentration
of MBP–NBD1 The optimum increases with increasing
MBP–NBD1 concentration: whereas at 1 lM of MBP–
NBD1 optimal stimulation is observed at 0.2 mM
S-decyl-glutathione, at 4 lM of MBP–NBD1, the optimal
stimulation is achieved at 0.3–0.4 mM of S-decyl-glutathi-one (Fig 4A) A similar concentration-dependence was found for MBP–NBD2 and NBD1 (Fig 4), even though the optimal concentrations are shifted
These results clearlydemonstrate that we are not dealing with a simple enzyme-substrate system As the presence of two allocrite-binding sites (a stimulatoryhigh-affinity, and
an inhibitorylow-affinitysite) on a NBD is unlikely, we reasoned that it could be a nonspecific, detergent-related effect As we were unable to find in the literature any indications at which concentration S-decyl-glutathione forms detergent micelles, this critical micelle concentration was determined bytaking advantage of the fact that the fluorescence of 1,6-dihydro-1,2,3-hexatriene is greatly
Fig 3 ATPase activity of MBP-LmrA-NBD is stimulated by
S-decyl-glutathione MBP–LmrA–NBD at 2 l M was incubated with 5 m M
ATP and different concentrations of S-decyl-glutathione in 50 m M
Tris/HCl, pH 7.5, 50 m M NaCl, 10 m M MgCl 2 , and 10 m M
dithio-threitol, for 30 min at room temperature The ATPase activitywas
determined as described in Materials and methods and calculated
relative to the activitymeasured in absence of surfactant Error bars
indicate standard deviations Each determination was performed at
least twice.
Fig 4 Concentration profile of the S-decyl-glutathione-stimulated ATPase activity depends on the concentration of the nucleotide-binding domain Four different concentrations of (A) MBP–NBD1 (1, 2, 3,
4 l M ), (B) MBP–NBD2 (1, 2, 3, 4 l M ) or (C) NBD1 (5, 10, 15, 20 l M )
in 50 m M Tris/HCl, pH 7.5, 50 m M NaCl, 10 m M MgCl 2 , and 10 m M
dithiothreitol, were incubated with 1 m M ATP for 30 min at room temperature The ATPase activities were determined in presence of S-decyl-glutathione as described in Materials and methods and normalized to the activityfound in absence of surfactant (100%) The relative ATPase activities at the indicated concentrations of surfactant are represented bycolumns with upward diagonallyhatched, black, horizontallyhatched or downward diagonallyhatched surface for the activities at the lowest to highest concentration of the NBDs, respectively Each activity was measured at least twice The bar represents the standard deviation.
Trang 6enhanced in hydrophobic environment, e.g upon formation
of micelles In Fig 5, the fluorescence of
1,6-dihydro-1,2,3-hexatriene in presence of different concentrations of
S-decyl-glutathione, decyl-maltoside and dodecyl-maltoside
are depicted The increase of fluorescence starting at
approximately0.15 mMof dodecyl-maltoside and the slight
increase starting at 1 mM of decyl-maltoside corresponds
with the critical micelle concentrations of these compounds,
respectively, 0.17 mM and 1.8 mM (values manufacturer)
The 1,6-dihydro-1,2,3-hexatriene-mediated fluorescence in
the presence of S-decyl-glutathione suggests that this
compound forms micelles alreadyat low concentrations
At the moment we cannot explain whythe
S-decyl-glutathione-induced fluorescence shows a relative minimum
at approximately0.8 mMof S-decyl-glutathione (Fig 5B)
As micelle formation could affect the ATPase activityof
the NBD, we tested the effects of surfactants with different
critical micelle concentrations The three surfactants
un-decyl-, dodecyl- and tridecyl-maltoside could stimulate the
ATPase activityof GST–NBD1 to a similar extent as
S-decyl-glutathione, but with different concentration optima:
0.1–0.5, 0.05–0.2 and 0–0.1 m , respectively(Fig 6) These
values correspond reasonablywell to the critical micelle con-centrations of these compounds: 0.6, 0.12 and 0.024 mM, respectively(values manufacturer) Similarly, Triton-X100 (critical micelle concentration value: 0.23 mM) stimulated the ATPase activityof MBP-NBD2 in a similar fashion as dodecyl-maltoside and S-decyl-glutathione (not shown) Thus, the stimulatoryeffect of S-decyl-glutathione or other surfactants on the ATPase activityof the MRP–NBDs seems to be related to micelle formation and not to be a specific, allocrite-mediated effect
D I S C U S S I O N
In this study, we show that the isolated nucleotide-binding domains of human MRP1 displaycomparable ATPase activities The Michaelis–Menten constants Km and Vmax for the GST-fusion proteins of NBD1 and NBD2 are
340 lM and 6.0 nmol PiÆmg)1Æmin)1, and 910 lM and 7.5 nmol PiÆmg)1Æmin)1, respectively These kinetic param-eters are in the same range of those observed for the isolated NBDs of the human MDR1 P-glycoprotein [42], the cystic fibrosis transmembrane conductance regulator [43] and prokaryotic ABC transporters [44] Furthermore, these results are in reasonable agreement with the results obtained with the isolated NBDs of MRP1 comprising an N-terminal His-tag [36], and to the values obtained with the reconsti-tuted MRP1, isolated from insect cells [37] or human tumour cells [38] The similarityof the basal ATPase activities of the two isolated domains does not seem to correlate to the differences found in photo-affinitylabelling experiments with MRP1 using 8-azido-adenosine nucleo-tides [45–47] ATP labelling occurred preferentiallyat NBD1, while ortho-vanadate-induced trapping of ADP occurred predominantlyat NBD2 However, such different
Fig 6 Stimulation of the ATPase activity of GST–NBD1 by undecyl-, dodecyl-, and tridecyl-maltoside In a buffer of 50 m M Tris/HCl,
pH 7.5, 50 m M NaCl, 10 m M MgCl 2 , and 10 m M dithiothreitol, 1 l M
of GST-NBD1 was incubated with 1 m M ATP in presence of different concentrations of n-undecyl-b- D -maltoside (A; upward diagonally hatched bars), n-dodecyl-b- D -maltoside (B; black bars) or n-tridecyl-b- D -maltoside (C; downward diagonallyhatched bars) for 30 min at room temperature ATPase activities were measured as described in Materials and methods and represented as activities relative to the activitymeasured in absence of the surfactants Experiments were performed at least in duplicate Error bars indicate the standard deviations.
Fig 5 Detection of micelle formation by domaltoside (A),
decyl-maltoside and S-decyl-glutathione (B) Different concentrations of
dodecyl-maltoside (A, d), decyl-maltoside (B; r) and
S-decyl-gluta-thione (B; j) were incubated with 5 l M 1,6-dihydro-1,2,3-hexatriene
in 50 m M Tris/HCl, pH 7.5, 50 m M NaCl, 10 m M MgCl 2 , and 10 m M
dithiothreitol, for 3 h at room temperature Thereafter, the
fluores-cence was determined using a 355-nm excitation wavelength and a
428-nm emission wavelength For everymeasurement, the fluorescence
was followed for at least 5 min in order to assure a stable value.
Trang 7behaviour mayresult from different contacts within the full
length MRP1 of the NBD(s) with other domains and/or the
lipidic bilayer
The hydrolysis of ATP by both NBDs of MRP1 was
dependent on divalent cations and was inhibited byATPase
inhibitors, such as ortho-vanadate and azide Our data
indicate that both NBDs of MRP1 are also able to
hydrolyze GTP This result is consistent with previous
studies on full length MRP1 protein, which demonstrated
the hydrolysis of ATP and GTP by purified MRP1 [15], and
the dependence on these nucleotides of MRP1-mediated
drug transport in plasma membrane vesicles [5]
FrequentlyMBP-fusion proteins are more stable than
GST-fusion proteins, in line with the postulation that the
MBP-moietyfunctions as an intramolecular chaperone [48]
We could observe a similar effect for the NBDs of MRP1,
although the differences in solubilityfor the two types
of fusion proteins were not verylarge The MBP-fusion
proteins however, appeared to be cleaved bythrombin less
efficientlythan the GST-fusion proteins, most likelydue to a
shielding effect of the MBP moiety The nonfused NBD1
appeared to be stable, but to have an ATPase activitythat is
one order of magnitude lower than that of the fused forms
of this domain As yet, we have no satisfactory explanation
for this A similar effect was observed for the NBD of P-gp,
which appeared to express a 100–1000-fold lower ATPase
activityas compared to P-gp full length It was suggested
that NBDs mayneed to interact with the membrane-bound
domains (i.e intracellular loops) to fold properly[23] In
addition, differences in the N- and C-terminal boundaries of
subcloned NBDs seem to affect the solubilityof the isolated
domains and mayalso influence the ATPase activity
Indeed, preliminarydata show significant differences in
basal ATPase activities of MBP-fused constructs of NBD1
of different length (not shown)
An important aspect of the mechanism, bywhich ABC
transporter proteins expel the allocrites from the cell, is how
the ATP hydrolysis is coupled to transport Stimulation of
ATPase activitybyallocrites has been used as an important
assayfor the elucidation of this coupling However, even
though the ATPase activitycan be stimulated bysome
allocrites, this stimulation is modest and not observed for all
allocrites In addition, the manystudies that have been
performed to reveal the drug-binding sites of MDR
transporters suggest that these binding sites are located in
the transmembrane segments Indeed, whereas modulators
of transport activity, e.g flavonoids, were shown to interact
with the NBDs P-gp or MRP proteins [49,50], no
interac-tions between allocrites and the NBDs of these transporters
were reported In contrast, the oleandomycin ABC
trans-porter OleB from Streptomyces antibioticus was suggested to
have an allocrite-binding site located on the ATP-binding
domain [31] Furthermore, the ATP-binding component of
the Escherichia coli ABC transporter KspTM involved in
the transport of polysialic acid, KpsT, was shown to
co-immunoprecipitate with the allocrite polysialic acid [32]
The latter examples encouraged us to measure the effects of
MRP-specific allocrites on the ATPase activityof the
isolated NBDs
We have tested several MRP-specific allocrites in their
abilityto stimulate the ATPase activityof NBD1 Most of
the tested compounds did not induce a significant
stimula-tion of the basal activity Remarkably, however, the high
affinity-allocrite S-decyl-glutathione showed a stimulatory effect, which was concentration dependent An optimal concentration was found around 100–300 lM, above which concentration the stimulatoryeffect decreased to zero (Fig 2) This effect is verysimilar to that observed with reconstituted MRP2: a 2.5-fold stimulation of the ATPase activitywas observed at 100 lM S-decyl-glutathione, whereas no stimulation could be measured at 1 mM [17] However, additional experiments cautioned us to interpret the observed effect as a demonstration of the presence of an allocrite-binding site on NBD1 First, we could obtain a similar stimulatoryeffect with NBD2, and with the NBD of the bacterial transporter LmrA (Fig 3) that is not supposed
to transport glutathione-conjugates Secondly, we noticed that the optimal stimulatoryconcentration of allocrite was dependent on the concentration of NBD (Fig 4) Taken together, these results pointed at a possiblynonspecific effect of S-decyl-glutathione
As S-decyl-glutathione comprises an alkyl chain, it may act as a surfactant After having alreadydetermined that S-decyl-glutathione is capable of forming micelles at low concentrations (Fig 5) we measured the effects of surfac-tants with different critical micelle concentration values on the ATPase activityof the NBDs (Fig 6) It was found that the stimulation of these compounds, which are not known
to be allocrites of MRP1, coincides with micelle formation Thus, the observed effect appears to be more surfactant-related than allocrite-surfactant-related
The shift in optimal stimulatoryconcentration of allocrite related to the NBD concentration can be rationalized by assuming a direct interaction between surfactant molecules and the protein, which would cause a decrease in free concentration of surfactant and therebya shift to a higher critical micelle concentration value Such an interaction was suggested between P-gp and detergents [20]
It is tempting to speculate that the surfactant-dependent stimulation maymimick the interaction between the NBD and lipid bilayer Indeed, the phospholipidic content of biomembranes affects the basal and the drug-stimulated ATPase activityof MRP1 [51] and P-gp [52], similar to the effects on the ATPase activityof ABCR [53] This is supported bythe close proximityof the NBDs of P-gp to the membrane surface observed byspectroscopic measurements [54,55] and electron microscopy[56], and bythe recently published crystal structure of the Escherichia coli ABC transporter MsbA [57] At this point we cannot explain the decrease in stimulatoryeffect at higher concentrations of surfactant Further work is required to elucidate these effects
In conclusion, the NBDs of MRP1 show comparable basal ATPase activities that can be stimulated bythe allocrite S-decyl-glutathione We could show, however, that this stimulation results not from an allocrite-specific effect, but from surfactant-mediated micelle formation Our results stronglysuggest that the stimulatoryaction of S-decyl-glutathione on the ATPase activityshould not be used as a signal for specific interaction between MRP and allocrite
A C K N O W L E D G E M E N T S
This investigation was supported bygrant RuG 96–1218 from the Dutch Cancer Societyand part of a joint GUIDE-GBB research programme at the Groningen University We would like to thank
Trang 8Sarina van der Zee, Patrycja Golon and Sylwia Chocholska for
technical assistance Furthermore we thank Piet Borst for the kind gift
of plasmid pJ3W, and Marjon Pasmooij, Sonja Albers and Heidi
Landmesser for purified LmrA-NBD, GlcV, and MalK, respectively.
H W v V was a fellow of the Netherlands Academyof Art and
Sciences.
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