Multidrug resistance-associated protein 4 is a bile transporter of Clonorchis sinensis simulated by in silico docking
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Multidrug resistance-associated protein 4 (MRP4) is a member of the C subfamily of the ABC family of ATP-binding cassette (ABC) transporters. MRP4 regulates ATP-dependent efflux of various organic anionic substrates and bile acids out of cells. Since Clonorchis sinensis lives in host’s bile duct, accumulation of bile juice can be toxic to the worm’s tissues and cells. Therefore, C. sinensis needs bile transporters to reduce accumulation of bile acids within its body.
We cloned MRP4 (CsMRP4) from C. sinensis and obtained a cDNA encoding an open reading frame of 1469 amino acids. Phylogenetic analysis revealed that CsMRP4 belonged to the MRP/SUR/CFTR subfamily. A tertiary structure of CsMRP4 was generated by homology modeling based on multiple structures of MRP1 and P-glycoprotein. CsMRP4 had two membrane-spanning domains (MSD1 & 2) and two nucleotide-binding domains (NBD1 & 2) as common structural folds. Docking simulation with nine bile acids showed that CsMRP4 transports bile acids through the inner cavity. Moreover, it was found that CsMRP4 mRNA was more abundant in the metacercariae than in the adults. Mouse immune serum, generated against the CsMRP4-NBD1 (24.9 kDa) fragment, localized CsMRP4 mainly in mesenchymal tissues and oral and ventral suckers of the metacercariae and the adults.
Our findings shed new light on MRPs and their homologs and provide a platform for further structural and functional investigations on the bile transporters and parasites’ survival.
KeywordsClonorchis sinensis Bile transporter MRP4 ABCC4 Structure Localization
5′-rapid amplification of the cDNA ends
Bile salts export pump
Cystic fibrosis transmembrane conductance regulator
Multidrug resistance-associated protein 4
Quantitative real-time PCR
Clonorchiasis is a major endemic disease affecting over 35 million people in Asian countries including Korea, China, Thailand and Vietnam [1, 2, 3]. Clonorchis sinensis infections are caused by the ingestion of raw or undercooked freshwater fish that harbor the metacercariae . Complications associated with the infection increase with an increase in the intensity and duration of the infection. Clonorchis sinensis has also been recognized by the World Health Organization as a biological carcinogen that can induce cholangiocarcinoma in humans .
C. sinensis migrates into the bile duct of the host and lives there. However, the bile duct can be regarded as an extreme environment, since accumulation of bile juice can be toxic to the worm’s tissues and cells . Among the various bile juice, lithocholic acid (LCA) has been proven to have a toxic effect on the survival of juvenile C. sinensis . Therefore, it is important that the influx and efflux of bile acids should be balanced to prevent bile intoxication in the worm’s body. In humans, there are many importers and exporters of bile juice circulation , such as apical sodium-dependent bile acid transporter (ASBT), Na+ taurocholate co-transporting polypeptide (NTCP), Multidrug resistance protein (MRP), bile salts export pump (BSEP), and organic solute transporter (OST). Therefore, we believe that C. sinensis needs these bile transporters to reduce accumulation of bile acids within its body.
MRPs belong to a subfamily of the ATP binding cassette (ABC) transporter family [9, 10]. In higher animals, MRP4 is a unidirectional and distinctive bilaterally localized transporter in polarized cells, such as baso-lateral membrane of hepatocytes  and choroid plexus epithelial cells [12, 13]. Such tissue-specific distribution suggests that MRP4 has multiple functions. MRP4 is responsible for pumping out a broad range of substrates, including bile acids, as well as for physiological regulation via transport of cyclic nucleotides out of cells .
Therefore in this study, we identified and characterized C. sinensis MRP4 (CsMRP4), the first MRP4 in trematodes, at the in silico, molecular, and biochemical levels. The structure of CsMRP4 was built using homology modeling, and the structural features and bile acid-binding affinities were investigated. CsMRP4 was found to be localized mainly in the mesenchymal tissues and oral suckers of C. sinensis adults and metacercariae.
Parasites and animals
Pungtungia herzi (Jinju, Korea), the second intermediate host of C. sinensis, was ground and digested as described by Dai et al. . Metacercariae were then collected from the saline-rinsed digestive leavings under a dissecting microscope. Next, New Zealand white rabbits (2.3 kg; Koatech, Seoul, Korea) were infected with 200 metacercariae per rabbit twice in 1 week. Adult C. sinensis were then recovered from the rabbit livers after 2 months and stored in a -80 °C freezer until use. Female 7 week-old BALB/c mice (Orient Bio Inc., Gyeonggi-do, Korea) were immunized with a bacterially-produced recombinant protein.
A putative MRP4 polypeptide sequence (GenBank ID: GAA49862.1) of C. sinensis was retrieved from the National Center for Biotechnology Information (NCBI) database. The coding DNA sequence (CDS) was obtained from the C. sinensis DNA scaffold (GenBank ID: DF142991.1) to which it belonged. DNA-walking was performed twice for CsMRP4-I and CsMRP4-II due to the long size (approximately 4 kb) of the products. Two sets of PCR primers were designed according to CDS and synthesized (Bioneer, Daejeon, Korea) (Additional file 1: Table S1). Total cDNA of C. sinensis was prepared as described previously , and 50 ng per reaction was used as the template for DNA-walking. PCR amplification was performed under the following conditions: pre-denaturation (94 °C for 5 min), amplification phase with 35 cycles (94 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min 15 s), and final extension (72 °C for 10 min). The PCR products were then purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and sequenced (Macrogen Inc., Seoul, Korea). The CsMRP4-I and CsMRP4-II sequences were used for assembling the putative CDS and translated into amino acid (aa) sequences. However, CsMRP4 was assumed to be incomplete at the 5′-end upon comparison with MRP4 of other species. Therefore, 5′-rapid amplification of the cDNA ends (5′-RACE) was carried out to obtain the entire CDS. Total cDNA of C. sinensis was synthesized using the SMARTer™ RACE cDNA amplification kit (Clontech, Mountain View, CA, USA) according to the manufacturer’s instructions. The missing 5′-end of CsMRP4 was amplified by RACE-PCR run using the 5′-RACE universal primer mix (UPM) and gene specific reverse primer (GSP). The PCR product was then confirmed using nested PCR, purified, and subjected to TOPO TA cloning (Invitrogen, Carlsbad, CA, USA). Through blue-white screening, the positive white colony was selected and reconfirmed by rapid colony PCR. Its plasmid DNA was extracted using the Plasmid Miniprep kit (Qiagen, Seoul, Korea) and sequenced (Macrogen Inc., Seoul, Korea). The primers used for RACE-PCR, DNA-walking, and multiple sequencing are listed in Additional file 1: Table S1.
In silico methods for characterizing sequence features
For CsMRP4, the isoelectric point (pI) and molecular weight (Mr) was estimated using the ExPASy ProtParam Tool (http://web.expasy.org/protparam/). CsMRP4 was blasted against UniProtKB/Swiss-Prot v. 2017_07 . Domain organization and residue annotation were conducted using the Conserved Domain Database (CDD)  and InterProScan v. 64 .
In order to confirm that CsMRP4 belongs to a MRP subfamily and to infer its phylogenetic relationship with the ABCC and ABCB subfamilies, 12 canonical ABCC proteins and 11 canonical ABCB proteins were retrieved from UniProtKB/Swiss-Prot v. 2017_07 . Multiple sequence alignment was performed using the L-INS-i method of MAFFT v. 7.299 . An evolutionary history was inferred by employing the neighbor-joining (NJ) method using MEGA v. 6.06 . All the positions containing gaps and missing data were eliminated.
Homology modeling and refinement
The standard protocol of YASARA Structure v. 17.6.5  was used to build the homology models of CsMRP4. To obtain these models, PSI-BLAST  was carried out against PDB entries (updated August, 2017) . After building the homology models for each template, the models were submitted to high-resolution energy minimization using a YASARA force field . The result was then validated to ensure that the refinement did not move the model in the wrong direction. Finally, a hybrid homology model was obtained by combining the best scoring parts of the four models. In addition, potential errors in the 3D models were evaluated using a Ramachandran plot  and ERRAT .
Structure-based function analysis
Structural conservation was calculated and visualized using ENDscript/ESPript v. 3.0  with the PDBAA95 database, E-value of 1e-12, and contact range of 2.7 Å. COACH  was used to predict ligand-binding sites in CsMRP4. SDF files for bile acids were retrieved from the PubChem database  as of August 2017 and transformed into the MOL2 format using OpenBabel . Bile acids were docked into CsMRP4 using PyRx v. 0.8 , which includes AutoGrid  and AutoDock Vina . A grid box extended to all membrane-spanning domains (MSDs) of CsMRP4; no information regarding the exact location of the binding sites of the various bile acids was available. Active site dimensions were set as the grid size of center X: 25.6 Å, center Y: -13.5 Å, and center Z: -6.5 Å, and 8 maximum exhaustiveness was calculated for each bile acid. All structure visualizations were carried out using UCSF CHIMERA v. 1.10.2  and PyMOL Molecular Graphics System v. 126.96.36.199 (Schrödinger, LLC., New York, NY, USA).
Quantitative measurement of CsMRP4 developmental expression
To evaluate the mRNA expression level in different developmental stages, quantitative real time PCR (Q-rt.-PCR) was performed. Primers were designed using Oligo-primer analysis software v. 6.71 (Molecular Biology Insights, Cascade, WA, USA) (Additional file 1: Table S1). Calcyphosine (CAP) and phosphoglycerate kinase (PGK) were employed as the reference genes . Q-rt.-PCR reaction mixtures were prepared in triplicate using LightCycler FastStart DNA Master SYBR Green I Kit (Roche, Mannheim, Germany), with each reaction containing 50 ng of total cDNA of the adults or metacercariae. Q-rt.-PCR was performed on LightCycler 2.0 (Roche, Penzberg, Germany) with the following thermal cycle parameters: pre-heating (95 °C for 15 min), 40 cycles of 95 °C for 10 s, 48 °C for 10 s, and 72 °C for 30 s. The relative transcription ratio was calculated according to the 2-ΔΔCt method .
Production of recombinant protein
A cDNA fragment encoding CsMRP4-NBD1 was amplified using PCR (Additional file 1: Table S1). The purified PCR product was then subcloned into pET23b and confirmed by colony PCR and restriction enzyme digestion. Plasmid DNA of the positive clone was extracted using the QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany) and sequenced (Macrogen, Seoul, Korea). The correct construct was then transformed in Escherichia coli BL21[DE3]pLysS (Novagen, San Diego, CA, USA) by heat-shock at 42 °C for 30 s and spread on LB/ampicillin/chloramphenicol agar. After overnight incubation at 37 °C, a single colony was picked from the LB plate and grown in LB/ampicillin liquid medium by shaking vigorously at 37 °C. The recombinant(r) CsMRP4-NBD1 was then induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (TaKaRa, Shiga, Japan) for 5 h. The bacteria were then harvested, and recombinant protein was purified as described previously .
Production of mouse immune serum
The rCsMRP4-NBD1 was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and cut off alone to obtain specific antigens for mouse immunization. The gel slice was equilibrated and homogenized in pre-cooled 1× PBS by complete grinding. The liquid homogenate containing rCsMRP4-NBD1 was then injected into BALB/c mice according to an immunization method . Blood was drawn from the eye and stored at room temperature for 1 to 2 h. The immune serum was obtained by centrifugation at 4000× rpm for 20 min. In order to examine the antibody titer in the immune serum against rCsMRP4-NBD1 and native CsMRP4, C. sinensis crude extracts were prepared using the Mem-PER Plus Membrane Protein Extraction Kit (Thermo scientific, Rockford, USA) following the manufacturer’s instructions. The crude antigen was then examined to determine its concentration using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) and then stored as aliquots at -70 °C until use.
The reactivity of the antibody was checked by western blot against recombinant protein and immuno-enhanced chemiluminescence (ECL) against native CsMRP4 in crude extracts. The rCsMRP4-NBD1 and C. sinensis crude antigens were loaded onto SDS gels for electrophoresis and then transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Seoul, Korea). The membranes were then blocked using 5% skim milk in PBS/0.05% Tween20, followed by incubation with mouse immune serum at 1:400 at 4 °C overnight and then with goat-anti-mouse-IgG alkaline phosphatase-conjugated antibody (Sigma-Aldrich, St. Louis, MO, USA) at 1:5000 for western blotting or with peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA) at 1:10,000 for ECL at room temperature for 2 h. Normal mouse serum was used as the negative control. The recombinant protein was visualized by color developing in BCIP/NBT (Sigma-Aldrich, St. Louis, MO, USA). The native CsMRP4 was detected using a ECL solution kit (Bio Sesang, Seoul, Korea) and visualized using ImageQuant LAS 4000 (GE Healthcare Bio-Sciences, Amersham, UK).
Paraffin block preparation and immunohistochemical staining were performed using our previously described methods . Mouse anti-NBD1 immune serum diluted at 1:200 served as the primary antibody. Normal mouse serum was used as the negative control. Dako EnVision + System-horseradish peroxidase-labeled polymer anti-mouse IgG (Dako Cytomation, Glostrup, Denmark) diluted at 1:400 was used as the secondary antibody.
Results and discussion
Identification and molecular characteristics
The complete coding cDNA sequence (4410 nt) of CsMRP4 was obtained through DNA-walking and 5′-RACE on the C. sinensis total cDNA. Its open reading frame was 1469 aa in length (Additional file 2: Figure S1 and Additional file 3: Figure S2). The Mr. of CsMRP4 was about 165.5 kDa and its pI was estimated to be 6.5. BLASTP was performed against the UniProtKB/Swiss-Prot database , which is a high-quality, manually annotated, and non-redundant protein database. Annotation information from NCBI non-redundant and UniProtKB/TrEMBL databases need to be further reviewed, since those contents were generated using in silico annotation or large-scale functional prediction. CsMRP4 is the closest to human MRP4 (HsMRP4) (UniProt ID: O15439) with an E-value of 5.3e-147 and identity of 44.2%, followed by HsMRP6 (UniProt ID: O95255) of 8.8e-89 and 38.4%, Mus musculus MRP3 (MmMRP3) (UniProt ID: B2RX12) of 5.1e-104 and 38.2%, and MmMRP5 (UniProt ID: Q9R1X5) of 2.9e-115 and 38.0%. CsMRP4 was significantly matched to multiple MRPs since the highest-scoring pairwise alignment was found predominantly in the NBD2 region, which is particularly conserved among ABC family transporters . As a subfamily of the ABC family of transporters, the MRP subfamily contains 12 members including MRP1-9, cystic fibrosis transmembrane conductance regulator (CFTR), sulfonylurea receptor 1 (SUR1), and SUR2 [9, 10]. The highest identity (39.0%) was observed between CsMRP4 and HsMRP4 in comparison with canonical human MRP/SUR/CFTR subfamily (Additional file 4: Table S2). Thus, this clone was designated as CsMRP4.
MRP-specific functional domains
Functional domains in CsMRP4 revealed diverse characteristics of ABC transporters. There were “ABC transporter type 1, transmembrane domain” (InterProScan ID: IPR011527) in two regions, aa64–378 and aa831–1162. Two domains, MSD1 and MSD2, consisted of six transmembrane α-helices each. As intracellular NBDs that linked with the MSDs, “ABCC_MRP_domain1” (CDD ID: cd03250) for NBD1 and “ABCC_MRP_domain2” (CDD ID: cd03244) for NBD2 were found in the aa407–603 and aa1174–1394 regions, respectively. CsMRP4 had a single four-domain organization of MSD1-NBD1-MSD2-NBD2, which is common to all short forms of the ABCC subfamily, such as MRP4, 5, 8, 9, and CFTR .
Like in typical MRPs, there were several conserved motifs in the NBD1 of CsMRP4, such as an ATP-binding site (441GCxKSSx26Qx78DDx31N585), ABC transporter signature motif (528LSGGQKARIG537), Walker A/P-loop (438GPVGCGKS445), Walker B (548FLLLDD553), D-loop (556AAVD559), Q-loop/lid (470YMPQ473), and H-loop/switch region (581LLVTNQL587). These motifs play pivotal roles in transporting substrates via conformational changes between outward-facing and inward-facing forms. Dimerization of two NBDs forms the nucleotide-binding site between the Walker A/P-loop, Walker B, and ABC signature motif. The bound ATP is hydrolyzed to provide energy in order to efflux endogenous and xenobiotic substrates from cells to the extracellular milieu .
3D homology model of CsMRP4
The final model proved highly accurate based on the following validation. The overall Z-score of the resulting hybrid model was -1.4 using internal quality evaluation of YASARA Structure. A Z-score indicates the number of standard deviations the model quality is away from the average high-resolution X-ray structure. Moreover, a Ramachandran plot  of the final model showed that 90.7% of all the residues were found in the most favored regions, 8.4% in additional allowed regions, and only 0.3% in disallowed regions (Additional file 5: Figure S3). These results indicated that the backbone dihedral angles were highly accurate. The ERRAT value, as an overall quality score, was 98.4% (Additional file 6: Figure S4). The final model of the CsMRP4 in PDB format can be found in Additional file 7.
Docking simulation for bile acid binding
Ligand and ligand-binding residues predicted using COACH
Consensus binding residues
W412, T420, V440, G441, C442, G443, K444, S445, S446, Q473
Y1185, A1192, T1212, G1213, A1214, G1215, K1216, S1217, S1218, V1227, Q1258, E1338, H1369
S1217, Q1258, D1337, E1338, V1367
L94, P98, M101, S348, L864, I1093, V1097
Y349, L353, I1093, I1118, V1122
Docking results between CsMRP4 and bile acids using AutoDock Vina
Binding energy (kcal/mol)
No. of configurations
Taurolithocholic acid (TLCA)a
Lithocholic acid (LCA)
Taurochenodeoxycholic acid (TCDCA)a
Chenodeoxycholic acid (CDCA)
Taurodeoxycholic acid (TDCA)a
Taurocholic acid (TCA)a
Glycocholic acid (GCA)a
Deoxycholic acid (DCA)
Cholic acid (CA)a
Developmental expression and tissue distribution
CsMRP4 was mainly localized in the mesenchymal tissues in our study. In trematodes, several transporters have been found to be expressed in mesenchymal tissues. BSEP and MRP1 of adult Fasciola hepatica have been shown to be localized in not only mesenchymal tissues but also the tegumental cell layer, implying that the two transporters facilitate the diffusion of bile salts and chemicals in flukes . CsMRP7, which might be involved in drug resistance, has also been found to be expressed in the mesenchymal tissues . It is therefore speculated that mesenchymal tissues may be regions of strategic importance for transporter functions throughout the body of flukes.
The function of CsMRP4 seems to be to export bile acids from the worm’s body, which is similar to the function of typical MRP4. C. sinensis adults and metacercariae immerse themselves in bile juice, which can exert toxic effects and impair the tissues and cells of the worm’ bodies. Bile acids have also been reported to decrease the locomotive cycles of juvenile F. hepatica and to provoke parasite death . Together, these data suggest that C. sinensis needs to dilute the high concentrations of bile acids in the interior of the body by pumping them out and that CsMRP4 plays a role in transporting bile acids in coordination with other bile acid exporters.
In summary, we cloned and characterized CsMRP4 using computational, molecular, and biochemical approaches in this study. In addition to structural similarities, sequence similarities were also found between CsMRP4 and human MRP4 (39% identity), and CsMRP4 was confirmed as belonging to the ABCC family. A reliable tertiary structure of CsMRP4 was also modeled and shown to have a common structural fold, MSD1-NBD1-MSD2-NBD2. When binding affinities of CsMRP4 with nine bile acids were tested through virtual docking simulation, the results indicated that CsMRP4 could be regarded as a bile transporter. The NBD2 of CsMRP4 was conserved more than NBD1, which was therefore used as a CsMRP4-specific antigen for subsequent immunohistochemistry experiments. In the metacercariae and adults of C. sinensis, CsMRP4 was found to be mainly distributed in mesenchymal tissues, which suggested that these tissues are regions of strategic importance for transporter functions throughout the fluke’s body. These findings suggest that CsMRP4 plays a role in exporting bile acids and inhibitors. The results from this study will also serve as a platform for further research on other bile transporters and homologues in flukes.
Ok-Kyoung Lim, faculty from the Department of Pathology, Chung-Ang University, contributed the immunohistochemical samples of C. sinensis worms.
This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (grant no. 2014R1A2A1A11051870).
Availability of data and materials
The data supporting the conclusions of this article are included within the article and its additional files.
SJH and WGY conceived and designed this project. JYL, FD, YL and WMS performed experiments. WGY and JHP analyzed data. WGY contributed to utilizing the analysis tools. FD, WGY and SJH wrote the paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was carried out in strict accordance with the national guidelines outlined by Korean Laboratory Animal Act (No. KCDC-122–14–2A) of the Korean Centers for Disease Control and Prevention (KCDC). Animal handling and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Chung-Ang University (Approval Number 2015–00005).
Consent for publication
The authors declare that they have no competing interests.
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