Abstract
Multidrug resistance-associated protein 7 (MRP7, ABCC10) is a C subfamily member of the ATP-binding cassette (ABC) superfamily. MRP7 is a lipophilic anion transporter that pumps endogenous and xenobiotic substrates from the cytoplasm to the extracellular milieu. Here, we cloned and characterized CsMRP7 as a novel ABC transporter from the Chinese liver fluke, Clonorchis sinensis. Full-length cDNA of CsMRP7 was 5174 nt, encoded 1636 amino acids (aa), and harbored a 147-bp 5′-untranslated region (5′-UTR) and 116-bp 3′-UTR. Phylogenetic analysis confirmed that CsMRP7 was closer to the ABCC subfamily than the ABCB subfamily. Tertiary structures of the N-terminal region (1–322 aa) and core region (323–1621 aa) of CsMRP7 were generated by homology modeling using glucagon receptor (PDB ID: 5ee7_A) and P-glycoprotein (PDB ID: 4f4c_A) as templates, respectively. CsMRP7 nucleotide-binding domain 2 (NBD2) was conserved more than NBD1, which was the sites of ATP binding and hydrolysis. Like typical long MRPs, CsMRP7 has an additional membrane-spanning domain 0 (MSD0) and cytoplasmic loop, along with a common structural fold consisting of MSD1-NBD1-MSD2-NBD2 as a single polypeptide assembly. MSD0, MSD1, and MSD2 consisted of TM1-7, TM8-13, and TM14-19, respectively. The CsMRP7 transcript was more abundant in the metacercariae than in the adult worms. Truncated NBD1 (39 kDa) and NBD2 (44 kDa) were produced in bacteria and mouse immune sera were raised. CsMRP7 was localized in the apical side of the intestinal epithelium, sperm in the testes and seminal receptacle, receptacle membrane, and mesenchymal tissue around intestine in the adult worm. These results provide molecular information and insights into structural and functional characteristics of CsMRP7 and homologs of flukes.
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Acknowledgements
This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (grant no. 2014R1A2A1A11051870). Ok-Kyoung Lim, faculty of the Department of Pathology, Chung-Ang University, contributed with immunohistochemical staining of C. sinensis adult worms.
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Fuhong Dai and Won Gi Yoo contributed equally to this study.
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Table S1
Primer sets used to amplify CsMRP7 cDNA fragments by PCR (PDF 70 kb)
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Table S2
Top 10 homologues of the N-terminal region of CsMRP7 (PDF 73 kb)
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Table S3
Top 10 homologues of the core region of CsMRP7 (PDF 72 kb)
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Fig. S1
Schematic diagram of strategy to clone full-length cDNA of CsMRP7 gene. Colored bars represent PCR fragments mentioned in Table 1. Arrows indicate forward and reverse primers. Gray is a putative CsMRP7 (GenBank Acc. ID: GAA30468). P1 fragment is an EST clone CSA23232. P4 fragment is an amplicon of 5′-RACE corresponding to closed star (−147 to 549 bp). Open star (2974–3126 bp) indicates the insertion sequence which is absent from MRP7 sequence. Brackets represent sequences overlapped with two amplicon fragments. (PDF 79 kb)
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Fig. S2
Evolutionary conservation on 3D structure of NBD1 (A) and NBD2 (B). Amino acid with highest conservation (score = 9) is presented as a maroon sphere. (PDF 591 kb)
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Fig. S3
Multiple alignments of NBD1 (A) and NBD2 (B) in CsMRP7 with those of parasites and mammals. α, α-helix; β, β-sheet. Green background, Walker A/P-loop; yellow, Q-loop/lid; light green, ABC transporter signature motif; orange, Walker B; maroon, D-loop; cyan, H-loop/switch region; red triangle, ATP-binding site. Red bold and red letters are identical and similar amino acid residues, respectively. GenBank acc. ID are as follows: AAA66477.1 of S. mansoni, KHN88859.1 of Toxocara canis, ERG81795.1 of Ascaris suum, XP_001892748.1 of Brugia malayi, AAD49436.1 of Onchocerca volvulus, EUB63724.1 of Echinococcus granulosus, ACI24156.1 of Toxoplasma gondii, AAP79578.1 of Leishmania major, CAC83020.1 of Trypanosoma brucei, AIJ50465.1 of Plasmodium falciparum, NP_502413.1 of Caenorhabditis elegans, NP_035206.2 of M. musculus, NP_001185863.1 of Homo sapiens. (PDF 221 kb)
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Fig. S4
Comparison of putative transmembrane topologies. MSDs were predicted using homology modeling and two topology predictors and visualized using IBS ver. 1.0. (A) MSD0, colored in pink, was predicted using MODELER, and MSD1 and MSD2 in green, were identified using SWISS-MODEL. MSDs of (B) and (C) were predicted using TMHMM and TOPCONS, respectively. A circled number indicates the order of TM helices and range of the helix was shown as amino acid numbers. Up- and down- arrows indicate extracellular or cytoplasmic side of the terminus. (PDF 164 kb)
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Fig. S5
Purification of recombinant truncated NBD1 and NBD2 proteins of CsMRP7. Abbreviations are: M, molecular marker (kDa); U, uninduced total lysate; T, induced total lysate; S, urea-treated clear supernatant; P, pellet; W, last washing; Elute1–8, 1st to 8th fraction eluted from a Ni-NTA column. Arrow indicates a target protein. (PDF 272 kb)
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Dai, F., Yoo, W.G., Lee, JY. et al. Molecular and structural characteristics of multidrug resistance-associated protein 7 in Chinese liver fluke Clonorchis sinensis . Parasitol Res 116, 953–962 (2017). https://doi.org/10.1007/s00436-016-5371-0
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DOI: https://doi.org/10.1007/s00436-016-5371-0