Characterization of a novel organic solute transporter homologue from Clonorchis sinensis - PubMed (original) (raw)

Characterization of a novel organic solute transporter homologue from Clonorchis sinensis

Yanyan Lu et al. PLoS Negl Trop Dis. 2018.

Abstract

Clonorchis sinensis is a liver fluke that can dwell in the bile ducts of mammals. Bile acid transporters function to maintain the homeostasis of bile acids in C. sinensis, as they induce physiological changes or have harmful effects on C. sinensis survival. The organic solute transporter (OST) transports mainly bile acid and belongs to the SLC51 subfamily of solute carrier transporters. OST plays a critical role in the recirculation of bile acids in higher animals. In this study, we cloned full-length cDNA of the 480-amino acid OST from C. sinensis (CsOST). Genomic analysis revealed 11 exons and nine introns. The CsOST protein had a 'Solute_trans_a' domain with 67% homology to Schistosoma japonicum OST. For further analysis, the CsOST protein sequence was split into the ordered domain (CsOST-N) at the N-terminus and disordered domain (CsOST-C) at the C-terminus. The tertiary structure of each domain was built using a threading-based method and determined by manual comparison. In a phylogenetic tree, the CsOST-N domain belonged to the OSTα and CsOST-C to the OSTβ clade. These two domains were more highly conserved with the OST α- and β-subunits at the structure level than at sequence level. These findings suggested that CsOST comprised the OST α- and β-subunits. CsOST was localized in the oral and ventral suckers and in the mesenchymal tissues abundant around the intestine, vitelline glands, uterus, and testes. This study provides fundamental data for the further understanding of homologues in other flukes.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Schematic representation of the genomic structure of CsOST.

5′ UTR (143 bp) located at exon I, exon II, and a part of exon III (100 bp), while 3′-UTR was located in the posterior region including exon XI (117 bp). The poly(A)-tail was 22 bp in length. The lengths of intron (shown as a solid line) and exon (shown as a colored box) are proportional to each size.

Fig 2. Sequence conservation of CsOST-N and other OST α-subunits.

CsOST-N is an N-terminal domain (Met1–Arg354) of CsOST. Residues that are 75% conserved among the alignment are shaded in light blue and those that are 100% conserved are highlighted in dark blue. The five consecutive cysteine residues are indicated with red arrowheads. The solid red boxes locate Arg-X-Arg (RXR) motif, and the dotted red boxes show Arg-Arg-Lysine (RRK) and Arg-Lys-Lys (RKK). Parentheses show pairwise sequence identity between CsOST-N and other OSTα subunits. Abbreviations are: CsOST-N, N-terminal regions of CsOST; HsOSTα, Homo sapiens OSTα; MmOSTα, Mus musculus OSTα; BtOSTα, Bos taurus OSTα; LeOSTα, Leucoraja erinacea OSTα; DrOSTα, Drosophila melanogaster OSTα; XtOSTα, and Xenopus tropicalis OSTα.

Fig 3. Comparison of the amino acid sequence of CsOST-C with OST β-subunits.

CsOST-C is a C-terminal domain (Arg355–Leu480) of the CsOST. Conservation is the same as depicted in Fig 2. The di-leucine (LL) residues are labeled with red and blue triangles. The solid boxes in red indicate an Arg-X-Arg (RXR) motif, such as RIR, RNR, and RTR. Parentheses show pairwise sequence identities between CsOST-C and other OSTβ subunits. Abbreviations are: CsOST-C, C-terminal regions of CsOST; HsOSTβ, H. sapiens OSTβ; MmOSTβ, M. musculus OSTβ; LeOSTβ, L. erinacea OSTβ; BtOSTβ, and B. taurus OSTβ.

Fig 4. Phylogenetic tree of CsOST-N and -C domains with canonical OSTα and β subunits.

Bootstrap values (1000 replicates) are shown next to the branches. UniProtKB/Swiss-Prot IDs [27] are shown in parentheses. CsOST-N and -C were indicated with closed and open arrowhead, respectively. Abbreviations are defined as in the legends of Figs 2 and 3.

Fig 5. Structural comparison of CsOST-N with OSTα subunits.

The 3D models of HsOSTα (red), MmOSTα (blue), and CsOST-N (green) are superimposed. Pairwise comparisons were performed with HsOSTα and MmOSTα (A), HsOSTα and CsOST-N (B), MmOSTα and CsOST-N (C). TM-score between the two superposed structures was calculated using TM-align [39]. Abbreviations are provided in the legend to Fig 2.

Fig 6. Structural comparison of the CsOST-C with OST β-subunits.

The 3D models of HsOSTβ (red), MmOSTβ (blue) and CsOST-N (green) were superposed on each other. Pairwise comparisons were performed with as follows: HsOSTβ and MmOSTβ (A), HsOSTβ and CsOST-C (B) and MmOSTβ and CsOST-C (C). TM-score between two superposed structures was calculated using TM-align [39]. Abbreviations are provided in the legend to Fig 3.

Fig 7. Relative mRNA level of CsOST and reactivity of mouse anti-CsOST-C immune serum.

(A) CsOST was expressed 2-fold more in the metacercaria than in the adult. (B) Mouse immune serum reacted specifically with native CsOST in the cytosolic and membrane fractions of C. sinensis. Abbreviations are: M, protein molecular marker; I, immune mouse serum; and N, normal mouse serum.

Fig 8. Distribution of CsOST in C. sinensis adults.

Panels A, C, E, G, and I display samples treated with mouse anti-CsOST serum at 1:100 dilution. Panels B, D, F, H, and J display samples incubated with normal mouse serum. Abbreviations are: I, intestine; MT, mesenchymal tissue; OS, oral sucker; T, testis; U, uterus; and VG, vitelline gland.

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Characterization of a novel organic solute transporter homologue from Clonorchis sinensis - PubMed (original) (raw)