Page 17 - U. Protein structure and function

Apical sodium-dependent bile acid transporter of Clonorchis sinensis acts as steroid
hormone transporter
Haneul Jung and Seok Ho Cha
Dept. of Parasitology and Tropical Medicine, Inha University, Incheon, Korea

Abstract

The main habitat of Clonorchis sinensis (Cs) is bile duct. Because of bile acid toxicity, their living environment may be extreme condition. Thus, to maintain their life they have to
control the bile acid with influx and efflux system. We, therefore, try to isolate and characterize the apical sodium-dependent bile acid transporter of Cs.
From NCBI database, apical sodium-dependent bile acid transporter (ARO50445.1, CsASBAT) was selected. The selected gene was isolated using from cDNA library screening. The
CsASBAT cDNA consists of 2031 base pairs which encoded a 546-amino acid residue protein. Hydropathy analysis showed 10 putative transmembrane-spanning domains with 4 N-
glycosylation and 3 protein kinase C motifs. When expressed in Xenopus oocytes, radiolabeled estrone sulfate in a time- and sodium-dependent manner. The CsASBAT showed no
exchange mode of estrone sulfate by efflux experiments. Concentration-dependency results showed saturable kinetics following Michaelis-Menten equation. Nonlinear regression
3
analyses yielded a Km value of 0.3 ± 0.03 µM for [ H]estrone sulfate. These results suggest that this transporter may acts as eliminatory system of freely entered estrogen by
forming sulfate conjugate with estrogen sulfotransferase.
AIM METHODS

★ cDNA was prepared from adult Clonorchis sinensis cDNA library screening.
To clarify the molecular properties of bile acid transporter of Clonorchis
sinensis. ★ The probe cDNA was prepared from adult Clonorchis sinensis total RNA using
reverse transcription-polymerase chain reaction.
★ cRNA was synthesized using mMESSAGE mMACHINE T7 kit (Invitrogen).
★ Substrate uptake was determined by Xenopus laevis oocyte expression system.
★ Synthesized cRNA (50 ng/oocyte) was injected by manually using micromanupilator.

RESULTS

Figure 1. Amino acid sequjence of ASBT and dendrogram of the Figure 2. The transport properties of CsASBT for [ 3 H]ES. A. The Figure 3. ASBT substrate specificity. The uptake rates of
related transporters. A. the amino acid sequence of ASBT. Bold uptake of 50 nM [ 3 H]ES in control oocyte or CsASBT expressing radiolabeled compounds (50 mM [ 14 C]arginine, 50 nM [ 3 H]ES,
numeric number and bar showed mabrane spanning domains by oocytes was measured at indicated CsASBT expression time. B. 5 µM [ 14 C]α-ketoglutarate, 10 µM [ 14 C]p-aminohippurate, 200 nM
TMpred analysis. *: N-linked glycosylation site, #: protein kinase C CsASBT-mediated [ 3 H]ES uptake was measured after various [ 3 H]taurocholic acid, and 10 µM [ 14 C]tetraethylammonium were
site. incubation time. C. effect of extracellular cation on [ 3 H]ES uptake measured in water injected control oocytes and ASBT expressing
in Xenopus laevis oocytes expressing CsASBT. D. effect of unlabled oocytes for 1 h (mean ± S.E.; n = 8-10).
ES on efflux of [ 3 H]ES CsASBT.

Figure 4. Concentration dependence of the CsASBT-mediated Figure 5. Evidence of Cs estrogen sulfotransferase. Total RNA was CONCLUSION
uptake of [ 3 H]ES. The uptake rates of [ 3 H]ES by control or CsASBT- isolated from adult Clonorchis sinensis. CsSLUT was confirmed by
expressing oocytes for 1 h were measured at variable RT-PCR.
concentrations (mean ± S.E; n = 8-10). CsASBT-mediated transport
was calculated by subtracting control oocyte transport velocity from We could determine that CsASBT gene reported as
those of CsASBT-expressing oocytes. Inset, Eadie-Hofstee plot. V,
velocity; v/s, velocity per concentration of ES. sodium dependent bile acid transporter act as a
sodium dependent estrone sulfate transporter and its
role is exclusion of freely entering estrogen by
estrogen sulfotransferase.his work may contribute
to understand the relationship between liver
estrogen metabolism and effect of estrogen
metabolites to liver fluke.

Contact information

shcha@inha.ac.kr