Cloning/characterization of the canine organic anion transporting polypeptide 1b4 (Oatp1b4) and classification of the canine OATP/SLCO members

Abstract

The human liver-specific organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are involved in the elimination of numerous xenobiotics and drugs. Although dogs are frequently used for toxicologic and pharmacokinetic characterization of novel drugs, nothing is known about their OATP1B1/1B3 ortholog. Therefore, we cloned and characterized the first canine organic anion transporting polypeptide from dog liver, termed Oatp1b4. The isolated Oatp1b4 cDNA comprises 3661 base pairs (bp) with an open reading frame of 2076bp, encoding a 692-amino acid protein with a molecular mass of approximately 85kDa. The Oatp1b4 gene is approximately 61kb long and has a similar organization as the human OATP1B1 and OATP1B3 with 13 exons identical in length. Northern blot analysis shows that Oatp1b4 is predominantly expressed in the liver. Oatp1b4 mediates sodium-independent transport of typical organic anions including bromosulfophthalein (BSP), [D-penicillamine(2,5)]enkephalin (DPDPE), estradiol-17beta-glucuronide (E17betaG), estrone-3-sulfate and taurocholate. In addition, Oatp1b4 transports the OATP1B3-specific substrate cholecystokinin octapeptide (CCK-8). Kinetic studies showed that Oatp1b4-mediated E17betaG and estrone-3-sulfate transports were monophasic with K(m) values of 5+/-1microM and 33+/-4microM, respectively. In conclusion, the cloned canine Oatp1b4 will provide additional molecular basis to further characterize the species difference of the OATP1B family members.

Fig. 1

(A) Phylogenetic analysis of OATP1B family members. Phylogenetic analysis was performed with the PHYLIP software package. (B) Amino acid sequence alignment of the canine Oatp1b4, human OATP1B1 and OATP1B3. The open reading frame of canine Oatp1b4 is 2076-bp long, encoding a 692-amino acid polypeptide. Conserved amino acids among these three OATPs are marked with asterisks on the top of them. The twelve putative transmembrane domains of Oatp1b4 predicted by TMPred (http://www.ch.embnet.org/software/TMPRED_form.html) are underlined. Potential N-linked glycosylation sites predicted by NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/) are marked with triangles. The consensus phosphorylation sites for PKC and PKA predicted by GPS 2.0 (Xue et al., 2008) are marked with diamonds.

Fig. 2

Tissue distribution of dog Oatp1b4 by Northern blot analysis. A commercially available dog 15-lane multiple-tissue Northern blot containing 20 μg of total RNA per lane was hybridized with [α-³²P]dCTP-labeled Oatp1b4 open reading frame as probe. The hybridization and wash conditions were described in MATERIALS AND METHODS.

Fig. 3

Immunoblot Analysis of Recombinant and Endogenous Oatp1b4. (A) Immunoblot analysis of recombinant Oatp1b4. The open reading frame of dog Oatp1b4 was cloned into the pcDNA5/FRT expression vector and was transiently transfected into HEK293 cells. HEK293 cells transfected with empty pcDNA5/FRT vector was served as negative control. Plasma membrane proteins were isolated by surface biotinylation and separated by SDS-polyacrylamide gel electrophoresis. Oatp1b4 was detected with K23 antibody which was raised against the C-terminus of human OATP1B1. (B) Immunoblot analysis of endogenous Oatp1b4 from dog liver. Dog liver membrane proteins were isolated as described in MATERIALS AND METHODS and seperated by SDS-polyacrylamide gel electrophoresis (15 μg of total membrane protein loaded). Oatp1b4 was detected with the K23 antibody.

Fig. 4

Oatp1b4-mediated uptake for (A) E17βG, (B) estrone-3-sulfate (E3S) and (C) taurocholate (TCA) with sodium-containing and sodium-free uptake buffers. Uptake of trace amount of substrates was measured at 37 °C for 1 min with empty pcDNA5/FRT vector and Oatp1b4-transfected HEK293 cells. Uptake results were normalized to total protein concentrations determined with the BCA assay.

Fig. 5

Time courses of uptake of (A) 0.1 μM and (B) 100 μM E17βG and (C) 0.1 μM and (D) 100 μM estrone-3-sulfate in empty pcDNA5/FRT vector-transfected (○) and Oatp1b4-transfected (●) HEK293 cells. Uptake was measured at 37 °C with sodium-containing buffer and was normalized to total protein concentration.

Fig. 6

Concentration-dependent uptake of (A) E17βG and (B) estrone-3-sulfate by dog Oatp1b4. Uptake was measure at various concentrations from 0.1 to 100 μM for E17βG and from 0.02 to 500 μM for estrone-3-sulfate. Uptake was conducted at 37 °C for 1 min with empty vector and Oatp1b4-expressing HEK293 cells. The net uptake was obtained by subtracting the uptake of cells transfected with empty vector from the uptake of Oatp1b4-expressing cells. The K_m values were obtained by fitting the uptake data to the Michaelis-Menten equation.