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Amino-terminal region of human organic anion transporting polypeptide 1B1 dictates transporter stability and substrate interaction
Toxicology and Applied Pharmacology 378 (2019) 114642
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Amino-terminal region of human organic anion transporting polypeptide 1B1 dictates transporter stability and substrate interaction Xuyang Wanga,1, Jie Chena,1, Shaopeng Xua,b, Chunxu Nia, Zihui Fanga, Mei Honga,b, a b
T
⁎
College of Life Sciences, South China Agricultural University, Guangzhou, China Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, South China Agricultural University, Guangzhou, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Amino-terminus Organic anion transporting polypeptides Protein stability Transmembrane domains Uptake function
Organic anion transporting polypeptides (OATPs) are key players of drug absorption, distribution and excretion due to their broad substrate specificity, wide tissue distribution and the involvement in drug-drug interaction. OATP1B1 is specifically localized at the basolateral membrane of human hepatocytes and serves a crucial role in the drug clearance from the body. Previous studies have shown that transmembrane domains (TMs) are essential for proper functions of OATPs. In the present study, site-directed mutagenesis was performed to study the TM1 and amino-terminus of OATP1B1. Two positively charged residues, K41 and K49, as well as a hydrophobic residue I46, in TM1 were identified to be important for the proper function of the transporter. K41A and K49A exhibited altered Km value at the high and low affinity binding sites of estrone-3- sulfate (ES), respectively; while alanine substitution of I46 showed altered Km and Vmax values for both binding components of ES. Additional replacement of K41 revealed that the positively charged property at this position is important for maintaining OATP1B1 protein level and function; while the specific side-group structure of lysine at position 49 is irreplaceable for the transporter activity. Conservative replacement of I46 with leucine also recovered the function of the transporter. In addition, studies of the amino-terminus of OATP1B1 revealed that residues ranging from 19 to 27 are essential for protein stability and substrate interaction. Therefore, the amino-terminal region, which includes TM1 and the amino-terminus of OATP1B1, is important for proper function of the membrane protein.
1. Introduction Organic anion transporting polypeptides (OATPs, gene symbol SLCO) are solute carrier family members and mediate sodium-independent transport of a wide range of compounds (Hagenbuch and Gui, 2008). Substrates of OATPs include bile salts, hormones and their conjugates, toxins and a variety of drugs. In addition to charged compounds, OATPs also transport uncharged drugs such as glycosides digoxin (Noé et al., 1997) and ouabain (Bossuyt et al., 1996). So far there are 12 members of the human OATP family cloned, including OATP1A2, 1B1, 1B3, 1B7, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1 and 6A1 (Hagenbuch and Meier, 2003; Nakanishi and Tamai, 2012), though SLCO1B7 was proposed to be a pseudogene because OATP1B7 is considered as non-functional (Stieger and Hagenbuch, 2014). OATP family members have wide tissue distributions- some are expressed
ubiquitously; while others are predominantly found in certain organs or tissues. In recent years, OATPs have been extensively recognized as the key determinants of drug absorption, distribution and excretion and found to be involved in various kinds of drug-drug interaction (DDI) (Shitara et al., 2005; Poirier et al., 2007). Although many clinically important drugs have been identified as substrates of OATPs, the underlying mechanisms of substrate binding and/or recognition remain largely unclear due to the lacking of high resolution crystal structures of mammalian drug transporters (Miyagawa et al., 2009). OATP1B1 is the major OATP located at the basolateral membrane of human hepatocytes and plays a crucial role in the drug clearance from the body (Noé et al., 2007; Shitara et al., 2013). Quite a few studies have demonstrated that transmembrane domains (TMs) are essential for the proper function of OATP1B1. Transmembrane domains 8 and 9 were found to be critical for substrate recognition of the transporter
Abbreviations: BSP, sulphobromophthalein; E2G, estradiol-17β-glucuronide; ES, estrone-3-sulfate; NHS-SS-biotin, sulfosuccinimidyl 2-(biotinamido)-ethyl-1, 3-dithiopropionate; OATP, organic anion transporting polypeptide; PBS, phosphate-buffered saline; TM, transmembrane domain. ⁎ Corresponding author at: College of Life Sciences, South China Agricultural University, Guangzhou, China. E-mail address: [email protected] (M. Hong). 1 These two authors contributed equally to the work. https://doi.org/10.1016/j.taap.2019.114642 Received 17 April 2019; Received in revised form 20 June 2019; Accepted 25 June 2019 Available online 27 June 2019 0041-008X/ © 2019 Elsevier Inc. All rights reserved.
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2.4. Uptake assay
(Miyagawa et al., 2009). Several amino acid residues along TM10 are important for substrate translocation and maintenance of the proper protein structure of OATP1B1 (Gui and Hagenbuch, 2009). Previous studies of OATP1B1 in our laboratory found out that four amino acids within TM2, i.e. D70, F73, E74, and G76 are crucial for the uptake of estrone-3-sulfate (ES) (Li et al., 2012). Two highly conserved tryptophan residues (W258 and W259) that are close to the extracellular border of TM6 were also identified to be essential for the uptake function of the transporter (Huang et al., 2013). In a more recent study, six critical amino acid residues in addition to R580, which have been reported by others to be essential for proper function of OATP1B1 (Weaver and Hagenbuch, 2010), were identified within the TM11 of the transporter. The identified residues are important for substrate interaction, stability and/or correct targeting of OATP1B1 (Hong et al., 2015). Computer-generated model of OATP1B3, an OATP family member that is highly homologous to OATP1B1, proposed that several TMs of the N-terminal half, including TM1, 2, 4 and 5, may face the substrate interaction pore (Meier-Abt et al., 2005). Previous study of OATP1B3 identified conserved positively charged residues K41 and R580, which located at transmembrane domain 1 and 11, respectively, to be critical for substrate uptake of the transporter (Glaeser et al., 2010). In addition, the N-terminus region of OATP1B3 was demonstrated to be important for membrane localization of the transporter. It was found that amino acid residues 12–28 may be indispensable for membrane trafficking of the transporter. However, individual residues or structural motifs that is responsible for such a process is unclear (Chun et al., 2017). In the present study, we performed alanine-scanning to study the TM1 of OATP1B1. It was found that two positively charged amino acid residues, K41 and K49, as well as the hydrophobic I46, are important for the uptake function of the transporter. Further studies revealed that these residues may be involved in substrate interaction. In addition, the N-terminus of OATP1B1 was investigated and a region ranges from residues 19–27 was identified to be essential for stability and substrate interaction of the transporter protein.
Cells in 48-well plate were used for transport function analysis as described before (Li et al., 2012). Briefly, cells were incubated with uptake solution containing [3H]ES or [3H] taurocholic acid at 37 °C for 2 min (1 min for kinetic analysis) and uptake was stopped by the icecold phosphate-buffered saline (PBS) solution. Cells were then washed with cold PBS, solubilized in 0.2 N NaOH, neutralized with 0.2 N HCl and the radioactivity of the cell lysate was measured with a liquid scintillation counter (Triathler-Hidex, Hidex, Turku, Finland). The uptake count was standardized by the amount of protein in each well. 2.5. Cell surface biotinylation and western blotting Cell surface protein level of OATP1B1 and the mutants was examined using the membrane-impermeable biotinylation reagent Sulfosuccinimidyl 2-(biotinamido)-ethyl-1, 3-dithiopropionate (NHSSS-biotin) as described before (Li et al., 2012). In brief, HEK293 cells expressing OATP1B1 or mutants were labeled with NHS-SS-biotin for two consecutive 20-min incubations. Cells were then dissolved with RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% NP-40, protease inhibitors phenylmethylsulfonyl fluoride, 200 μg/ml, leupeptin, 3 μg/ ml, pH 7.4), and the biotin-labeled proteins were precipitated with streptavidin-agarose beads. The streptavidin-agarose beads bound proteins were then released in 4× Laemmli buffer at 55 °C and loaded onto a 7.5% SDS-polyacrylamide electrophoresis gel, transferred electrophoretically to a polyvinylidene difluoride membrane (Millipore, Billerica, MA) and detected with anti-HA antibody (1:1000 dilution, Beyotime Biotechnology, Inc., Jiangsu, China). 2.6. Homology modeling Escherichia coli glycerol-3-phosphate transporter (PDB: 1pw4) was used as the template for homology modeling of OATP1B1. The structure of OATP1B1 was modeled with the web-based protein structure prediction service Swiss-model (https://www.swissmodel.expasy.org/).
2. Materials and methods
2.7. Statistical analysis
2.1. Materials
Student's t-test was utilized for comparisons between two sets of data. One-way analysis of variance (ANOVA) with Bonferroni's post hoc test was carried out in cases where there are more than two groups were compared. Differences between means are regarded as significant if p < 0.05.
[3H]Estrone-3-sulfate (ES) and [3H]taurocholic acid were purchased from PerkinElmer Life Sciences (Waltham, MA). Sulfosuccinimidyl 2(biotinamido) -ethyl-1, 3-dithiopropionate (NHS-SS-biotin) and streptavidin-agarose beads were from Thermo Fisher Scientific (Waltham, MA). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO) except otherwise stated.
3. Results 3.1. Alanine-scanning of OATP1B1 transmembrane domain 1
2.2. Site-directed mutagenesis
To identify the critical amino acid residues within TM1 of OATP1B1, we first individually substituted the residues along the predicted TM1 with alanine. Amino acid residues in TM1 were chosen according to the Kyte-Doolittle hydrophobicity scale (Fig. 1). Hence 18 out of the 22 residues from position 28 to 49 of OATP1B1 were mutated to alanine, and four alanine residues (A32, A33, A40, and A45) located in TM1 were replaced with valine. Uptake function of estrone-3-sulfate (ES), an OATP prototypic substrate found to be transported by all OATPs (König, 2011; Yamaguchi et al., 2010), was measured. As shown in Fig. 2, though quite a few alanine-substituted mutants exhibited significant change in uptake function, only mutation of the positively charged K41 and K49, and the hydrophobic I46, resulted in > 50% decrease of the ES transport function. In addition, we performed transport assay with another prototypical substrate taurocholate, which was found to be transported by major OATPs including OATP1A2, 1B1, 1B3 (König, 2011) and 2B1 (Fang et al., 2018). It was found that K41A and K49A as well as I46A also
Mutants were generated with QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA) according to the manufacturer's instructions. The pReceiver M07 vector containing SLCO1B1 cDNA and 3-HA tags at the C-terminus was purchased from Genecopoeia (Rockville, MD) and used as the mutagenesis template. All mutant sequences were confirmed by full length sequencing (Thermo Fisher Scientific). 2.3. Cell culture and transfection of plasmid constructs into cells HEK293 cells were grown in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum at 37 °C and 5% CO2. Confluent cells in 48-well or 12-well plate were transfected with DNA plasmid using LipofectAMINE 2000 reagent (Thermo Fisher Scientific). Transfected cells were incubated for 48 h at 37 °C and then used for transport assay or cell surface biotinylation. 2
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Fig. 1. Putative transmembrane domain 1 of human organic anion transporting polypeptide family members. Multiple sequence alignment of 11 OATP family members was performed with ClustalW. Location of the putative TM1 was predicted with Kyte-Doolittle hydrophobicity scale. The corresponding sequences of TM1 were in bold. Residues from position 19 to 27 at the amino-terminus of OATP1B1 were underlined. Only partial sequences were shown here.
3.3. Kinetic analysis of mutants K41A, K49A and I46A
showed significantly decreased taurocholate uptake. In particular, taurocholate uptake by K41A was reduced to < 15% of that by the wild-type OATP1B1 (Fig. 3). We therefore further investigated these three residues in the following studies.
Next we wanted to investigate whether alanine replacement of these three residues affects the interaction of OATP1B1 with its substrates. Two binding sites for ES were reported in OATP1B1 (Noé et al., 2007; Gui and Hagenbuch, 2009; Li et al., 2012). Therefore, ES uptake function was examined at concentrations ranged from 0.01 to 20 μM. Since mutation of both K41 and K49 partially affected protein level of the transporter, we normalized the uptake with cell surface level of the mutants relative to wild-type to better represent their effect on the interaction of OATP1B1 and ES. As shown in Table 1, all mutants retained a biphasic kinetics for ES uptake. K41A exhibited a significant reduction of both Km and Vmax values compared to wild-type OATP1B1 in the high binding affinity site. The Km value for low affinity binding site of ES was significantly increased for K49A; while the Vmax was decreased in both high and low affinity components of the mutant. I46A showed significant change in Km for both binding components and Vmax of the mutant was significantly altered as well.
3.2. Protein expression of mutants with reduced transport activity Since OATP1B1 is a membrane protein, it needs to be properly targeted to the cell surface to exert its function. We therefore examined cell surface level of the mutants and found out that K41A exhibited ~40% of the cell surface protein abundance of wild-type OATP1B1, and plasma membrane level of K49A was reduced ~25%. On the other hand, protein level of I46A was comparable to that of wild-type (Fig. 4A). Total protein expression was also analyzed. As shown in Fig. 4B, total protein level correlated well with cell surface protein level of the mutants. These results suggested that mutation of K41 affected protein level of the transporter. However, the dramatically reduced transport function of the three alanine substituents could not be solely explained by altered cell surface protein abundance. Because all three mutants still exhibited significantly reduced ES and taurocholate uptake after normalized with cell surface transporter protein level (Supplementary Fig. 1 and Supplementary Fig. 2).
3.4. Estrone-3-sulfate uptake by additional mutants To investigate whether the side chain structure of the mutants is
Fig. 2. Estrone-3-sulfate uptake by TM1 mutants. Uptake of ES (50 nM) by HEK293 cells expressing OATP1B1 or its alanine-substituted mutants was measured at 37 °C at a 2 min interval. Net uptake was calculated by subtracting the uptake of cells transfected with empty vector from cells expressing wild-type OATP1B1 or mutants. The results represent data from three independent experiments, each with duplicate measurements. The results shown are means ± S.D. (n = 3). Statistical analysis was performed with Student's t-test. Asterisks indicate significant difference compared to wild-type OATP1B1 (p < 0.05). 3
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Fig. 3. Taurocholate uptake by K41A, K49A and I46A. Uptake of taurocholate (1 μM or 5 μM) by HEK293 cells transfected with empty vector or expressing OATP1B1, K41A, K49A and I46A was measured at 37 °C at a 2 min interval. The results represent data from three independent experiments, each with duplicate measurements. The results shown are means ± S.D. (n = 3). Student's t-test was performed for statistical analysis. Asterisks indicate significant difference compared to wild-type OATP1B1 (p < 0.05).
Fig. 4. Protein level of mutants with significantly decreased uptake function. A. Cell surface protein level of OATP1B1, K41A, K49A and I46A. Cells were biotinylated, and the biotin-labeled cell surface proteins were precipitated with streptavidin agarose beads, separated by SDS-PAGE, followed by western blotting with anti-HA antibody (1:1000 dilution). Same blot was probed with integrin antibody as surface protein loading control. A representative blot was shown. The intensity of protein bands was quantified with Image J and calculated relative to the wild-type. The results shown are means ± S.D. (n = 3). B. Total protein level of mutants compared to OATP1B1. Cells were lysed with RIPA buffer, separated by SDS-PAGE, and detected with anti-HA antibody. The band intensity was quantified with Image J and calculated relative to the wild-type. The results shown are means ± S.D. (n = 3). Statistical analysis was performed with Student's t-test. Asterisks indicate significant difference compared to wild-type OATP1B1 (p < 0.05).
3.5. N-terminus truncation of OATP1B1
important for the proper function of OATP1B1, we further substituted these residues with other amino acids. When K41 was replaced with arginine (K41R), the function and cell surface protein level were partially recovered. On the other hand, when the positively charged residue was substituted with the negatively charged glutamic acid (K41E), the function and protein level were similar to that of K41A. Replacing K49 with either negatively or positively charged residue, however, significantly reduced transport function even compared to that of K49A, though protein level of all the K49 mutants was comparable to wild-type OATP1B1 (Fig. 5A&B). In addition, conservative replacement of I46 with leucine also partially recovered ES uptake by the transporter (Fig. 5A).
It was shown in a previous report that a construct containing the Nterminal 50 amino acid residues of OATP1B1, of which includes only the N-terminus and TM1 of the transporter, could be detected on the cell surface membrane, suggesting that the region is important for membrane localization (Chun et al., 2017). Here, we wanted to pinpoint whether there is/are specific region(s) within the N-terminus sequence that dictate the fate of the transporter. As shown in Fig. 6A, truncation of the first 18 amino acid residues (DEL-18, aa2-18) did not affect ES uptake by the transporter; while a deletion of the first 27 residues (DEL-27, aa2-27) resulted in dramatic reduction of OATP1B1 transport function. Further analysis revealed that the total and cell surface protein level of DEL-27 was significantly reduced (Fig. 6B). 4
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cycloheximide chase method was applied (Fig. 6C). Reduced abundance of the transporter protein was partially recovered with the treatment of MG132, a peptide-aldehyde proteasome inhibitor (Fig. 7A), but not by lysosomal inhibitors Bafilomycin A1 and ammonia chloride (data not shown), suggesting the absence of aa 2–27 may decrease stability of the transporter protein. Uptake of ES by DEL-27 also increased significantly after MG132 treatment, which implicated that the recovered transporter is partially functional (Fig. 7B).
Table 1 Kinetic parameters of estrone-3- sulfate uptake by OATP1B1 wild-type and the mutants.
OATP1B1 K41A K49A I46A DEL-YCNGL
Km (μM)
Vmax (pmol/mg protein/min)
Clint (Vmax/Km) (μl/ mg protein/min)
0.20 ± 0.04 13.5 ± 0.1 ↓0.12* ± 0.03 14.6 ± 3.8 0.17 ± 0.08 ↑27.6* ± 7.4 ↓0.06* ± 0.01 ↑50.0* ± 10.3 ↓0.12* ± 0.01 12.5 ± 0.8
20.3 ± 3.5 185 ± 16 ↓12.1* ± 1.2 212 ± 23 ↓10.7* ± 3.4 ↓107* ± 8 ↓2.13* ± 0.30 ↓117* ± 23 ↓8.08* ± 1.56 ↓74.4* ± 3.4
102 13.7 101 14.5 ↓62.9 ↓3.88 ↓35.5 ↓2.34 ↓67.3 ↓5.95
3.6. Insertion of an unconventional region recovered function and cell surface protein level of the transporter Since the deletion of residues 2–18 did not affect uptake function of OATP1B1 (Fig. 6A), we then focused our further studies on amino acid residues 19–27, expecting that one or more domains within this region may be important for uptake function and/or protein level of OATP1B1. When the sequence of aa19–27 was analyzed, a region ranging from 23 to 27 with the sequence of YCNGL shows similarity to the conventional sorting sequence YXXϕ (ϕ stands for hydrophobic residues)of transmembrane proteins (Bonifacino and Traub, 2003). Therefore, we inserted this region back into the DEL-27 mutant and found out that the insertion of YCNGL (INS-YCNGL) recovered uptake function to > 60% of that of wild-type OATP1B1; while the deletion of this region (DELYCNGL) from OATP1B1 resulted in an effect on ES uptake similar to that of DEL-27 (Fig. 8A). Since the dramatically reduced function was mainly due to the significantly reduced protein abundance of DEL-27 (Fig. 6), we next wanted to see whether YCNGL is important for the
Estrone-3-sulfate uptake was measured at concentrations ranged from 0.01 to 20 μM for OATP1B1 wild-type and mutants at 37 °C at a 1-min interval and normalized with cell surface level of the transporter (protein abundance of OATP1B1-WT is considered as 1, and the relative protein level of mutants compared to that of wild-type was used for the normalization). Transporter kinetic parameters were determined with nonlinear regression of Michaelis–Menten equation incorporated in GraphPad Prism 5. The results shown are means ± S.D. (n = 5). Statistical analysis was performed with Student's t-test. Asterisks indicate values significantly different (p < 0.05) from that of OATP1B1 wild-type.
Further, it was found that DEL-27 exhibited a significantly higher degradation rate compared to wild-type OATP1B1-WT when
Fig. 5. Uptake function and cell surface protein level of additional mutants of K41, K49 and I46. A. ES uptake by additional mutants of K41, K49 and I46. Uptake of 50 nM ES was measured at 37 °C at a 2 min interval. The results represent data from three independent experiments, each with duplicate measurements. The results shown are means ± S.D. (n = 3). ANOVA was performed for statistical analysis. Different letters indicate significant difference among mutants of the same amino acid residue (p < 0.05). B. Cell surface protein analysis of additional mutants. Biotin labeling and protein analysis were carried out as described in Fig. 4A. A representative blot was shown. The intensity of protein bands was quantified with Image J and calculated relative to the wild-type. The results shown are means ± S.D. (n = 3). ANOVA was performed for statistical analysis. Different letters indicate significant difference among OATP1B1-WT and mutants (p < 0.05). 5
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Fig. 6. N-terminus truncation of OATP1B1. A. Estrone-3-sulfate uptake by DEL-18 and DEL-27. Uptake of 50 nM ES was measured at 37 °C at a 2 min interval. The results represent data from three experiments, each with duplicate measurements. The results shown are means ± S.D. (n = 3). B. Representative blots for cell surface (left panel) and total protein (right panel) level of DEL-27. The intensity of protein bands was quantified with Image J and calculated relative to the wild-type (lower panels). The results shown are means ± S.D. (n = 3). Cells were labeled with biotin and the proteins were extracted and analyzed as described in Fig. 4. Statistical analysis was performed with Student's t-test. Asterisks indicate significant difference compared to wild-type OATP1B1 (p < 0.05). C. Cyclohexamidechase analysis of DEL-27. Cells expressing wild-type OATP1B1 and DEL-27 were treated with 100 μg/ml of cycloheximide, collected at the indicated time points and analyzed as described before. The intensity of protein bands was quantified with Image J and calculated relative to the original protein level. The results shown are means ± S.D. (n = 3). Statistical analysis was performed with Student's t-test. Asterisks indicate significant difference compared to wild-type OATP1B1 (p < 0.05).
To further evaluate the effect of YCNGL on interaction of OATP1B1 with its substrate, kinetic analysis of DEL-YCNGL was carried out for estrone-3-sulfate uptake. As shown in Table 1, Km and Vmax values of ES high affinity binding site were significantly altered in DEL-YCNGL; while for the low affinity binding component of ES, only Vmax was significantly reduced. In addition, deletion or insertion of YCNGL affected uptake of taurocholate, another prototypic substrate of OATP1B1, in a similar manner to that of ES (Fig. 8E), implicating the importance of the region in the transport of multiple substrates. To rule out the possibility that the effect of YCNGL on OATP1B1 is merely due to the deletion of the region, we simultaneously mutated these five amino acid residues to alanine, similar effects on uptake function and protein level was observed for the quintuple mutant (Supplementary Fig. 3).
protein level of OATP1B1 as well. We found that the insertion of YCNGL on a DEL-27 background partially increased protein abundance, and the deletion of this region (DEL-YCNGL) exhibited a similar protein level to the insertion mutant (INS-YCNGL) (Fig. 8B), suggesting that though this region is crucial for proper function of OATP1B1, there may be other region(s) within the sequence of 19–27 that is/are important for OATP1B1 protein level. Further analysis of aa19–27 revealed a charged cluster (KKTR) located from position 19 to 22. Since cluster of positively charged amino acids has been proposed to be involved in proteinprotein interactions (Chun et al., 2017), deletion and insertion mutants of this region were generated. However, unlike YCNGL, the insertion of KKTR to DEL-27 did not recover the uptake function; while its deletion from wild-type OATP1B1 only resulted in around 30% reduction of ES uptake (Fig. 8A). Protein level of DEL-KKTR showed a similar reduction, which seemed to correlate with its decreased function (Fig. 8C). When uptake function of DEL-YCNGL and DEL-KKTR was normalized with cell surface protein level, transport activity of DEL-KKTR was comparable to that of wild-type OATP1B1; while DEL-YCNGL still exhibited < 40% function (Fig. 8D). These data suggested that YCNGL may be important for both uptake function and protein abundance of OATP1B1; while KKTR only affects protein level of the transporter.
4. Discussion The N-terminal region has been demonstrated to be important for membrane localization of OATP1B3 (Chun et al., 2017) and it was suggested that TM1 may be part of the substrate translocation pore of OATP1B3 according to a computer predicted model (Meier-Abt et al., 6
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Fig. 7. DEL-27 treated with proteasome inhibitor MG132. A. Total (upper left panels) and cell surface (upper right panels) protein level of OATP1B1 and DEL-27 after MG132 treatment. A representative blot was shown. The intensity of protein bands was quantified with Image J and calculated relative to the wild-type (lower panels). B. Estrone-3-sulfate uptake by OATP1B1 and DEL-27 after MG132 treatment. Cells expressing OATP1B1 or DEL-27 were treated with 5 μM MG132 for 12 h before the transporter protein was analyzed as described in Fig. 4 or ES uptake was measured as described in Fig. 2. Statistical analysis was performed with Student's t-test. Asterisks indicate significant difference compared to untreated control (p < 0.05).
this residue may be involved in the interaction with the substrate during the uptake process. However, Km of K41A is similar to that of the wild-type transporter for the low affinity binding site of ES (13.5 ± 0.1 μM for OATP1B1-WT vs 14.6 ± 3.8 μM for K41A, p = 0.64), indicating that K41 may only be involved in one of the substrate translocation pathways of OATP1B1. The replacement of K49 with arginine, however, did not rescue the transporter uptake function, suggesting that the charged property as well as the structural characteristics of lysine side-chain is important for OATP1B1 activity. Km value of the low binding affinity component of ES is increased in K49A, which implicated that the residue is involved in substrate interaction at the binding site. Vmax for both high and low affinity binding sites were significantly reduced in K49A, even after uptake function was normalized with cell surface protein level of the transporter, indicating that the position is important for substrate turn-over of OATP1B1 as well. Homology modeling (Biasini et al., 2014) of OATP1B1 using E.coli glycerol-3-phosphate transporter (PDB: 1pw4) as the template suggested that K41 and K49 may indeed directly interact with estrone-3sulfate (Fig. 9A). It should be noted that quite a few transporters with
2005). OATP1B1 is highly homologous to OATP1B3, thus may share some structural similarity with the transporter. In addition, previous studies of others and us have demonstrated that essential amino acid residues for substrate transport lined-up along multiple transmembrane domains of OATP1B1. In the present study, alanine-scanning was applied to study the residues within the putative TM1 of OATP1B1 and two lysine residues, i.e. K41 and K49, along with I46 was found to be important for substrate uptake of the transporter. Lysine 41 was identified before in a study of OATP1B3 to be important for the uptake of sulphobromophthalein (BSP) and pravastatin (Glaeser et al., 2010). The positively charge residue is conserved among OATP1 family members (Fig. 1). Further analysis demonstrated that conservative replacement of lysine with arginine partially recovered the cell surface protein abundance and the uptake function, suggesting that a positively charged residue at this position is important for maintaining the proper protein level of OATP1B1. In addition, kinetic analysis showed that alanine substitution of K41 altered interaction of the transporter and substrate at the high affinity binding site of ES (0.20 ± 0.04 μM for OATP1B1-WT vs 0.12 ± 0.03 μM for K41A, p = 0.04), suggesting that 7
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Fig. 8. Identification of the N-terminus essential regions for protein stability and uptake function of OATP1B1. A. Estrone-3-sulfate uptake by mutants within the region of aa19–27 at the N-terminus of OATP1B1. Uptake of ES (50 nM) by HEK293 cells expressing OATP1B1 or mutants was measured at 37 °C at a 2 min interval. Net uptake of mutants was compared to that of OATP1B1 wild-type and the relative percentage was presented. The results represent data from three independent experiments, each with duplicate measurements. The results shown are means ± S.D. (n = 3). B&C. Representative blots for cell surface and total protein level of YCNGL or KKTR mutants (left panels). The intensity of protein bands was quantified with Image J and calculated relative to the wild-type (right panels). The results shown are means ± S.D. (n = 3). Cells were labeled with biotin and the proteins were extracted and analyzed as described in Fig. 4. ANOVA was performed for statistical analysis. Different letters indicate significant difference between each other (p < 0.05). D. Estrone-3-sulfate uptake by mutants normalized with the corresponding cell surface transporter protein level. Net uptake of mutants was compared to that of OATP1B1 wild-type and the relative percentage was presented. ANOVA was performed for statistical analysis. Different letters indicate significant difference between each other (p < 0.05). E. Uptake of taurocholate by YCNGL mutants. Uptake of taurocholate (1 μM) was measured at 37 °C at a 2 min interval. Net uptake of mutants was compared to that of OATP1B1 wild-type and the relative percentage was presented. The results represent data from three independent experiments, each with duplicate measurements. The results shown are means ± S.D. (n = 3). ANOVA was performed for statistical analysis. Different letters indicate significant difference between each other (p < 0.05).
The identification of only three essential amino acid residues within TM1 of OATP1B1 is kind of unexpected. It has been proposed that TM1, along with TM2, 4 and 5 of the N-terminal half may be involved in the formation of the substrate interaction pocket and play an important role for the transport activity of OATP family members OATP1B3 and 2B1 (Meier-Abt et al., 2005). Indeed, a recent study by our own identified nine essential residues within the TM1 of OATP2B1 (Fang et al., 2018). Interestingly, Q62 in OATP2B1, which corresponds to K41 in OATP1B1, was also found to be an irreplaceable residue for the proper function of OATP2B1 and alanine substitution of the residue resulted in marginal but significant change of Km. Except for the OATP1 transporters, a polar, non-charged glutamine residue is found in all the other human OATPs. The replacement of K41 with glutamine also resulted in significant reduction of ES uptake (data not shown). We speculated that the corresponding residue of other OATPs may be situated at a different position relative to the substrate translocation pathway and hence play a different role for the uptake by these transporters. Further studies will be needed to clarify such an issue. Although estradiol-17β-glucuronide (E2G) is a sensitive in vitro OATP1B1 probe substrate (Izumi et al.,
their structures depicted have a comparable identity with OATP1B1 (~15%) to that of the E.coli glycerol-3-phosphate transporter, including the human glucose transporter family member 1 (PDB: 4pyp). However, homology regions of the glycerol-3-phosphate transporter cover the most extended area, i.e. from TM1-TM12 of OATP1B1, and it was therefore chosen to serve as the template for the present study. Besides the two positively charged amino acid residues, a dramatic reduction of ES and taurocholate uptake was also found for I46A, although protein abundance of the mutant is comparable to that of the wild-type transporter. When I46 was replaced with leucine, an amino acid with a similar side group structure, uptake was significantly increased, implicating the importance of the side chain structural feature at the position. Kinetic analysis revealed that Km and Vmax values were altered in both high and low affinity binding sites of ES, which suggested that I46 is involved in the substrate interaction of both components. Isoleucine is only found in OATP1B1 and 1B3 among the 11 human OATP members. Since alanine replacement of the residue exhibited such a significant effect, we speculate that it may play a substrate-specific role for these transporters. 8
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Fig. 9. Homology modeling of OATP1B1. A. Interaction of K41 and K49 of OATP1B1 with estrone-3-sulfate. B. Structural comparison of OATP1B1 wild-type and the DEL-YCNGL mutant. The structure of OATP1B1 was modeled with the web-based protein structure prediction service Swiss-model (https://www.swissmodel.expasy. org/) using E. coli glycerol-3-phosphate transporter (PDB: 1pw4) as the template.
showed a minor effect on OATP1B1 function; while the deletion of residues 2–27 (DEL-27) dramatically decreased protein level of the transporter and treatment of MG132 partially recovered its protein abundance as well as uptake function, suggesting the region of residues 19–27 is important for proper function of OATP1B1. To pinpoint the specific essential region(s), sequence of 19–27 was analyzed and it was found that residues18–22 contain a cluster of positively charged residues KKTR, and residues 19–27 possess a sequence of YCNGL that is similar to a conservative sorting motif YXXϕ. Further analysis demonstrated that the KKTR region may only be important for protein level of OATP1B1; while the YCNGL domain not only affects protein abundance but is also involved in substrate interaction of the transporter. However, unlike the N-terminus region of OATP1B3, which plays a crucial role in membrane trafficking, neither the KKTR nor the YCNGL domain seemed to affect targeting of OATP1B1. Because cell surface protein level of the deletion mutants correlated well with the mature form protein level in the total cell lysate (Fig. 8B & C). However, it should be noted that western blotting is a semi-quantitative quantification method for the analysis of protein level and further investigation will be needed to clarify the issue. The YCNGL sequence is unique for OATP1B1 (though the KKTR region is shared by OATP1B3) (Fig. 1) and hence may be important for specific substrate recognition as well. The altered Km value for the high affinity binding component and reduced Vmax for both binding sites of ES suggested that the YCNGL region may be essential for multiple binding components of OATP1B1. We speculated that the domain may play a role in maintaining a proper structure for substrate binding of the transporter. Homology modeling implicated that with YCNGL deleted, a dramatic position alteration would be observed for extracellular loops 2 and 3 of OATP1B1 (Fig. 9B). In summary, investigation of the putative TM1 of OATP1B1 identified two positively charged residues at position 41 and 49 along with a hydrophobic I46 residue that are important for substrate binding and/ or maintaining the protein level of the transporter; while within the N-
2013) and commonly used in the studies of OATP1B1, estrone-3-sulfate was selected as the prior substrate in our present study because OATP1B1 has two binding sites for ES (Noé et al., 2007; Gui and Hagenbuch, 2009; Li et al., 2012) and the analysis of the effect of mutations on both binding sites can help us more thoroughly evaluate the residues that are responsible for the uptake of different substrates. Since E2G and low concentration of ES (0.1 μM) has been found to competitively inhibit each other (Izumi et al., 2013)), suggesting that E2G may share the high affinity binding site of ES in the uptake process of OATP1B1, it may be likely that K41A and I46A, both of which were shown to affect the Km of the high affinity component of ES, may exhibit altered Km for E2G as well. Additionally, based on the kinetic parameters obtained in our study, it may also be possible that inhibitors of OATP1B1 such as rifampin and cyclosporine A would have a higher IC50 for K41A and I46A because binding affinity for ES was increased in the alanine mutants. In should be noted that besides the three residues identified in our present study, H54, which was predicted to be located at the extracellular entry of the putative TM1 of OATP1B1, was also demonstrated to be involved in the interaction with substrates such as bromosulfophthalein (BSP), estradiol-17β-glucuronide (E2G)], taurocholate and pravastatin (Gruetz et al., 2016). However, the TM1 in this study was predicted with an OATP1B1 model that was established based on the closely related OATP1B3 (Meier-Abt et al., 2005) and ranges from residue 30 to 54, which is different from the location of the putative TM1 of our study. Although alanine replacement of K41, K49 or I46 dramatically reduced the uptake function of OATP1B1, there is still a significant amount of transporter protein on the cell surface. Since the N-terminal region ranging from amino acid residue 1 to 50 of OATP1B1 was proposed to be essential for its membrane targeting (Chun et al., 2017), we truncated the first 18 or 27 residues of OATP1B1 to identify the specific region that may be important for the cell surface protein level of the transporter. The deletion of amino acid residues 2–18 (DEL-18) only 9
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terminus of the protein, the KKTR region is important for protein abundance, and YCNGL was found to be essential for substrate interaction as well as maintaining the protein level. The identification of crucial residues and/or motifs of OATP1B1 may help us better understand the therapeutics significance of OATP1B1 transmembrane domains and the underlying molecular and cellular mechanisms for the absorption, distribution and disposition of different drugs by the transporter. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.taap.2019.114642.
10.1080/00498250801986951. Hagenbuch, B., Meier, P.J., 2003. The superfamily of organic anion transporting polypeptides. Biochim. Biophys. Acta 1609, 1–18. Hong, W., Wu, Z., Fang, Z., Huang, J., Huang, H., Hong, M., 2015. Amino acid residues in the putative transmembrane domain 11 of human organic anion transporting polypeptide 1B1 dictate transporter substrate binding, stability, and trafficking. Mol. Pharm. 12, 4270–4276. https://doi.org/10.1021/acs.molpharmaceut.5b00466. Huang, J., Li, N., Hong, W., Zhan, K., Yu, X., Huang, H., Hong, M., 2013. Conserved tryptophan residues within putative transmembrane domain 6 affect transport function of organic anion transporting polypeptide 1B1. Mol. Pharmacol. 84, 521–527. https://doi.org/10.1124/mol.113.085977. Izumi, S., Nozaki, Y., Komori, T., Maeda, K., Takenaka, O., Kusano, K., Yoshimura, T., Kusuhara, H., Sugiyama, Y., 2013. Substrate-dependent inhibition of organic anion transporting polypeptide 1B1: comparative analysis with prototypical probe substrates estradiol-17β-glucuronide, estrone-3-sulfate, and sulfobromophthalein. Drug Metab. Dispos. 41, 1859–1866. https://doi.org/10.1124/dmd.113.052290. König, J., 2011. Uptake transporters of the human OATP family: molecular characteristics, substrates, their role in drug-drug interactions, and functional consequences of polymorphisms. Handb. Exp. Pharmacol. 201, 1–28. https://doi.org/10.1007/978-3642-14541-4_1. Li, N., Hong, W., Huang, H., Lu, H., Lin, G., Hong, M., 2012. Identification of amino acids essential for Estrone-3-sulfate transport within transmembrane domain 2 of organic anion transporting polypeptide 1B1. PLoS ONE 7, e36647. https://doi.org/10.1371/ journal.pone.0036647. Meier-Abt, F., Mokrab, Y., Mizuguchi, K., 2005. Organic anion transporting polypeptides of the OATP/SLCO superfamily: identification of new members in nonmammalian species, comparative modeling and a potential transport mode. J. Membr. Biol. 208, 213–227. Miyagawa, M., Maeda, K., Aoyama, A., Sugiyama, Y., 2009. The eighth and ninth transmembrane domains in organic anion transporting polypeptide 1B1 affect the transport kinetics of estrone-3-sulfate and estradiol-17beta-D-glucuronide. J. Pharmacol. Exp. Ther. 329, 551–557. https://doi.org/10.1124/jpet.108.148411. Nakanishi, T., Tamai, I., 2012. Genetic polymorphisms of OATP transporters and their impact on intestinal absorption and hepatic disposition of drugs. Drug Metab. Pharmacokinet. 27, 106–121. Noé, B., Hagenbuch, B., Stieger, B., Meier, P.J., 1997. Isolation of a multi-specific organic anion and cardiac glycoside transporter from rat brain. Proc. Natl. Acad. Sci. U. S. A. 94, 10346–10350. Noé, J., Portmann, R., Brun, M.E., Funk, C., 2007. Substrate-dependent drug-drug interactions between gemfibrozil, fluvastatin and other organic anion-transporting peptide (OATP) substrates on OATP1B1, OATP2B1, and OATP1B3. Drug Metab. Dispos. 35, 1308–1314. Poirier, A., Funk, C., Lavé, T., Noé, J., 2007. New strategies to address drug-drug interactions involving OATPs. Curr. Opin. Drug. Discov. Dev. 10, 74–83. Shitara, Y., Sato, H., Sugiyama, Y., 2005. Evaluation of drug-drug interaction in the hepatobiliary and renal transport of drugs. Annu. Rev. Pharmacol. Toxicol. 45, 689–723. Shitara, Y., Maeda, K., Ikejiri, K., Yoshida, K., Horie, T., Sugiyama, Y., 2013. Clinical significance of organic anion transporting polypeptides (OATPs) in drug disposition: their roles in hepatic clearance and intestinal absorption. Biopharm. Drug Dispos. 34, 45–78. https://doi.org/10.1002/bdd.1823. Stieger, B., Hagenbuch, B., 2014. Organic anion transporting polypeptides. Curr. Top. Membr. 73, 205–232. https://doi.org/10.1016/B978-0-12-800223-0.00005-0. Weaver, Y.M., Hagenbuch, B., 2010. Several conserved positively charged amino acids in OATP1B1 are involved in binding or translocation of different substrates. J. Membr. Biol. 236, 279–290. https://doi.org/10.1007/s00232-010-9300-3.
Acknowledgements This work was supported by the National Natural Science Foundation of China [grant number U1832101, 81373473&11335011] and Guangzhou Science and Technology Program [grant number 201707010412]. References Biasini, M., Bienert, S., Waterhouse, A., Arnold, K., Studer, G., Schmidt, T., Kiefer, F., Cassarino, T.G., Bertoni, M., Bordoli, L., Schwede, T., 2014. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258. https://doi.org/10.1093/nar/gku340. Bonifacino, J.S., Traub, L.M., 2003. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72, 395–447. Bossuyt, X., Müller, M., Meier, P.J., 1996. Multispecific amphipathic substrate transport by an organic anion transporter of human liver. J. Hepatol. 25, 733–738. Chun, S.E., Thakkar, N., Oh, Y., Park, J.E., Han, S., Ryoo, G., Hahn, H., Maeng, S.H., Lim, Y.R., Han, B.W., Lee, W., 2017. The N-terminal region of organic anion transporting polypeptide 1B3 (OATP1B3) plays an essential role in regulating its plasma membrane trafficking. Biochem. Pharmacol. 131, 98–105. https://doi.org/10.1016/j.bcp. 2017.02.013. Fang, Z., Huang, J., Chen, J., Xu, S., Xiang, Z., Hong, M., 2018. Transmembrane domain 1 of human organic anion transporting polypeptide 2B1 is essential for transporter function and stability. Mol. Pharmacol. 94, 842–849. https://doi.org/10.1124/mol. 118.111914. Glaeser, H., Mandery, K., Sticht, H., Fromm, M.F., König, J., 2010. Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3. Br. J. Pharmacol. 159, 698–708. https:// doi.org/10.1111/j.1476-5381.2009.00568.x. Gruetz, M., Sticht, H., Glaeser, H., Fromm, M.F., König, J., 2016. Analysis of amino acid residues in the predicted transmembrane pore influencing transport kinetics of the hepatic drug transporter organic anion transporting polypeptide 1B1 (OATP1B1). Biochim. Biophys. Acta 1858, 2894–2902. https://doi.org/10.1016/j.bbamem.2016. 08.018. Gui, C., Hagenbuch, B., 2009. Role of transmembrane domain 10 for the function of organic anion transporting polypeptide 1B1. Protein Sci. 18, 2298–2306. https://doi. org/10.1002/pro.240. Hagenbuch, B., Gui, C., 2008. Xenobiotic transporters of the human organic anion transporting polypeptides (OATP) family. Xenobiotica 38, 778–801. https://doi.org/
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Amino-terminal region of human organic anion transporting polypeptide 1B1 dictates transporter stability and substrate interaction Xuyang Wanga,1, Jie Chena,1, Shaopeng Xua,b, Chunxu Nia, Zihui Fanga, Mei Honga,b, a b
T
⁎
College of Life Sciences, South China Agricultural University, Guangzhou, China Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, South China Agricultural University, Guangzhou, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Amino-terminus Organic anion transporting polypeptides Protein stability Transmembrane domains Uptake function
Organic anion transporting polypeptides (OATPs) are key players of drug absorption, distribution and excretion due to their broad substrate specificity, wide tissue distribution and the involvement in drug-drug interaction. OATP1B1 is specifically localized at the basolateral membrane of human hepatocytes and serves a crucial role in the drug clearance from the body. Previous studies have shown that transmembrane domains (TMs) are essential for proper functions of OATPs. In the present study, site-directed mutagenesis was performed to study the TM1 and amino-terminus of OATP1B1. Two positively charged residues, K41 and K49, as well as a hydrophobic residue I46, in TM1 were identified to be important for the proper function of the transporter. K41A and K49A exhibited altered Km value at the high and low affinity binding sites of estrone-3- sulfate (ES), respectively; while alanine substitution of I46 showed altered Km and Vmax values for both binding components of ES. Additional replacement of K41 revealed that the positively charged property at this position is important for maintaining OATP1B1 protein level and function; while the specific side-group structure of lysine at position 49 is irreplaceable for the transporter activity. Conservative replacement of I46 with leucine also recovered the function of the transporter. In addition, studies of the amino-terminus of OATP1B1 revealed that residues ranging from 19 to 27 are essential for protein stability and substrate interaction. Therefore, the amino-terminal region, which includes TM1 and the amino-terminus of OATP1B1, is important for proper function of the membrane protein.
1. Introduction Organic anion transporting polypeptides (OATPs, gene symbol SLCO) are solute carrier family members and mediate sodium-independent transport of a wide range of compounds (Hagenbuch and Gui, 2008). Substrates of OATPs include bile salts, hormones and their conjugates, toxins and a variety of drugs. In addition to charged compounds, OATPs also transport uncharged drugs such as glycosides digoxin (Noé et al., 1997) and ouabain (Bossuyt et al., 1996). So far there are 12 members of the human OATP family cloned, including OATP1A2, 1B1, 1B3, 1B7, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1 and 6A1 (Hagenbuch and Meier, 2003; Nakanishi and Tamai, 2012), though SLCO1B7 was proposed to be a pseudogene because OATP1B7 is considered as non-functional (Stieger and Hagenbuch, 2014). OATP family members have wide tissue distributions- some are expressed
ubiquitously; while others are predominantly found in certain organs or tissues. In recent years, OATPs have been extensively recognized as the key determinants of drug absorption, distribution and excretion and found to be involved in various kinds of drug-drug interaction (DDI) (Shitara et al., 2005; Poirier et al., 2007). Although many clinically important drugs have been identified as substrates of OATPs, the underlying mechanisms of substrate binding and/or recognition remain largely unclear due to the lacking of high resolution crystal structures of mammalian drug transporters (Miyagawa et al., 2009). OATP1B1 is the major OATP located at the basolateral membrane of human hepatocytes and plays a crucial role in the drug clearance from the body (Noé et al., 2007; Shitara et al., 2013). Quite a few studies have demonstrated that transmembrane domains (TMs) are essential for the proper function of OATP1B1. Transmembrane domains 8 and 9 were found to be critical for substrate recognition of the transporter
Abbreviations: BSP, sulphobromophthalein; E2G, estradiol-17β-glucuronide; ES, estrone-3-sulfate; NHS-SS-biotin, sulfosuccinimidyl 2-(biotinamido)-ethyl-1, 3-dithiopropionate; OATP, organic anion transporting polypeptide; PBS, phosphate-buffered saline; TM, transmembrane domain. ⁎ Corresponding author at: College of Life Sciences, South China Agricultural University, Guangzhou, China. E-mail address: [email protected] (M. Hong). 1 These two authors contributed equally to the work. https://doi.org/10.1016/j.taap.2019.114642 Received 17 April 2019; Received in revised form 20 June 2019; Accepted 25 June 2019 Available online 27 June 2019 0041-008X/ © 2019 Elsevier Inc. All rights reserved.
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2.4. Uptake assay
(Miyagawa et al., 2009). Several amino acid residues along TM10 are important for substrate translocation and maintenance of the proper protein structure of OATP1B1 (Gui and Hagenbuch, 2009). Previous studies of OATP1B1 in our laboratory found out that four amino acids within TM2, i.e. D70, F73, E74, and G76 are crucial for the uptake of estrone-3-sulfate (ES) (Li et al., 2012). Two highly conserved tryptophan residues (W258 and W259) that are close to the extracellular border of TM6 were also identified to be essential for the uptake function of the transporter (Huang et al., 2013). In a more recent study, six critical amino acid residues in addition to R580, which have been reported by others to be essential for proper function of OATP1B1 (Weaver and Hagenbuch, 2010), were identified within the TM11 of the transporter. The identified residues are important for substrate interaction, stability and/or correct targeting of OATP1B1 (Hong et al., 2015). Computer-generated model of OATP1B3, an OATP family member that is highly homologous to OATP1B1, proposed that several TMs of the N-terminal half, including TM1, 2, 4 and 5, may face the substrate interaction pore (Meier-Abt et al., 2005). Previous study of OATP1B3 identified conserved positively charged residues K41 and R580, which located at transmembrane domain 1 and 11, respectively, to be critical for substrate uptake of the transporter (Glaeser et al., 2010). In addition, the N-terminus region of OATP1B3 was demonstrated to be important for membrane localization of the transporter. It was found that amino acid residues 12–28 may be indispensable for membrane trafficking of the transporter. However, individual residues or structural motifs that is responsible for such a process is unclear (Chun et al., 2017). In the present study, we performed alanine-scanning to study the TM1 of OATP1B1. It was found that two positively charged amino acid residues, K41 and K49, as well as the hydrophobic I46, are important for the uptake function of the transporter. Further studies revealed that these residues may be involved in substrate interaction. In addition, the N-terminus of OATP1B1 was investigated and a region ranges from residues 19–27 was identified to be essential for stability and substrate interaction of the transporter protein.
Cells in 48-well plate were used for transport function analysis as described before (Li et al., 2012). Briefly, cells were incubated with uptake solution containing [3H]ES or [3H] taurocholic acid at 37 °C for 2 min (1 min for kinetic analysis) and uptake was stopped by the icecold phosphate-buffered saline (PBS) solution. Cells were then washed with cold PBS, solubilized in 0.2 N NaOH, neutralized with 0.2 N HCl and the radioactivity of the cell lysate was measured with a liquid scintillation counter (Triathler-Hidex, Hidex, Turku, Finland). The uptake count was standardized by the amount of protein in each well. 2.5. Cell surface biotinylation and western blotting Cell surface protein level of OATP1B1 and the mutants was examined using the membrane-impermeable biotinylation reagent Sulfosuccinimidyl 2-(biotinamido)-ethyl-1, 3-dithiopropionate (NHSSS-biotin) as described before (Li et al., 2012). In brief, HEK293 cells expressing OATP1B1 or mutants were labeled with NHS-SS-biotin for two consecutive 20-min incubations. Cells were then dissolved with RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% NP-40, protease inhibitors phenylmethylsulfonyl fluoride, 200 μg/ml, leupeptin, 3 μg/ ml, pH 7.4), and the biotin-labeled proteins were precipitated with streptavidin-agarose beads. The streptavidin-agarose beads bound proteins were then released in 4× Laemmli buffer at 55 °C and loaded onto a 7.5% SDS-polyacrylamide electrophoresis gel, transferred electrophoretically to a polyvinylidene difluoride membrane (Millipore, Billerica, MA) and detected with anti-HA antibody (1:1000 dilution, Beyotime Biotechnology, Inc., Jiangsu, China). 2.6. Homology modeling Escherichia coli glycerol-3-phosphate transporter (PDB: 1pw4) was used as the template for homology modeling of OATP1B1. The structure of OATP1B1 was modeled with the web-based protein structure prediction service Swiss-model (https://www.swissmodel.expasy.org/).
2. Materials and methods
2.7. Statistical analysis
2.1. Materials
Student's t-test was utilized for comparisons between two sets of data. One-way analysis of variance (ANOVA) with Bonferroni's post hoc test was carried out in cases where there are more than two groups were compared. Differences between means are regarded as significant if p < 0.05.
[3H]Estrone-3-sulfate (ES) and [3H]taurocholic acid were purchased from PerkinElmer Life Sciences (Waltham, MA). Sulfosuccinimidyl 2(biotinamido) -ethyl-1, 3-dithiopropionate (NHS-SS-biotin) and streptavidin-agarose beads were from Thermo Fisher Scientific (Waltham, MA). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO) except otherwise stated.
3. Results 3.1. Alanine-scanning of OATP1B1 transmembrane domain 1
2.2. Site-directed mutagenesis
To identify the critical amino acid residues within TM1 of OATP1B1, we first individually substituted the residues along the predicted TM1 with alanine. Amino acid residues in TM1 were chosen according to the Kyte-Doolittle hydrophobicity scale (Fig. 1). Hence 18 out of the 22 residues from position 28 to 49 of OATP1B1 were mutated to alanine, and four alanine residues (A32, A33, A40, and A45) located in TM1 were replaced with valine. Uptake function of estrone-3-sulfate (ES), an OATP prototypic substrate found to be transported by all OATPs (König, 2011; Yamaguchi et al., 2010), was measured. As shown in Fig. 2, though quite a few alanine-substituted mutants exhibited significant change in uptake function, only mutation of the positively charged K41 and K49, and the hydrophobic I46, resulted in > 50% decrease of the ES transport function. In addition, we performed transport assay with another prototypical substrate taurocholate, which was found to be transported by major OATPs including OATP1A2, 1B1, 1B3 (König, 2011) and 2B1 (Fang et al., 2018). It was found that K41A and K49A as well as I46A also
Mutants were generated with QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA) according to the manufacturer's instructions. The pReceiver M07 vector containing SLCO1B1 cDNA and 3-HA tags at the C-terminus was purchased from Genecopoeia (Rockville, MD) and used as the mutagenesis template. All mutant sequences were confirmed by full length sequencing (Thermo Fisher Scientific). 2.3. Cell culture and transfection of plasmid constructs into cells HEK293 cells were grown in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum at 37 °C and 5% CO2. Confluent cells in 48-well or 12-well plate were transfected with DNA plasmid using LipofectAMINE 2000 reagent (Thermo Fisher Scientific). Transfected cells were incubated for 48 h at 37 °C and then used for transport assay or cell surface biotinylation. 2
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Fig. 1. Putative transmembrane domain 1 of human organic anion transporting polypeptide family members. Multiple sequence alignment of 11 OATP family members was performed with ClustalW. Location of the putative TM1 was predicted with Kyte-Doolittle hydrophobicity scale. The corresponding sequences of TM1 were in bold. Residues from position 19 to 27 at the amino-terminus of OATP1B1 were underlined. Only partial sequences were shown here.
3.3. Kinetic analysis of mutants K41A, K49A and I46A
showed significantly decreased taurocholate uptake. In particular, taurocholate uptake by K41A was reduced to < 15% of that by the wild-type OATP1B1 (Fig. 3). We therefore further investigated these three residues in the following studies.
Next we wanted to investigate whether alanine replacement of these three residues affects the interaction of OATP1B1 with its substrates. Two binding sites for ES were reported in OATP1B1 (Noé et al., 2007; Gui and Hagenbuch, 2009; Li et al., 2012). Therefore, ES uptake function was examined at concentrations ranged from 0.01 to 20 μM. Since mutation of both K41 and K49 partially affected protein level of the transporter, we normalized the uptake with cell surface level of the mutants relative to wild-type to better represent their effect on the interaction of OATP1B1 and ES. As shown in Table 1, all mutants retained a biphasic kinetics for ES uptake. K41A exhibited a significant reduction of both Km and Vmax values compared to wild-type OATP1B1 in the high binding affinity site. The Km value for low affinity binding site of ES was significantly increased for K49A; while the Vmax was decreased in both high and low affinity components of the mutant. I46A showed significant change in Km for both binding components and Vmax of the mutant was significantly altered as well.
3.2. Protein expression of mutants with reduced transport activity Since OATP1B1 is a membrane protein, it needs to be properly targeted to the cell surface to exert its function. We therefore examined cell surface level of the mutants and found out that K41A exhibited ~40% of the cell surface protein abundance of wild-type OATP1B1, and plasma membrane level of K49A was reduced ~25%. On the other hand, protein level of I46A was comparable to that of wild-type (Fig. 4A). Total protein expression was also analyzed. As shown in Fig. 4B, total protein level correlated well with cell surface protein level of the mutants. These results suggested that mutation of K41 affected protein level of the transporter. However, the dramatically reduced transport function of the three alanine substituents could not be solely explained by altered cell surface protein abundance. Because all three mutants still exhibited significantly reduced ES and taurocholate uptake after normalized with cell surface transporter protein level (Supplementary Fig. 1 and Supplementary Fig. 2).
3.4. Estrone-3-sulfate uptake by additional mutants To investigate whether the side chain structure of the mutants is
Fig. 2. Estrone-3-sulfate uptake by TM1 mutants. Uptake of ES (50 nM) by HEK293 cells expressing OATP1B1 or its alanine-substituted mutants was measured at 37 °C at a 2 min interval. Net uptake was calculated by subtracting the uptake of cells transfected with empty vector from cells expressing wild-type OATP1B1 or mutants. The results represent data from three independent experiments, each with duplicate measurements. The results shown are means ± S.D. (n = 3). Statistical analysis was performed with Student's t-test. Asterisks indicate significant difference compared to wild-type OATP1B1 (p < 0.05). 3
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Fig. 3. Taurocholate uptake by K41A, K49A and I46A. Uptake of taurocholate (1 μM or 5 μM) by HEK293 cells transfected with empty vector or expressing OATP1B1, K41A, K49A and I46A was measured at 37 °C at a 2 min interval. The results represent data from three independent experiments, each with duplicate measurements. The results shown are means ± S.D. (n = 3). Student's t-test was performed for statistical analysis. Asterisks indicate significant difference compared to wild-type OATP1B1 (p < 0.05).
Fig. 4. Protein level of mutants with significantly decreased uptake function. A. Cell surface protein level of OATP1B1, K41A, K49A and I46A. Cells were biotinylated, and the biotin-labeled cell surface proteins were precipitated with streptavidin agarose beads, separated by SDS-PAGE, followed by western blotting with anti-HA antibody (1:1000 dilution). Same blot was probed with integrin antibody as surface protein loading control. A representative blot was shown. The intensity of protein bands was quantified with Image J and calculated relative to the wild-type. The results shown are means ± S.D. (n = 3). B. Total protein level of mutants compared to OATP1B1. Cells were lysed with RIPA buffer, separated by SDS-PAGE, and detected with anti-HA antibody. The band intensity was quantified with Image J and calculated relative to the wild-type. The results shown are means ± S.D. (n = 3). Statistical analysis was performed with Student's t-test. Asterisks indicate significant difference compared to wild-type OATP1B1 (p < 0.05).
3.5. N-terminus truncation of OATP1B1
important for the proper function of OATP1B1, we further substituted these residues with other amino acids. When K41 was replaced with arginine (K41R), the function and cell surface protein level were partially recovered. On the other hand, when the positively charged residue was substituted with the negatively charged glutamic acid (K41E), the function and protein level were similar to that of K41A. Replacing K49 with either negatively or positively charged residue, however, significantly reduced transport function even compared to that of K49A, though protein level of all the K49 mutants was comparable to wild-type OATP1B1 (Fig. 5A&B). In addition, conservative replacement of I46 with leucine also partially recovered ES uptake by the transporter (Fig. 5A).
It was shown in a previous report that a construct containing the Nterminal 50 amino acid residues of OATP1B1, of which includes only the N-terminus and TM1 of the transporter, could be detected on the cell surface membrane, suggesting that the region is important for membrane localization (Chun et al., 2017). Here, we wanted to pinpoint whether there is/are specific region(s) within the N-terminus sequence that dictate the fate of the transporter. As shown in Fig. 6A, truncation of the first 18 amino acid residues (DEL-18, aa2-18) did not affect ES uptake by the transporter; while a deletion of the first 27 residues (DEL-27, aa2-27) resulted in dramatic reduction of OATP1B1 transport function. Further analysis revealed that the total and cell surface protein level of DEL-27 was significantly reduced (Fig. 6B). 4
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cycloheximide chase method was applied (Fig. 6C). Reduced abundance of the transporter protein was partially recovered with the treatment of MG132, a peptide-aldehyde proteasome inhibitor (Fig. 7A), but not by lysosomal inhibitors Bafilomycin A1 and ammonia chloride (data not shown), suggesting the absence of aa 2–27 may decrease stability of the transporter protein. Uptake of ES by DEL-27 also increased significantly after MG132 treatment, which implicated that the recovered transporter is partially functional (Fig. 7B).
Table 1 Kinetic parameters of estrone-3- sulfate uptake by OATP1B1 wild-type and the mutants.
OATP1B1 K41A K49A I46A DEL-YCNGL
Km (μM)
Vmax (pmol/mg protein/min)
Clint (Vmax/Km) (μl/ mg protein/min)
0.20 ± 0.04 13.5 ± 0.1 ↓0.12* ± 0.03 14.6 ± 3.8 0.17 ± 0.08 ↑27.6* ± 7.4 ↓0.06* ± 0.01 ↑50.0* ± 10.3 ↓0.12* ± 0.01 12.5 ± 0.8
20.3 ± 3.5 185 ± 16 ↓12.1* ± 1.2 212 ± 23 ↓10.7* ± 3.4 ↓107* ± 8 ↓2.13* ± 0.30 ↓117* ± 23 ↓8.08* ± 1.56 ↓74.4* ± 3.4
102 13.7 101 14.5 ↓62.9 ↓3.88 ↓35.5 ↓2.34 ↓67.3 ↓5.95
3.6. Insertion of an unconventional region recovered function and cell surface protein level of the transporter Since the deletion of residues 2–18 did not affect uptake function of OATP1B1 (Fig. 6A), we then focused our further studies on amino acid residues 19–27, expecting that one or more domains within this region may be important for uptake function and/or protein level of OATP1B1. When the sequence of aa19–27 was analyzed, a region ranging from 23 to 27 with the sequence of YCNGL shows similarity to the conventional sorting sequence YXXϕ (ϕ stands for hydrophobic residues)of transmembrane proteins (Bonifacino and Traub, 2003). Therefore, we inserted this region back into the DEL-27 mutant and found out that the insertion of YCNGL (INS-YCNGL) recovered uptake function to > 60% of that of wild-type OATP1B1; while the deletion of this region (DELYCNGL) from OATP1B1 resulted in an effect on ES uptake similar to that of DEL-27 (Fig. 8A). Since the dramatically reduced function was mainly due to the significantly reduced protein abundance of DEL-27 (Fig. 6), we next wanted to see whether YCNGL is important for the
Estrone-3-sulfate uptake was measured at concentrations ranged from 0.01 to 20 μM for OATP1B1 wild-type and mutants at 37 °C at a 1-min interval and normalized with cell surface level of the transporter (protein abundance of OATP1B1-WT is considered as 1, and the relative protein level of mutants compared to that of wild-type was used for the normalization). Transporter kinetic parameters were determined with nonlinear regression of Michaelis–Menten equation incorporated in GraphPad Prism 5. The results shown are means ± S.D. (n = 5). Statistical analysis was performed with Student's t-test. Asterisks indicate values significantly different (p < 0.05) from that of OATP1B1 wild-type.
Further, it was found that DEL-27 exhibited a significantly higher degradation rate compared to wild-type OATP1B1-WT when
Fig. 5. Uptake function and cell surface protein level of additional mutants of K41, K49 and I46. A. ES uptake by additional mutants of K41, K49 and I46. Uptake of 50 nM ES was measured at 37 °C at a 2 min interval. The results represent data from three independent experiments, each with duplicate measurements. The results shown are means ± S.D. (n = 3). ANOVA was performed for statistical analysis. Different letters indicate significant difference among mutants of the same amino acid residue (p < 0.05). B. Cell surface protein analysis of additional mutants. Biotin labeling and protein analysis were carried out as described in Fig. 4A. A representative blot was shown. The intensity of protein bands was quantified with Image J and calculated relative to the wild-type. The results shown are means ± S.D. (n = 3). ANOVA was performed for statistical analysis. Different letters indicate significant difference among OATP1B1-WT and mutants (p < 0.05). 5
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Fig. 6. N-terminus truncation of OATP1B1. A. Estrone-3-sulfate uptake by DEL-18 and DEL-27. Uptake of 50 nM ES was measured at 37 °C at a 2 min interval. The results represent data from three experiments, each with duplicate measurements. The results shown are means ± S.D. (n = 3). B. Representative blots for cell surface (left panel) and total protein (right panel) level of DEL-27. The intensity of protein bands was quantified with Image J and calculated relative to the wild-type (lower panels). The results shown are means ± S.D. (n = 3). Cells were labeled with biotin and the proteins were extracted and analyzed as described in Fig. 4. Statistical analysis was performed with Student's t-test. Asterisks indicate significant difference compared to wild-type OATP1B1 (p < 0.05). C. Cyclohexamidechase analysis of DEL-27. Cells expressing wild-type OATP1B1 and DEL-27 were treated with 100 μg/ml of cycloheximide, collected at the indicated time points and analyzed as described before. The intensity of protein bands was quantified with Image J and calculated relative to the original protein level. The results shown are means ± S.D. (n = 3). Statistical analysis was performed with Student's t-test. Asterisks indicate significant difference compared to wild-type OATP1B1 (p < 0.05).
To further evaluate the effect of YCNGL on interaction of OATP1B1 with its substrate, kinetic analysis of DEL-YCNGL was carried out for estrone-3-sulfate uptake. As shown in Table 1, Km and Vmax values of ES high affinity binding site were significantly altered in DEL-YCNGL; while for the low affinity binding component of ES, only Vmax was significantly reduced. In addition, deletion or insertion of YCNGL affected uptake of taurocholate, another prototypic substrate of OATP1B1, in a similar manner to that of ES (Fig. 8E), implicating the importance of the region in the transport of multiple substrates. To rule out the possibility that the effect of YCNGL on OATP1B1 is merely due to the deletion of the region, we simultaneously mutated these five amino acid residues to alanine, similar effects on uptake function and protein level was observed for the quintuple mutant (Supplementary Fig. 3).
protein level of OATP1B1 as well. We found that the insertion of YCNGL on a DEL-27 background partially increased protein abundance, and the deletion of this region (DEL-YCNGL) exhibited a similar protein level to the insertion mutant (INS-YCNGL) (Fig. 8B), suggesting that though this region is crucial for proper function of OATP1B1, there may be other region(s) within the sequence of 19–27 that is/are important for OATP1B1 protein level. Further analysis of aa19–27 revealed a charged cluster (KKTR) located from position 19 to 22. Since cluster of positively charged amino acids has been proposed to be involved in proteinprotein interactions (Chun et al., 2017), deletion and insertion mutants of this region were generated. However, unlike YCNGL, the insertion of KKTR to DEL-27 did not recover the uptake function; while its deletion from wild-type OATP1B1 only resulted in around 30% reduction of ES uptake (Fig. 8A). Protein level of DEL-KKTR showed a similar reduction, which seemed to correlate with its decreased function (Fig. 8C). When uptake function of DEL-YCNGL and DEL-KKTR was normalized with cell surface protein level, transport activity of DEL-KKTR was comparable to that of wild-type OATP1B1; while DEL-YCNGL still exhibited < 40% function (Fig. 8D). These data suggested that YCNGL may be important for both uptake function and protein abundance of OATP1B1; while KKTR only affects protein level of the transporter.
4. Discussion The N-terminal region has been demonstrated to be important for membrane localization of OATP1B3 (Chun et al., 2017) and it was suggested that TM1 may be part of the substrate translocation pore of OATP1B3 according to a computer predicted model (Meier-Abt et al., 6
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Fig. 7. DEL-27 treated with proteasome inhibitor MG132. A. Total (upper left panels) and cell surface (upper right panels) protein level of OATP1B1 and DEL-27 after MG132 treatment. A representative blot was shown. The intensity of protein bands was quantified with Image J and calculated relative to the wild-type (lower panels). B. Estrone-3-sulfate uptake by OATP1B1 and DEL-27 after MG132 treatment. Cells expressing OATP1B1 or DEL-27 were treated with 5 μM MG132 for 12 h before the transporter protein was analyzed as described in Fig. 4 or ES uptake was measured as described in Fig. 2. Statistical analysis was performed with Student's t-test. Asterisks indicate significant difference compared to untreated control (p < 0.05).
this residue may be involved in the interaction with the substrate during the uptake process. However, Km of K41A is similar to that of the wild-type transporter for the low affinity binding site of ES (13.5 ± 0.1 μM for OATP1B1-WT vs 14.6 ± 3.8 μM for K41A, p = 0.64), indicating that K41 may only be involved in one of the substrate translocation pathways of OATP1B1. The replacement of K49 with arginine, however, did not rescue the transporter uptake function, suggesting that the charged property as well as the structural characteristics of lysine side-chain is important for OATP1B1 activity. Km value of the low binding affinity component of ES is increased in K49A, which implicated that the residue is involved in substrate interaction at the binding site. Vmax for both high and low affinity binding sites were significantly reduced in K49A, even after uptake function was normalized with cell surface protein level of the transporter, indicating that the position is important for substrate turn-over of OATP1B1 as well. Homology modeling (Biasini et al., 2014) of OATP1B1 using E.coli glycerol-3-phosphate transporter (PDB: 1pw4) as the template suggested that K41 and K49 may indeed directly interact with estrone-3sulfate (Fig. 9A). It should be noted that quite a few transporters with
2005). OATP1B1 is highly homologous to OATP1B3, thus may share some structural similarity with the transporter. In addition, previous studies of others and us have demonstrated that essential amino acid residues for substrate transport lined-up along multiple transmembrane domains of OATP1B1. In the present study, alanine-scanning was applied to study the residues within the putative TM1 of OATP1B1 and two lysine residues, i.e. K41 and K49, along with I46 was found to be important for substrate uptake of the transporter. Lysine 41 was identified before in a study of OATP1B3 to be important for the uptake of sulphobromophthalein (BSP) and pravastatin (Glaeser et al., 2010). The positively charge residue is conserved among OATP1 family members (Fig. 1). Further analysis demonstrated that conservative replacement of lysine with arginine partially recovered the cell surface protein abundance and the uptake function, suggesting that a positively charged residue at this position is important for maintaining the proper protein level of OATP1B1. In addition, kinetic analysis showed that alanine substitution of K41 altered interaction of the transporter and substrate at the high affinity binding site of ES (0.20 ± 0.04 μM for OATP1B1-WT vs 0.12 ± 0.03 μM for K41A, p = 0.04), suggesting that 7
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Fig. 8. Identification of the N-terminus essential regions for protein stability and uptake function of OATP1B1. A. Estrone-3-sulfate uptake by mutants within the region of aa19–27 at the N-terminus of OATP1B1. Uptake of ES (50 nM) by HEK293 cells expressing OATP1B1 or mutants was measured at 37 °C at a 2 min interval. Net uptake of mutants was compared to that of OATP1B1 wild-type and the relative percentage was presented. The results represent data from three independent experiments, each with duplicate measurements. The results shown are means ± S.D. (n = 3). B&C. Representative blots for cell surface and total protein level of YCNGL or KKTR mutants (left panels). The intensity of protein bands was quantified with Image J and calculated relative to the wild-type (right panels). The results shown are means ± S.D. (n = 3). Cells were labeled with biotin and the proteins were extracted and analyzed as described in Fig. 4. ANOVA was performed for statistical analysis. Different letters indicate significant difference between each other (p < 0.05). D. Estrone-3-sulfate uptake by mutants normalized with the corresponding cell surface transporter protein level. Net uptake of mutants was compared to that of OATP1B1 wild-type and the relative percentage was presented. ANOVA was performed for statistical analysis. Different letters indicate significant difference between each other (p < 0.05). E. Uptake of taurocholate by YCNGL mutants. Uptake of taurocholate (1 μM) was measured at 37 °C at a 2 min interval. Net uptake of mutants was compared to that of OATP1B1 wild-type and the relative percentage was presented. The results represent data from three independent experiments, each with duplicate measurements. The results shown are means ± S.D. (n = 3). ANOVA was performed for statistical analysis. Different letters indicate significant difference between each other (p < 0.05).
The identification of only three essential amino acid residues within TM1 of OATP1B1 is kind of unexpected. It has been proposed that TM1, along with TM2, 4 and 5 of the N-terminal half may be involved in the formation of the substrate interaction pocket and play an important role for the transport activity of OATP family members OATP1B3 and 2B1 (Meier-Abt et al., 2005). Indeed, a recent study by our own identified nine essential residues within the TM1 of OATP2B1 (Fang et al., 2018). Interestingly, Q62 in OATP2B1, which corresponds to K41 in OATP1B1, was also found to be an irreplaceable residue for the proper function of OATP2B1 and alanine substitution of the residue resulted in marginal but significant change of Km. Except for the OATP1 transporters, a polar, non-charged glutamine residue is found in all the other human OATPs. The replacement of K41 with glutamine also resulted in significant reduction of ES uptake (data not shown). We speculated that the corresponding residue of other OATPs may be situated at a different position relative to the substrate translocation pathway and hence play a different role for the uptake by these transporters. Further studies will be needed to clarify such an issue. Although estradiol-17β-glucuronide (E2G) is a sensitive in vitro OATP1B1 probe substrate (Izumi et al.,
their structures depicted have a comparable identity with OATP1B1 (~15%) to that of the E.coli glycerol-3-phosphate transporter, including the human glucose transporter family member 1 (PDB: 4pyp). However, homology regions of the glycerol-3-phosphate transporter cover the most extended area, i.e. from TM1-TM12 of OATP1B1, and it was therefore chosen to serve as the template for the present study. Besides the two positively charged amino acid residues, a dramatic reduction of ES and taurocholate uptake was also found for I46A, although protein abundance of the mutant is comparable to that of the wild-type transporter. When I46 was replaced with leucine, an amino acid with a similar side group structure, uptake was significantly increased, implicating the importance of the side chain structural feature at the position. Kinetic analysis revealed that Km and Vmax values were altered in both high and low affinity binding sites of ES, which suggested that I46 is involved in the substrate interaction of both components. Isoleucine is only found in OATP1B1 and 1B3 among the 11 human OATP members. Since alanine replacement of the residue exhibited such a significant effect, we speculate that it may play a substrate-specific role for these transporters. 8
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Fig. 9. Homology modeling of OATP1B1. A. Interaction of K41 and K49 of OATP1B1 with estrone-3-sulfate. B. Structural comparison of OATP1B1 wild-type and the DEL-YCNGL mutant. The structure of OATP1B1 was modeled with the web-based protein structure prediction service Swiss-model (https://www.swissmodel.expasy. org/) using E. coli glycerol-3-phosphate transporter (PDB: 1pw4) as the template.
showed a minor effect on OATP1B1 function; while the deletion of residues 2–27 (DEL-27) dramatically decreased protein level of the transporter and treatment of MG132 partially recovered its protein abundance as well as uptake function, suggesting the region of residues 19–27 is important for proper function of OATP1B1. To pinpoint the specific essential region(s), sequence of 19–27 was analyzed and it was found that residues18–22 contain a cluster of positively charged residues KKTR, and residues 19–27 possess a sequence of YCNGL that is similar to a conservative sorting motif YXXϕ. Further analysis demonstrated that the KKTR region may only be important for protein level of OATP1B1; while the YCNGL domain not only affects protein abundance but is also involved in substrate interaction of the transporter. However, unlike the N-terminus region of OATP1B3, which plays a crucial role in membrane trafficking, neither the KKTR nor the YCNGL domain seemed to affect targeting of OATP1B1. Because cell surface protein level of the deletion mutants correlated well with the mature form protein level in the total cell lysate (Fig. 8B & C). However, it should be noted that western blotting is a semi-quantitative quantification method for the analysis of protein level and further investigation will be needed to clarify the issue. The YCNGL sequence is unique for OATP1B1 (though the KKTR region is shared by OATP1B3) (Fig. 1) and hence may be important for specific substrate recognition as well. The altered Km value for the high affinity binding component and reduced Vmax for both binding sites of ES suggested that the YCNGL region may be essential for multiple binding components of OATP1B1. We speculated that the domain may play a role in maintaining a proper structure for substrate binding of the transporter. Homology modeling implicated that with YCNGL deleted, a dramatic position alteration would be observed for extracellular loops 2 and 3 of OATP1B1 (Fig. 9B). In summary, investigation of the putative TM1 of OATP1B1 identified two positively charged residues at position 41 and 49 along with a hydrophobic I46 residue that are important for substrate binding and/ or maintaining the protein level of the transporter; while within the N-
2013) and commonly used in the studies of OATP1B1, estrone-3-sulfate was selected as the prior substrate in our present study because OATP1B1 has two binding sites for ES (Noé et al., 2007; Gui and Hagenbuch, 2009; Li et al., 2012) and the analysis of the effect of mutations on both binding sites can help us more thoroughly evaluate the residues that are responsible for the uptake of different substrates. Since E2G and low concentration of ES (0.1 μM) has been found to competitively inhibit each other (Izumi et al., 2013)), suggesting that E2G may share the high affinity binding site of ES in the uptake process of OATP1B1, it may be likely that K41A and I46A, both of which were shown to affect the Km of the high affinity component of ES, may exhibit altered Km for E2G as well. Additionally, based on the kinetic parameters obtained in our study, it may also be possible that inhibitors of OATP1B1 such as rifampin and cyclosporine A would have a higher IC50 for K41A and I46A because binding affinity for ES was increased in the alanine mutants. In should be noted that besides the three residues identified in our present study, H54, which was predicted to be located at the extracellular entry of the putative TM1 of OATP1B1, was also demonstrated to be involved in the interaction with substrates such as bromosulfophthalein (BSP), estradiol-17β-glucuronide (E2G)], taurocholate and pravastatin (Gruetz et al., 2016). However, the TM1 in this study was predicted with an OATP1B1 model that was established based on the closely related OATP1B3 (Meier-Abt et al., 2005) and ranges from residue 30 to 54, which is different from the location of the putative TM1 of our study. Although alanine replacement of K41, K49 or I46 dramatically reduced the uptake function of OATP1B1, there is still a significant amount of transporter protein on the cell surface. Since the N-terminal region ranging from amino acid residue 1 to 50 of OATP1B1 was proposed to be essential for its membrane targeting (Chun et al., 2017), we truncated the first 18 or 27 residues of OATP1B1 to identify the specific region that may be important for the cell surface protein level of the transporter. The deletion of amino acid residues 2–18 (DEL-18) only 9
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terminus of the protein, the KKTR region is important for protein abundance, and YCNGL was found to be essential for substrate interaction as well as maintaining the protein level. The identification of crucial residues and/or motifs of OATP1B1 may help us better understand the therapeutics significance of OATP1B1 transmembrane domains and the underlying molecular and cellular mechanisms for the absorption, distribution and disposition of different drugs by the transporter. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.taap.2019.114642.
10.1080/00498250801986951. Hagenbuch, B., Meier, P.J., 2003. The superfamily of organic anion transporting polypeptides. Biochim. Biophys. Acta 1609, 1–18. Hong, W., Wu, Z., Fang, Z., Huang, J., Huang, H., Hong, M., 2015. Amino acid residues in the putative transmembrane domain 11 of human organic anion transporting polypeptide 1B1 dictate transporter substrate binding, stability, and trafficking. Mol. Pharm. 12, 4270–4276. https://doi.org/10.1021/acs.molpharmaceut.5b00466. Huang, J., Li, N., Hong, W., Zhan, K., Yu, X., Huang, H., Hong, M., 2013. Conserved tryptophan residues within putative transmembrane domain 6 affect transport function of organic anion transporting polypeptide 1B1. Mol. Pharmacol. 84, 521–527. https://doi.org/10.1124/mol.113.085977. Izumi, S., Nozaki, Y., Komori, T., Maeda, K., Takenaka, O., Kusano, K., Yoshimura, T., Kusuhara, H., Sugiyama, Y., 2013. Substrate-dependent inhibition of organic anion transporting polypeptide 1B1: comparative analysis with prototypical probe substrates estradiol-17β-glucuronide, estrone-3-sulfate, and sulfobromophthalein. Drug Metab. Dispos. 41, 1859–1866. https://doi.org/10.1124/dmd.113.052290. König, J., 2011. Uptake transporters of the human OATP family: molecular characteristics, substrates, their role in drug-drug interactions, and functional consequences of polymorphisms. Handb. Exp. Pharmacol. 201, 1–28. https://doi.org/10.1007/978-3642-14541-4_1. Li, N., Hong, W., Huang, H., Lu, H., Lin, G., Hong, M., 2012. Identification of amino acids essential for Estrone-3-sulfate transport within transmembrane domain 2 of organic anion transporting polypeptide 1B1. PLoS ONE 7, e36647. https://doi.org/10.1371/ journal.pone.0036647. Meier-Abt, F., Mokrab, Y., Mizuguchi, K., 2005. Organic anion transporting polypeptides of the OATP/SLCO superfamily: identification of new members in nonmammalian species, comparative modeling and a potential transport mode. J. Membr. Biol. 208, 213–227. Miyagawa, M., Maeda, K., Aoyama, A., Sugiyama, Y., 2009. The eighth and ninth transmembrane domains in organic anion transporting polypeptide 1B1 affect the transport kinetics of estrone-3-sulfate and estradiol-17beta-D-glucuronide. J. Pharmacol. Exp. Ther. 329, 551–557. https://doi.org/10.1124/jpet.108.148411. Nakanishi, T., Tamai, I., 2012. Genetic polymorphisms of OATP transporters and their impact on intestinal absorption and hepatic disposition of drugs. Drug Metab. Pharmacokinet. 27, 106–121. Noé, B., Hagenbuch, B., Stieger, B., Meier, P.J., 1997. Isolation of a multi-specific organic anion and cardiac glycoside transporter from rat brain. Proc. Natl. Acad. Sci. U. S. A. 94, 10346–10350. Noé, J., Portmann, R., Brun, M.E., Funk, C., 2007. Substrate-dependent drug-drug interactions between gemfibrozil, fluvastatin and other organic anion-transporting peptide (OATP) substrates on OATP1B1, OATP2B1, and OATP1B3. Drug Metab. Dispos. 35, 1308–1314. Poirier, A., Funk, C., Lavé, T., Noé, J., 2007. New strategies to address drug-drug interactions involving OATPs. Curr. Opin. Drug. Discov. Dev. 10, 74–83. Shitara, Y., Sato, H., Sugiyama, Y., 2005. Evaluation of drug-drug interaction in the hepatobiliary and renal transport of drugs. Annu. Rev. Pharmacol. Toxicol. 45, 689–723. Shitara, Y., Maeda, K., Ikejiri, K., Yoshida, K., Horie, T., Sugiyama, Y., 2013. Clinical significance of organic anion transporting polypeptides (OATPs) in drug disposition: their roles in hepatic clearance and intestinal absorption. Biopharm. Drug Dispos. 34, 45–78. https://doi.org/10.1002/bdd.1823. Stieger, B., Hagenbuch, B., 2014. Organic anion transporting polypeptides. Curr. Top. Membr. 73, 205–232. https://doi.org/10.1016/B978-0-12-800223-0.00005-0. Weaver, Y.M., Hagenbuch, B., 2010. Several conserved positively charged amino acids in OATP1B1 are involved in binding or translocation of different substrates. J. Membr. Biol. 236, 279–290. https://doi.org/10.1007/s00232-010-9300-3.
Acknowledgements This work was supported by the National Natural Science Foundation of China [grant number U1832101, 81373473&11335011] and Guangzhou Science and Technology Program [grant number 201707010412]. References Biasini, M., Bienert, S., Waterhouse, A., Arnold, K., Studer, G., Schmidt, T., Kiefer, F., Cassarino, T.G., Bertoni, M., Bordoli, L., Schwede, T., 2014. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258. https://doi.org/10.1093/nar/gku340. Bonifacino, J.S., Traub, L.M., 2003. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72, 395–447. Bossuyt, X., Müller, M., Meier, P.J., 1996. Multispecific amphipathic substrate transport by an organic anion transporter of human liver. J. Hepatol. 25, 733–738. Chun, S.E., Thakkar, N., Oh, Y., Park, J.E., Han, S., Ryoo, G., Hahn, H., Maeng, S.H., Lim, Y.R., Han, B.W., Lee, W., 2017. The N-terminal region of organic anion transporting polypeptide 1B3 (OATP1B3) plays an essential role in regulating its plasma membrane trafficking. Biochem. Pharmacol. 131, 98–105. https://doi.org/10.1016/j.bcp. 2017.02.013. Fang, Z., Huang, J., Chen, J., Xu, S., Xiang, Z., Hong, M., 2018. Transmembrane domain 1 of human organic anion transporting polypeptide 2B1 is essential for transporter function and stability. Mol. Pharmacol. 94, 842–849. https://doi.org/10.1124/mol. 118.111914. Glaeser, H., Mandery, K., Sticht, H., Fromm, M.F., König, J., 2010. Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3. Br. J. Pharmacol. 159, 698–708. https:// doi.org/10.1111/j.1476-5381.2009.00568.x. Gruetz, M., Sticht, H., Glaeser, H., Fromm, M.F., König, J., 2016. Analysis of amino acid residues in the predicted transmembrane pore influencing transport kinetics of the hepatic drug transporter organic anion transporting polypeptide 1B1 (OATP1B1). Biochim. Biophys. Acta 1858, 2894–2902. https://doi.org/10.1016/j.bbamem.2016. 08.018. Gui, C., Hagenbuch, B., 2009. Role of transmembrane domain 10 for the function of organic anion transporting polypeptide 1B1. Protein Sci. 18, 2298–2306. https://doi. org/10.1002/pro.240. Hagenbuch, B., Gui, C., 2008. Xenobiotic transporters of the human organic anion transporting polypeptides (OATP) family. Xenobiotica 38, 778–801. https://doi.org/
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