Illuminating the Function of the Orphan Transporter, SLC22A10 in Humans and Other Primates

Abstract

SLC22A10 is classified as an orphan transporter with unknown substrates and function. Here we describe the discovery of the substrate specificity and functional characteristics of SLC22A10. The human SLC22A10 tagged with green fluorescent protein was found to be absent from the plasma membrane, in contrast to the SLC22A10 orthologs found in great apes. Estradiol-17β-glucuronide accumulated in cells expressing great ape SLC22A10 orthologs (over 4-fold, p<0.001). In contrast, human SLC22A10 displayed no uptake function. Sequence alignments revealed two amino acid differences including a proline at position 220 of the human SLC22A10 and a leucine at the same position of great ape orthologs. Site-directed mutagenesis yielding the human SLC22A10-P220L produced a protein with excellent plasma membrane localization and associated uptake function. Neanderthal and Denisovan genomes show human-like sequences at proline 220 position, corroborating that SLC22A10 were rendered nonfunctional during hominin evolution after the divergence from the pan lineage (chimpanzees and bonobos). These findings demonstrate that human SLC22A10 is a unitary pseudogene and was inactivated by a missense mutation that is fixed in humans, whereas orthologs in great apes transport sex steroid conjugates.

Figure 1.

Analysis of the phylogenetic tree, plasma membrane expression of SLC22A10, and uptake of organic anion substrates of the human SLC22 family. A. Multiple sequence alignments were performed with reference amino acid sequences for each anion transporter from humans and rodents, using the Clustal Omega Multiple Sequence Alignment program (https://www.ebi.ac.uk/Tools/msa/clustalo/). The dendrogram was generated from the output of the Clustal Omega alignment. B. Localization of human SLC22A10 conjugated to green fluorescent protein (GFP) was examined in HEK293 cells using high-content imaging and cellular staining with the plasma membrane marker wheat germ agglutinin (WGA). The results showed no colocalization of GFP-tagged SLC22A10 with WGA. C. Uptake of various radiolabeled organic anions, which are typical substrates of organic anion transporters in the SLC22A family, was assessed. Uptake was performed 48 hours after transient transfection of plasmids encoding human SLC22A10, GFP expression vector, and one other member in the SLC22A family as a positive control. Accumulation of substrates inside cells was determined after 15 minutes. Figure shows a representative plot from one experiment (mean ± S.D. from three replicate wells). The experiments were repeated at least one time and showed similar results. Multiple comparisons using one-way analysis of variance followed by Dunnett’s two-tailed test were performed. HEK293 cells transiently transfected with the GFP vector served as the control. The fold uptake of the substrate, relative to the control cells, was plotted based on one representative experiment conducted in triplicate wells (mean ± s.d.). The statistical significance for all the cells transfected with organic anion transporters SLC22A6, SLC22A8, or SLC2224 is p<0.001.

Figure 2.

Localization to the plasma membrane, uptake, and sequence comparison of human SLC22A10 were examined in comparison with SLC22A10 from great apes (chimpanzee, bonobo, gorilla and orangutan). A. This figure shows the plasma membrane localization of SLC22A10 orthologs from great apes, which were conjugated to green fluorescent protein (GFP) in HEK293 cells. The GFP tag is located at the N-terminus of SLC22A10. Confocal imaging revealed that the 552 amino acid isoforms of SLC22A10 from chimpanzee, bonobo, gorilla, and orangutan primarily colocalized with wheat germ agglutinin (WGA) on the plasma membrane of the cell. In contrast, the 540 amino acid isoform of SLC22A10 from bonobo, chimpanzee, and gorilla showed no colocalization of GFP-tagged SLC22A10 with WGA on the plasma membrane, suggesting intracellular localization in the cytoplasm. B. The uptake of [³H]-estradiol-17b-glucuronide was determined in HEK293 cells overexpressing either a GFP expression vector or SLC22A10 expression vectors containing sequences from various primates including human, chimpanzee, bonobo, gorilla, and orangutan. SLC22A10 orthologs from chimpanzee, bonobo, gorilla, and orangutan expressing the longer isoform (552 amino acids) significantly accumulated [³H]-estradiol-17b-glucuronide. Please refer to the “Statistical Analysis“ section for details on the statistical methods used to determine the significance of each cell transfected with the different SLC22A10 orthologs. C. Sequence alignments of the last exon of SLC22A10 in human, chimpanzee, bonobo, gorilla, and orangutan are shown. In humans, the frequency of the A-allele insertion is significantly greater (98%) than in chimpanzees (2.5%) and is not present in available sequences from bonobos, gorillas, or orangutans. The A-allele insertion results in the expression of human SLC22A10 with 541 amino acids, while bonobo, gorilla, orangutan and the majority of chimpanzees are predicted to express isoforms of SLC22A10 with 552 amino acids.

Figure 3.

A single mutation of proline to leucine at amino acid position 220 of human SLC22A10 significantly enhances the accumulation of [³H]-estradiol-17b-glucuronide in HEK293. A. The amino acid sequence alignment of human SLC22A10 and SLC22A10 from other great apes (chimpanzee, bonobo and gorilla) shows that only the amino acids at positions 18 and 220 differ between the human ortholog and orthologs from great apes. Additionally, there are several amino acid differences starting at position 533. B. The uptake of [³H]-estradiol-17b-glucuronide in HEK293 cells transiently transfected with plasmids encoding human SLC22A10 with reference amino acids or amino acids that are similar to those found in other great apes, namely SLC22A10-p.M18I and SLC22A10-p.P220L. A chimeric protein consisting of the first 533 amino acids of human SLC22A10 and the last 19 amino acids of chimpanzee SLC22A10 (534–552) was also evaluated, but did not significantly accumulate [³H]-estradiol-17b-glucuronide compared to the chimeric protein with p.P220L. The fold uptake of the substrate, relative to the control (GFP) cells, was plotted based on one representative experiment conducted in triplicate wells (mean ± s.d.). The statistical significance for cells transfected with SLC22A10 #4 (Human SLC2210 p.P220L (541 aa)), #5 (Human SLC2210 p.P220L (1–533) + Pt SLC22A10 (534 – 552)) and #6 (Chimp SLC2210 (552 aa)) is p<0.001. C. This figure shows the plasma membrane localization of SLC22A10 conjugated to green fluorescent protein (GFP) in HEK293 cells. The GFP tag is located at the N-terminus of SLC22A10. Confocal imaging revealed that human SLC22A10-p.P220L localizes primarily to the plasma membrane of the cell, while there was no localization to the plasma membrane in cells expressing a chimeric protein or chimpanzee SLC22A10 with proline at the 220 amino acid position.

Figure 4.

SLC22A10 of chimpanzees, bonobos, orangutans, and gibbons are predicted to have shorter isoforms expressing 533 or 538 amino acids. A. A comparison of the SLC22A10 amino acid sequence of humans, chimpanzees, bonobos, and orangutans, which express 533 (chimpanzee, orangutan, gibbon), 538 (bonobo), 540 (bonobo, chimpanzee), or 541 (human) amino acids, shows that the major differences are at the end of the SLC22A10 sequence. B. Confocal imaging revealed that SLC22A10 from chimpanzees and bonobos (isoforms expressing 533 or 538 amino acids) primarily localize to the plasma membrane of the cell, whereas weaker localization was observed for orangutan SLC22A10 (533 amino acids) to the plasma membrane of the cell. GFP conjugated to SLC22A10 was used for this experiment. C. The uptake of [³H]-estradiol-17b-glucuronide in HEK293 cells was observed after transient transfection of plasmids encoding human SLC22A10 with reference amino acids or SLC22A10 with reference amino acids of other great apes with different isoforms. The results showed that SLC22A10 isoforms expressing 533 and 552 amino acids significantly accumulate the substrate. However, weaker substrate accumulation was observed in cells transfected with the bonobo SLC22A10 isoform expressing 538 amino acids.

Figure 5.

This figure presents information about the transport mechanism and kinetics of chimpanzee SLC22A10. A. The uptake of seven steroid glucuronides and two steroids in HEK293 cells stably transfected with chimpanzee SLC22A10 (552 amino acids) was measured using LC/MS-MS to determine the accumulation of the compounds. B. The effect of pH on accumulation of [³H]-estradiol-17b-glucuronide in HEK293 cells stably transfected with chimpanzee SLC22A10 isoforms expressing 533 and 552 amino acids was investigated. C. The effect of sodium and chloride on accumulation of [³H]-estradiol-17b-glucuronide in HEK293 cells stably transfected with 533 and 552 amino acid isoforms of chimpanzee SLC22A10 was investigated. D. The effects of trans-stimulation of [³H]-estradiol-17b-glucuronide uptake by chimpanzee SLC22A10 was determined. Uptake was trans-stimulated by preloading the cells with 2 mM of butyrate, glutaric acid, alpha-ketoglutarate, or succinic acid for 2 hours, and then measuring the uptake of [³H]-estradiol-17b-glucuronide after 15 minutes. The data are presented as mean ± S.D. and were normalized by setting the uptake of SLC22A10-expressing cells trans-stimulated by HBSS to 1.0. Trans-stimulation of [³H]-estradiol-17b-glucuronide by glutaric acid was observed for both isoforms of chimpanzee SLC22A10. E. The kinetics of [³H]-estradiol-17b-glucuronide uptake for chimpanzee SLC22A10 isoforms expressing 533 and 552 amino acids were analyzed. The uptake rate was evaluated at 5 minutes and the data were fit to a Michaelis-Menten equation. To fit the kinetic curve to a Michaelis-Menten equation, the concentration of estradiol-17b-glucuronide is set up to 10 μM. The figure shows a representative plot from one experiment. All experiments were repeated once, in triplicate and showed similar results.