Biochemical and Phylogenetic Analysis of Organic Anion Transporting Polypeptides

Abstract

Organic Anion Transporting Polypeptides (Human: OATPs, all others: Oatps), are sodium-independent membrane transporters predicted to have 12-transmembrane domains. Oatps/OATPs mediate the transport of a broad range of endo- and xenobiotics including bile salts, hormones and their conjugates, statins, thyroid hormones, prostaglandins, and anticancer agents. Oatps have varying tissue expression profiles with some Oatps having a very discrete tissue-specific expression pattern while other Oatps are ubiquitously expressed throughout multiple tissues. The functional diversity displayed by Oatps/OATPs has made it difficult to understand their overall physiological role. The research presented here takes a two-pronged approach to elucidate the evolved physiological role of Oatps. The first approach investigates the active site structure of a specific Oatp, Oatp1c1, through transport inhibition and the second approach involves a comprehensive phylogenetic analysis of the Oatp family. Oatp1c1 is expressed predominantly in the blood-brain barrier and the blood-cerebrospinal fluid barrier cells with minimal expression in specialized cells of the eye and gonads. Oatp1c1 has a narrower range of recognizable substrates compared to other Oatps. However, Oatp1c1 is a high affinity thyroxine transporter with the lowest Km¬ yet identified (90nM-340nM) of all transporters, indicating an important role in thyroid hormone homeostasis. Previous research suggests Oatp1c1 contains multiple binding sites based on the biphasic uptake curves of T4 and E2-17β-G as well as T4 and E2-17β-G cis-inhibition values differing from their Km¬s. Unpublished data from the Rumbley Laboratory was used to characterize the Oatp1c1 T4 uptake site by calculating IC50 values for multiple inhibitors of T4 uptake. However, the Oatp1c1 E2-17β-G uptake site is not fully characterized. In this study, HEK293 cells were transiently transfected with a Gateway expression vector, pEF-DEST 51, containing the Oatp1c1 gene sequence and uptake of 3H-E2-17β-G was assessed. Oatp1c1 expressing cells were found to uptake 3H-E2-17β-G 4-fold over empty vector transfected cells and uptake was shown in a time dependent manner. Next, multiple inhibitors were used to test their ability to inhibit the uptake of 3H-E2-17β-G and preliminary IC50 values were calculated and compared to the IC50 values calculated for the same inhibitors against the uptake of T4. Data presented here supports the idea of Oatp1c1 possessing a single flexible binding site capable of binding multiple substrate molecules simultaneously. However, the presence of multiple substrate binding sites capable of communication was not ruled out. Oatps/OATPs are promiscuous membrane transporters and are highly conserved proteins present in all metazoans sequenced to date but are absent in all other organisms including plants, bacteria and yeast. Sequence alignments of Homo sapiens OATPs with that of primitive organisms have shown Oatps/OATPs have a high level of evolutionary conservation. Understanding why such primitive organisms like Monosiga, Trichoplax and Nematostella possess Oatps may allow clarification of the evolved physiological role of Oatps/OATPs. Due to the high level of Oatps/OATPs conservation among all metazoans, understanding the physiological purpose these transport proteins serve is important, especially since Oatps/OATPs are present in such a diverse range of organisms. For comprehensive phylogenetic analysis of the Oatp family, H. sapiens OATP4A1 protein sequence was used as the query sequence for BLASTp searches completed against organisms with complete or nearly complete genomes. Multiple sequence alignments of all 1264 sequences were completed using MAFFT, QuickProbs and Clustal Omega. These alignments were compared to one another and subsequently submitted to FastTree 2.0 and MrBayes 3.2 for phylogenetic analysis. Amphimedon queenslandica was identified as the first metazoan to possess Oatp protein sequences more closely related to the Oatp4 family than to any other indicating the Oatp4 family was likely the first to evolve. Oatps/OATPs where shown to cluster into seven divergent families, with the seventh labeled as the Uncharacterized Oatp cluster. The Uncharacterized Oatp cluster emerges with the Bilaterian split of Protostomia and Deuterostomia but is lost somewhere in the chordate lineage before the emergence of vertebrates. Combined, both research studies will aid in the understanding of the evolved biological role of Oatps/OATPs and potentially lead to rational drug design of specifically targeted pharmaceutics.