Structure-function studies of SLC15 peptide transporters
The proton coupled oligopeptide transporter (POT) family of proteins includes mammalian PepT1 (SLC15A1; high capacity, low affinity) and PepT2 (SLC15A2; low capacity, high affinity) transporters which are involved in the co-transport of protons and di- and tri- peptides and drugs similar in structure (peptidomimetics). PepT1, and to a lesser extent PepT2, have therefore gained attention due to their role in determining the absorption, retention and elimination of transported drugs. In this study three novel prodrug-carriers namely Amino-Isobutynyl-Alanyl-Serine (Aib), Cyclo-Leucyl-Serine (cLu) and Alanyl-[ψCS-NH]-Serine (Thio) were tested for PepT1 mediated transport in PepT1 expressing Xenopus laevis oocytes and differentiated Caco-2 cells grown on Transwell® inserts. The cLu- carrier was found to exhibit higher binding affinity towards PepT1 compared to Aib- and Thio- carriers and to transport the conjugated drugs both in the oocyte and Caco-2 assays. The Aib-and Thio- carriers exhibited similar affinities towards PepT1 and were also found to be transported. Therefore all three carriers were shown to have the potential to deliver a range of conjugated drugs/compounds with differing physicochemical properties through PepT1. Homology models for PepT1/2 have been proposed due to the lack of crystal structure data available for mammalian peptide transporters, based on recent crystal structures of the bacterial POT family members. Although offering invaluable insights, all the bacterial transporters lack the mammalian large extracellular domain (ECD) between transmembrane (TMD) 9 and 10. Human PepT1 (hPepT1) and hPepT2 mutants with their ECD deleted (ECDΔ) or swapped (hPepT1-ECD2, hPepT2-ECD1) were tested for functional effects. The hPepT1-ECDΔ and hPepT1-ECD2 transporters were found not to express, showing a critical structural role for the ECD in hPepT1. In contrast, hPepT2-ECDΔ and hPepT2-ECD1 were found to function as the wild type hPepT2. In a collaborative project, Dr Simon Newstead and colleagues (Department of Biochemistry, Oxford University), crystallised the ECD of mouse PepT1 and rat PepT2. Despite the ECD having a thyroxin binding fold, no evidence of thyroxin hormone interaction with the ECD was found. However hPepT2-ECDΔ mediated transport of [3H]-D-Phe-L-Gln was significantly reduced compared to wild type at extracellular pH 6.5, the pH prevalent in the kidney where PepT2 is predominantly expressed, suggesting that the ECD could function like a pH sensor by allowing hPepT2 to perform at its maximum capacity. Previous point mutation studies on PepT1 have identified several key conserved residues that play critical structure-functional roles. Mutation of the analogous residues in PepT2 indicated that the amino acid residues tested (Y42, Y94, Y188, R301, W313, and E622) played a similar role in PepT2, suggesting functional/structural as well as sequence homology. Finally, rat peptide histidine transporter (ratPHT1, SLC15A4), has been reported to transport L-histidine in addition to di- and tri- peptides, and to be expressed on the neuronal lysosomal membrane. Surface expression studies of FLAG epitope tagged PHT1 protein confirmed that the protein localised to the plasma membrane of oocytes but no evidence for transport of [3H]-L-Histidine or any other amino acid (electrophysiology) was found. Therefore the exact function of the PHT1 transporter still remains unclear. Together, these results presented here demonstrate novel findings into the structurefunction relationship of SLC15 peptide transport proteins.
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