A single amino acid Gly-tag enables metal-free protein purification.
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ABSTRACT:
SUBMITTER: Purushottam L
PROVIDER: S-EPMC8163197 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND AND PURPOSE The intestinal proton-coupled amino acid transporter, SLC36A1, transports zwitterionic ?-amino acids and drugs such as vigabatrin, gaboxadol and ?-aminolevulinic acid. We hypothesize that SLC36A1 might also transport some dipeptides. The aim of the present study was to investigate SLC36A1-mediated transport of Gly-Gly and Gly-Gly mimetics, and to investigate Gly-Sar transport via SLC36A1 and the proton-coupled dipeptide/tripeptide transporter, SLC15A1 in Caco-2 cells. EXPERIMENTAL APPROACH Transport of a compound via SLC36A1 was determined by its ability to induce an increase in the inward current of two-electrode voltage clamped SLC36A1 cRNA-injected Xenopus laevis oocytes. SLC36A1-mediated L-[³H]Pro uptake in Caco-2 cells was measured in the absence and presence of Gly-Gly or Gly-Sar. In addition, apical [¹?C]Gly-Sar uptake was measured in the absence and presence of the SLC36A1 inhibitor 5-hydroxy-L-tryptophan (5-HTP) or the SLC15A1 inhibitor L-4,4'-biphenylalanyl-L-proline (Bip-Pro). KEY RESULTS In SLC36A1-expressing oocytes, an inward current was induced by Gly-Sar, Gly-Gly, ?-aminolevulinic acid, ?-aminoethylglycine, ?-aminopentanoic acid, GABA, Gly and Pro, whereas Val, Leu, mannitol, 5-HTP and the dipeptides Gly-Ala, Gly-Pro and Gly-Phe did not evoke currents. In Caco-2 cell monolayers, the apical uptake of 30?mM Gly-Sar was inhibited by 20 and 22% in the presence of 5-HTP or Bip-Pro, respectively, and by 48% in the presence of both. CONCLUSION AND IMPLICATIONS Our results suggest that whereas Gly-Gly amid bond bioisosteres are widely accepted by the hPAT1 carrier, dipeptides in general are not; and therefore, Gly-Sar might structurally define the size limit of dipeptide transport via SLC36A1.
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