Functional Characterization of Human Peptide/Histidine Transporter 1 in Stably Transfected MDCK Cells

Abstract

The proton-coupled oligopeptide transporter PHT1 (SLC15A4), which facilitates cross-membrane transport of histidine and small peptides from inside the endosomes or lysosomes to cytosol, plays an important role in intracellular peptides homeostasis and innate immune responses. However, it remains a challenge to elucidate functional properties of the PHT1 transporter because of its subcellular localization. The purpose of this study was to resort hPHT1 protein from the subcellular to outer cell membrane of MDCK cells stably transfected with human PHT1 mutants, and to characterize its functional activity in these cells. Using this model, the functional activity of hPHT1 was evaluated by cellular uptake studies with d-l-histidine, GlySar, and the bacterial peptidoglycan products MDP and Tri-DAP. We found that the disruption of two dileucine motifs was indispensable for hPHT1 transporter being preferentially targeting to plasma membranes. hPHT1 showed high affinity for d-l-histidine and low affinity for GlySar, with K values of 16.3 ± 1.9 μM and 1.60 ± 0.30 mM, respectively. Moreover, the bacterial peptidoglycan components MDP and Tri-DAP were shown conclusively to be hPHT1 substrates. The uptake of MDP by hPHT1 was inhibited by di/tripeptides and peptide-like drugs, but not by glycine and acyclovir. The functional activity of hPHT1 was also pH-dependent, with an optimal cellular uptake in buffer pH 6.5. Taken together, we established a novel cell model to evaluate the function of hPHT1 in vitro, and confirmed that MDP and Tri-DAP were substrates of hPHT1. Our findings suggest that PHT1 may serve as a potential target for reducing the immune responses and for drug treatment of inflammatory diseases.

Regulation and biological role of the peptide/histidine transporter SLC15A3 in Toll-like receptor-mediated inflammatory responses in macrophage

“…SLC15A4, another POT family member, is also localized in endosomes and lysosomes, and has similar transport properties with SLC15A3 9 , 10 . Several studies have reported that SLC15A4 is closely associated with inflammatory diseases such as diabetes, systemic lupus erythematosus and inflammatory bowel disease 11 – 14 .…”

Determination of intracellular anlotinib, osimertinib, afatinib and gefitinib accumulations in human brain microvascular endothelial cells by liquid chromatography/tandem mass spectrometry

“…A non-volatile SDS solution (0.1%) has been applied in previous studies for the preparation of cell extracts. [23][24][25] In our preliminary experiments, the applicability of SDS as the presence of halogen, but the exact mechanism was not clear.…”

“…However, to decrease volatilization at room temperature, lysing of cells with organic solvents and further scraping of cells from plates are usually conducted on ice, a procedure that is elaborate and time‐consuming. A non‐volatile SDS solution (0.1%) has been applied in previous studies for the preparation of cell extracts 23–25 . In our preliminary experiments, the applicability of SDS (0.025%, 0.05% and 0.1%), sodium acetate (0.2 M) solution and methanol (100%) to lyse cells was evaluated.…”

Functional Characterization of Peptide Transporters in Bovine Mammary Epithelial Cells

“…β-Ala-Lys-AMCA uptake peaked at pH 6.5, which was consistent with previous studies that the maximal uptake of Gly-Sar was observed at pH 6.5 in PC-3 cells, MDCK cells, and LLC-PK1 cells transfected with PepT2 from different species. 2,18,19 Our transport-kinetics assay showed that the transport of β-Ala-Lys-AMCA in BMECs fit into Michaelis−Menten type kinetics and had a K m of 82 ± 18 μM and a V max of 124 ± 11 pmol/min per milligram of protein, which are similar to previously reported values in mouse splenic macrophages (K m = 75.5 μM and V max = 25.4 pmol/min per milligram of protein). 20 These low K m and V max values suggest that the transport of β-Ala-Lys-AMCA in BMECs is mainly mediated by PepT2, which is a highaffinity and low-capacity transporter.…”