hPepT1 mediates bacterial tripeptide fMLP uptake in human monocytes.
Here, we examined hPepT1 expression in the monocytic cell line, KG-1. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that hPepT1 is expressed in KG-1 cells, while cDNA cloning and direct sequencing confirmed the sequence of KG-1 hPepT1 (accession number, AY634368). Immunoblotting of cell lysates from KG-1 cells or macrophages isolated from human peripheral blood revealed a approximately 100 kDa immunoreactive band mainly present in the membrane fraction. Uptake experiments showed that the transport of 20 microM radiolabeled Gly-Sarcosine ([14C]Gly-Sar) in KG-1 cells was Na+, Cl- dependent and disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS)-sensitive. In addition, hPepT1 activity was likely to be coupled to a Na+/H+ exchanger, as evidenced by the fact that [14C]Gly-Sar uptake was not affected by the absence of Na+ when cells were incubated at low pH (5.2). Interestingly, hPepT1-mediated transport was reduced in KG-1 cells incubated at low pH as it was also observed in nonpolarized Caco2-BBE cells. This pattern of pH-dependence is due to a disruption of the driving force of hPepT1-mediated transport events. This was supported by our finding that nonpolarized cells, Caco2-BBE cells and KG-1 cells, have an increased permeability to H+ when compared to polarized Caco2-BBE cells. Finally, we showed that hPepT1 is responsible for transporting fMLP into undifferentiated and differentiated (macrophage-like) KG-1 cells. Together, these results show that hPepT1 is expressed in nonpolarized immune cells, such as macrophages, where the transporter functions best at the physiological pH 7.2. Furthermore, we provide evidence for hPepT1-mediated fMLP transport, which might constitute a novel immune cell activation pathway during intestinal inflammation.
Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
Laetitia
L
Charrier
Adel
A
Driss
Yutao
Y
Yan
Vivienne
V
Nduati
Jan-Michael
JM
Klapproth
Shanthi V
SV
Sitaraman
Didier
D
Merlin
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D001693:Biological Transport, Active; D018938:Caco-2 Cells; D002454:Cell Differentiation; D002460:Cell Line; D002463:Cell Membrane Permeability; D016764:Cell Polarity; D004151:Dipeptides / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D009000:Monocytes / Q000166:cytology / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D000074011:Peptide Transporter 1; D011522:Protons; D012964:Sodium / Q000378:metabolism; D027981:Symporters / Q000096:biosynthesis / Q000502:physiology*
D004151:Dipeptides; D000074011:Peptide Transporter 1; D011522:Protons; C082186:SLC15A1 protein, human; D027981:Symporters; C004194:glycylsarcosine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid