The functional evaluation of human peptide/histidine transporter 1 (hPHT1) in transiently transfected COS-7 cells
Introduction
The Proton-coupled Oligopeptide Transporter (POT) superfamily comprises peptide transporters that utilize a proton-dependent mechanism for the transmembrane transport of primarily di- and tripeptide-based substrates (Herrera-Ruiz and Knipp, 2003, Daniel and Kottra, 2004). POT family members have been reported in mammals, bacteria, fungi, yeast and plants (Meredith and Boyd, 2000). A large number of genes that encode components of oligopeptide transport systems in bacteria have been cloned and sequenced. In contrast, a minimal number of eukaryotic and mammalian peptide transporters have been reported (Meredith and Boyd, 2000). In humans, the primary POT isoforms studied have been PepT1 (SLC15A1) and PepT2 (SLC15A2), both of which have been widely demonstrated to actively transport numerous peptide-based substrates including di- and tripeptides derived from the 20 proteinogenic l-amino acids (Herrera-Ruiz and Knipp, 2003, Daniel and Kottra, 2004). Comparatively, PepT1 is mainly expressed in epithelial cells in the intestine, S1 segment of renal proximal tubular cells, the liver and in bile duct epithelial cells (Shen et al., 1999, Herrera-Ruiz et al., 2001, Knutter et al., 2002, Herrera-Ruiz and Knipp, 2003), while PepT2 is primarily expressed in the S2 and S3 segments of the apical membranes in kidney tubular cells, brain astrocytes, epithelial cells of choroids plexus, mammary gland and bronchial epithelial cells (Berger and Hediger, 1999, Groneberg et al., 2001, Groneberg et al., 2002, Izzedine et al., 2005).
Recently, two additional putative peptide/histidine transporters (PHT1 [SLC15A4] and PHT2 [SLC15A3]) and their different splice variants were identified in the human genome and databases (Botka et al., 2000, Herrera-Ruiz and Knipp, 2003, Knipp and Herrera-Ruiz, 2004). Mammalian PHT1 was originally cloned from a rat brain cDNA library and characterized in rat tissues, demonstrating a high affinity for histidine and the histidine-containing dipeptide, carnosine (Yamashita et al., 1997). The rPHT1 cDNA had an open reading frame of 1719 bp, encoding a 572 amino acid protein with a predicted molecular mass of 64.9 kDa, 12 predicted transmembrane domains (TMD) and the POT family member signatures. Yamashita et al. (1997) also analyzed the rPHT1 mRNA expression in several tissues by Northern blot analysis, with expression shown primarily in the brain, eye, lung and spleen. Additional in situ hybridization studies using a 35S-labeled cRNA probe revealed rPHT1 localization in several regions of the rat brain, such as the hippocampus, cerebellum and pontine nucleus, and in lower levels of the cerebral cortex, brain stem, thalamus and hypothalamus. Moreover, RT-PCR and Southern blot analyses have also demonstrated rPHT1 mRNA expression throughout the rat GI-tract and placenta (Herrera-Ruiz et al., 2001). Overall, the reported putative hPHT1 sequence (GI:21717816) is 86.5% identical to the rPHT1 isoform and 48.4% identical to rPHT2. Further comparison of the hPHT1 protein sequence with other proteins revealed that hPHT1 contains conservative domains found in several protein sequences relevant to the POT family (Botka et al., 2000, Herrera-Ruiz and Knipp, 2003, Daniel and Kottra, 2004).
While the functional analysis of rPHT1 has been characterized, the identity and functional characterization of the human orthologue have not yet been widely studied. Our objective was to clone and characterize the human orthologue of the peptide/histidine transporter 1 (hPHT1) as a potential intestinal transporter of peptide-based nutrients and/or drugs. To accomplish this, the full length human PHT1 sequence was amplified from the human placental trophoblast BeWo cell line, inserted into a pcDNA3.1–V5/His TOPO® plasmid and transiently transfected into COS-7 cells, which were subsequently used to characterize the uptake kinetics of [3H]histidine and [3H]carnosine, as well as study the uptake of [3H]valacyclovir and [3H]glycylsarcosine (GlySar), in general. Several experiments have shown that COS-7 cells can be used to express biologically active cell-surface and secreted proteins (Rose and Bergmann, 1982, Hosokawa et al., 1997, Sakai et al., 2000). We also generated a rabbit polyclonal antibody to hPHT1 and investigated its expression in the stomach, colon and small intestinal segments of three Asian male intestinal tissue protein lysates as well as its cellular localization in the small intestine. Thus, the complete cDNA cloning of human PHT1, its expression in the gastrointestinal tract and the determination of its functional activity are reported below. These results may serve as a template for further assessing the relative physiological significance of this transporter.
Section snippets
Materials
TRIzol® Reagent was obtained from Sigma Chemical Company (St. Louis, MO). Reverse transcriptase-polymerase chain reaction kits, LipofectAMINE® Plus and OPTIMEM were obtained from Invitrogen, Inc. (Carlsbad, CA). The long and accurate polymerase enzyme was obtained from Panvera (Madison, WI). The North2South Biotin Random Prime Kit used in the preparation of DNA probes and the North2South Chemiluminescent Nucleic Acid Hybridization and Detection Kit used for Southern analysis were purchased from
PHT1 protein expression
Invitrogen was contracted to generate a specific rabbit polyclonal antibody to the hPHT1 epitope RSRANGVPTSRRA. The rabbit hPHT1 polyclonal antisera was tested by ELISA analysis and determined to have a high titer for the hPHT1 (Invitrogen, data not shown). In Fig. 1a, the Western blot analysis using the hPHT1 polyclonal antibody reveals a band corresponding to an approximate molecular weight of 83 kDa in a small intestinal protein lysate sample, which was highly specific. Western blot analysis
Discussion
Peptide/histidine transporter 1 was originally cloned from a rat brain library (Yamashita et al., 1997). The rat PHT1 was found to be structurally related to members of the proton-dependent oligopeptide transporter superfamily, where homology with the PepT-like transporters was relatively low (Yamashita et al., 1997). The presence of a human orthologue of rPHT1 was first identified by Botka et al. (2000) and Herrera-Ruiz et al. (2001). The present study describes the expression pattern of hPHT1
Acknowledgements
We would like to thank Stephen Carl and Refika Pakunlu for their technical assistance in preparing this manuscript.
References (27)
- et al.
Localization of the peptide transporter PEPT2 in the lung: implications for pulmonary oligopeptide uptake
Am. J. Pathol.
(2001) - et al.
Current perspectives on established and putative mammalian oligopeptide transporters
J. Pharm. Sci.
(2003) - et al.
Cloning and characterization of a brain-specific cationic amino acid transporter
J. Biol. Chem.
(1997) - et al.
Selective release of peptides from lysosomes
J. Biol. Chem.
(1993) - et al.
Expression from cloned cDNA of cell-surface, secreted forms of the glycoprotein of vesicular stomatitis virus in eukaryotic cells
Cell
(1982) - et al.
Involvement of the actin cytoskeleton in the regulation of serotonin transporter (SET) activity: possible mechanism underlaying SET regulation by protein kinase C
Neurochem. Int.
(2000) - et al.
Targeting of membrane proteins to endosomes and lysosomes
Trends Cell. Biol.
(1994) - et al.
An active mechanism for completion of the final stage of protein degradation in the liver, lysosomal transport of dipeptides
J. Biol. Chem.
(1997) - et al.
Expression of PPAR, RXR isoforms and fatty acid transporting proteins in the rat and human gastrointestinal tracts
J. Pharm. Sci.
(2005) - et al.
Cloning and functional expression of a brain peptide/histidine transporter
J. Biol. Chem.
(1997)
Distribution of peptide transporter PEPT2 mRNA in the rat nervous system
Anat. Embryol. (Berl.)
Delineation of human peptide transporter 1 (hPepT1)-mediated uptake and transport of substrates with varying transporter affinities utilizing stably transfected hPepT1/Madin–Darby canine kidney clones and Caco-2 cells
J. Pharmacol. Exp. Ther.
Human proton/oligopeptide transporter (POT) genes: identification of putative human genes using bioinformatics
AAPS PharmSci
Cited by (0)
- 1
These authors contributed equally to the preparation of this manuscript.