Dipeptide Transport Research Articles

Characterization of an oligopeptide transporter in renal lysosomes

Renal lysosomes play a major role in catabolism of plasma proteins. Final products of this catabolism include dipeptides and tripeptides that must be exported to the cytosol for hydrolysis. The aim of the present study was to determine whether an oligopeptide transporter is present in the renal lysosomal membrane that could mediate this export. The existence of an oligopeptide transporter was probed with the uptake of glycylglutamine (Gly-Gln) by membrane vesicles prepared from renal lysosomes. Kinetic analysis showed the presence of a single transporter with a K m of 8.77 mM for the uptake of Gly-Gln. The Gly-Gln uptake was energized by the imposition of an inwardly directed proton gradient (pH out 5.0/pH in 7.3) and membrane potential (outside positive/inside negative) resulting in overshoot. The Gly-Gln uptake was inhibited by the presence of dipeptides and tripeptides, but not amino acids. Western blot analysis of lysosomal membrane proteins with Pept-1 (an oligopeptide transporter) antibody as the probe showed the presence of an immunoreactive protein. This immunoreaction was abolished when the antiserum was preabsorbed with the Pept-1 epitope (0.5 μg/ml). In conclusion, the present data show the existence of a low-affinity dipeptide transporter in the renal lysosomal membrane that appears to belong to the Pept family of transporters. The function of this transporter appears to be to prevent accumulation of dipeptides in renal lysosomes.

Meeting future challenges in topical ocular drug delivery:: Development of an air-interfaced primary culture of rabbit conjunctival epithelial cells on a permeable support for drug transport studies

The purpose of this study was to develop and characterize a functional air-interfaced primary culture of rabbit conjunctival epithelial cells grown on a permeable support for drug transport studies. Conjunctival epithelial cells from the pigmented rabbit were isolated, seeded at 1.2×10 6 cells cm −2 on permeable Transwell filters, and cultured at the air interface using a modified PC-1 medium. Conjunctival epithelial cell layers showed a transepithelial resistance of 1.1±0.1 kΩ cm 2, a potential difference of 17.0±0.5 mV, and an equivalent short-circuit current ( I eq) of 16.1±0.4 μA cm −2. The I eq was reduced by 35% using 0.01 mM bumetanide, 66% using 0.1 mM ouabain, 46% using 2 mM barium chloride (all three in the basolateral fluid), and 63% using 0.3 mM NPAA in the apical fluid, consistent with active Cl −-secretion across the conjunctival epithelial barrier. Amiloride-sensitive Na + channels were absent. The permeability of the cell layers to polar solutes decreased with increased solute size, and the calculated equivalent pore size was about 8.0 nm. The Papp of β-blockers varied with lipophilicity in a sigmoidal fashion. Uridine transport showed temperature sensitivity and directionality, favoring transport in the apical-to-basolateral direction. Apical l-carnosine uptake was reduced by 46% in the absence of an inwardly directed proton gradient, and lowering the temperature to 4°C abolished direction-dependent l-carnosine uptake. Furthermore, uptake was inhibited by 73% using apical 10 mM glycyl sarcosine (a dipeptide transporter substrate) and by 60% using 1 mM l-valacyclovir (a dipeptide prodrug). In conclusion, a functional air-interfaced primary culture of rabbit conjunctival epithelial cell layers was established. This air-interfaced primary culture model may be useful for studying passive and active transport processes for ion and solute translocation in the mammalian conjunctival epithelial barrier in a defined experimental setting.

Absorption of glucose, amino acids, and dipeptides by the intestines of Atlantic salmon (Salmo salar L.).

Rates of glucose, amino acid and dipeptide absorption by the intestine of Atlantic salmon (Salmo salar L.) were measured in vitro at 10 °C as functions of concentration and region using intact tissues (everted sleeve method). Salmon (weight range 300–1300 g) fed a commercial, extruded salmon feed, were kept at 12–13 °C in freshwater. Maximum rates (Vmax) of glucose and dipeptide transport were low compared to most amino acid Vmax values. There was a declining proximal-to-distal gradient of absorption along the post-gastric intestinal tract. A saturable component of absorption was not evident for proline and glycyl-proline in the distal intestine, and glycyl-sarcosine in any region. `Apparent diffusion', which may include low affinity, high capacity carrier systems and carrier-independent influx, appears to account for the majority of total uptake at higher concentrations of amino acids and dipeptides. There was competition between the dipeptides for transporter sites in the pyloric ceca and mid intestine, suggesting a common carrier. There was also indication of hydrolysis of these dipeptides by brush border membrane enzymes.

Enhancement of Transport of D-Melphalan Analogue by Conjugation with L-Glutamate across Bovine Brain Microvessel Endothelial Cell Monolayers

In this paper, the L-glutamate (L-Glu) transport system was targeted to improve the delivery of a model compound, p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD), through the blood-brain barrier (BBB) in vitro cell culture model. D-MOD is an analogue of an antitumor agent D-melphalan. To target the L-Glu transport system, D-MOD was conjugated to L-Glu to give D-MOD-L-Glu conjugate. D-MOD and D-MOD-L-Glu transport properties were evaluated using the bovine brain microvessel endothelial cell (BBMEC) monolayers. The results suggest that D-MOD-L-Glu conjugate permeates through the BBMEC monolayers more readily than the parent D-MOD. The improvement of transport may be due to the recognition of D-MOD-L-Glu by the L-Glu transport system. The transport mechanism was evaluated using several different experiments including: (a) concentration-dependent studies; (b) temperature-dependent studies; (c) substrate inhibition studies; and (d) metabolic inhibitor studies. The D-MOD-L-Glu transport was inhibited by the change of temperature from 37°C to 4°C. At higher concentrations, the transport of D-MOD-L-Glu reached plateau due to saturation. Furthermore, some amino acids (i.e., L-Glu, L-Asp, D-Asp, and L-Gln) inhibited the transport of D-MOD-L-Glu; presumably the conjugate was competing with these amino acids for the same transport system. Metabolic inhibitors (i.e., 2,4-dinitrophenol and sodium azide) suppressed the transport of the conjugate. However, the conjugate was not transported by monocarboxylic acid, dipeptide and neutral amino acid transporters. In conclusion, the L-Glu transport system can be utilized to facilitate a non-permeable drug across the BBB by conjugating the drug with L-Glu amino acid.

Roundtable discussion: Cephalosporins in the 1990s and beyond

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Biopharmaceutics of transmucosal peptide and protein drug administration: role of transport mechanisms with a focus on the involvement of PepT1

Non-invasive delivery of peptide and protein drugs will soon become a reality. This is due partly to a better understanding of the endogenous transport mechanisms, including paracellular transport, endocytosis, and carrier-mediated transport of mucosal routes of peptide and protein drug administration. This paper focuses on work related to the elucidation of structure-function, intracellular trafficking, and regulation of the intestinal dipeptide transporter, PepT1.

Characteristics of dipeptide transport in pig jejunum in vitro.

Characteristics of dipeptide transport in pig jejunum were investigated in vitro by applying the Ussing-chamber technique and mucosal uptake studies. Addition of both glycyl-L-glutamine and glycyl-L-sarcosine (20 mmol.l-1) to the mucosal buffer solution significantly increased the short-circuit current by 2.60 +/- 0.15 and 1.57 +/- 0.20 mu eq.cm-2.h-1, respectively. Concentration-dependent changes in short-circuit current followed Michaelis-Menten kinetics with similar affinity constants for both dipeptides. From unidirectional flux rates for radiolabelled glycyl-L-sarcosine, a net flux rate for glycyl-L-sarcosine of 49.8 +/- 6.7 nmol.cm-2.h-1 was calculated. In mucosal uptake experiments, the apical influx of 14C-labelled glycyl-L-sarcosine into isolated porcine mucosa was pH dependent and significantly inhibited by glycyl-L-glutamine. Moreover, RT-PCR studies with primers derived from rabbit PepT1 identified two PCR fragments of identical size to rabbit PepT1 from pig intestinal mRNA preparations. In conclusion, our studies revealed key features of mammalian intestinal peptide transporters and give evidence for a PepT1-like transporter in the pig jejunum that could significantly contribute to the overall amino acid absorption from the gut.

VanX, a bacterial D-alanyl-D-alanine dipeptidase: resistance, immunity, or survival function?

The zinc-containing D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase VanX has been detected in both Gram-positive and Gram-negative bacteria, where it appears to have adapted to at least three distinct physiological roles. In pathogenic vancomycin-resistant enterococci, vanX is part of a five-gene cluster that is switched on to reprogram cell-wall biosynthesis to produce peptidoglycan chain precursors terminating in D-alanyl-D-lactate (D-Ala-D-lactate) rather than D-Ala-D-Ala. The modified peptidoglycan exhibits a 1, 000-fold decrease in affinity for vancomycin, accounting for the observed phenotypic resistance. In the glycopeptide antibiotic producers Streptomyces toyocaensis and Amylocatopsis orientalis, a vanHAX operon may have coevolved with antibiotic biosynthesis genes to provide immunity by reprogramming cell-wall termini to D-Ala-D-lactate as antibiotic biosynthesis is initiated. In the Gram-negative bacterium Escherichia coli, which is never challenged by the glycopeptide antibiotics because they cannot penetrate the outer membrane permeability barrier, the vanX homologue (ddpX) is cotranscribed with a putative dipeptide transport system (ddpABCDF) in stationary phase by the transcription factor RpoS (sigma(s)). The combined action of DdpX and the permease would permit hydrolysis of D-Ala-D-Ala transported back into the cytoplasm from the periplasm as cell-wall crosslinks are refashioned. The D-Ala product could then be oxidized as an energy source for cell survival under starvation conditions.

Glycylsarcosine uptake in rabbit renal brush border membrane vesicles isolated from outer cortex or outer medulla: evidence for heterogeneous distribution of oligopeptide transporters.

Studies were initially performed in rabbit brush border membrane vesicles (BBMV) prepared from whole cortex plus outer medulla. In these studies using combined tissues, two distinct peptide/H+transport systems were found for glycylsarcosine (GlySar) uptake, with one representing a low-affinity/high-capacity system (Vm1 = 974 pmol/mg/10 sec and Km1 = 4819 microM) and the other a high-affinity/low-capacity system (Vm2 = 220 pmol/mg/10 sec and Km2 = 96 microM). Thus, under linear conditions, the high-affinity transporter accounted for about 92% of the total transport of dipeptide. To better define the regional heterogeneity of peptide transporter activity in kidney, subsequent studies were performed in vesicles prepared from separately harvested outer cortical and outer medullary tissue. In BBMV studies prepared from outer cortex, two saturable components were revealed for GlySar transport (low-affinity/high-capacity transport system: Vm1 = 1921 pmol/mg/10 sec and Km1 = 11714 microM; high-affinity/low-capacity transport system: Vm2 = 143 pmol/mg/10 sec and Km2= 138 microM). However, in BBMV studies prepared from outer medulla, only one saturable component was revealed for GlySar transport (high-affinity/low-capacity transport system:Vm2= 168 pmol/mg/10 sec and Km2 = 230 microM). Overall, these studies support the contention that peptides are handled sequentially in kidney (ie, first by low-affinity transporter PEPT1, and then by high-affinity transporter PEPT2) and that PEPT2 is primarily responsible for the renal reabsorption of peptides and peptidomimetics.

Functional and molecular expression of intestinal oligopeptide transporter (Pept-1) after a brief fast

The intestinal oligopeptide transporter, cloned as Pept-1, has major roles in the assimilation of dietary proteins and absorption of peptidomimetic medications. The initial aim of the present experiment was to investigate whether the functional expression of this transporter is affected by dietary intake. Functional expression was determined as the rate of uptake of glycylglutamine (GlyGln) by brush-border membrane vesicles (BBMVs) prepared from the jejunum of fed and fasted rats. Surprisingly, the rate of dipeptide uptake was greatly increased after 1 day of fasting. The subsequent aim of the experiment became the investigation of the mechanism of this alteration in transport, which showed that 1 day of fasting increased (1) the maximal GlyGln uptake (V max) by twofold without changing the K m of GlyGln uptake by BBMVs, (2) the amount of intestinal oligopeptide transporter (Pept-1) protein by threefold in the brush-border membrane, and (3) the abundance of Pept-1 mRNA by threefold in the intestinal mucosa. We conclude that 1 day of fasting increases dipeptide transport in rat intestine by increasing the population of Pept-1 in the brush-border membrane. The mechanism appears to be an increase in Pept-1 gene expression.

A novel role for carbonic anhydrase: cytoplasmic pH gradient dissipation in mouse small intestinal enterocytes.

1. The spatial and temporal distribution of intracellular H+ ions in response to activation of a proton-coupled dipeptide transporter localized at the apical pole of mouse small intestinal isolated enterocytes was investigated using intracellular carboxy-SNARF-1 fluorescence in combination with whole-cell microspectrofluorimetry or confocal microscopy. 2. In Hepes-buffered Tyrode solution, application of the dipeptide Phe-Ala (10 mM) to a single enterocyte reduced pHi locally in the apical submembranous space. After a short delay (8 s), a fall of pHi occurred more slowly at the basal pole. 3. In the presence of CO2/HCO3--buffered Tyrode solution, the apical and basal rates of acidification were not significantly different and the time delay was reduced to 1 s or less. 4. Following application of the carbonic anhydrase inhibitor acetazolamide (100 microM) in the presence of CO2/HCO3- buffer, addition of Phe-Ala once again produced a localized apical acidification that took 5 s to reach the basal pole. Basal acidification was slower than at the apical pole. 5. We conclude that acid influx due to proton-coupled dipeptide transport can lead to intracellular pH gradients and that intracellular carbonic anhydrase activity, by facilitating cytoplasmic H+ mobility, limits their magnitude and duration.

Cellular and molecular mechanisms of dietary regulation on rat intestinal H +/peptide transporter PepT1

Background & Aims: Dietary regulation is one of the most important factors of intestinal peptide transport. However, the cellular and molecular mechanisms of dietary regulation of the intestinal peptide transport system remain unknown. This study investigated the molecular mechanism of transcriptional activation of intestinal peptide transporter (PepT1) gene by the dietary protein. The promoter region of the rat PepT1 gene was isolated and characterized. Methods: PepT1 messenger RNA levels were determined by Northern blot analysis. In transient transfection experiments, effects of amino acid and dipeptide on luciferase activity were investigated. Results: The proximal promoter region of the rat PepT1 gene has a TATA-like box and a GC box sequence. The luciferase activities of the clone −351 RPT-LUC responded to particular amino acids (phenylalanine, arginine, and lysine) and dipeptides (Gly-Sar, Gly-Phe, Lys-Phe, and Asp-Lys). An AP-1 binding site and an amino acid–responsible element were present at −295 and −277 nucleotides relative to the transcription start site in this region. Conclusions: These results suggest that the up-regulation of dipeptide transport activity by dietary protein is caused by transcriptional activation of the PepT1 gene by selective amino acids and dipeptides in the diet. GASTROENTEROLOGY 1999;116:354-362

Evidence for overlapping substrate specificity between large neutral amino acid (LNAA) and dipeptide (hPEPT1) transporters for PD 158473, an NMDA antagonist.

The objective of this research was to investigate the substrate specificity of large neutral amino acid carrier (LNAA) and di/tripeptide (hPEPT1) transporters with respect to PD 158473, an NMDA antagonist. Cellular uptake studies were carried out using two types of Chinese Hamster Ovary (CHO). CHO-K1 cells represent the wild type with inherent large neutral amino acid (LNAA) activity. CHO-PEPT1 cells were generated by stable transfection of hPEPT1 gene into CHO cells. Therefore, these cells possess both LNAA activity and di/tripeptide transporter activities as a result of the transfection. Cellular uptake of PD 158473 was quantified using a HPLC method previously developed in our laboratory. The utility of the CHO-PEPT1 cell model was demonstrated by determining the uptake kinetics of Gly-Sar, a prototypical dipeptide transporter substrate. Uptake kinetics of PD 158473 displayed two carrier-mediated transport components in CHO-PEPT1 cells, while in CHO-K1 cells the relationship was consistent with classic one component Michaelis-Menten kinetics. These results confirmed the affinity of PD 158473 for both LNAA and di/tripeptide transporters. Further, results from inhibition experiments using these two cell types indicate that the high affinity-low capacity system was the LNAA carrier and the low affinity-high capacity carrier was the di/tripeptide transporter. This study demonstrates overlapping substrate specificity between LNAA carrier and di/tripeptide transporter (hPEPT1) for PD 158473, an amino acid analog. Establishing Structure Transport Relationship (STR) for this overlap will aid in a design strategy for increasing oral absorption or targeting specific drugs to selected tissues.

Conjugation of dipeptide to fluorescent dyes enhances its affinity for a dipeptide transporter (PEPT1) in human intestinal Caco-2 cells.

Dipeptide transporters in small intestine have a very wide substrate specificity, so that the transporter sometimes serves as a carrier for peptide-like compounds. We have synthesized dipeptide analogues conjugated at an epsilon-amino group of Lys in Val-Lys or Lys-Sar with fluorescent compounds such as fluorescein isothiocyanate and coumarin-3-carboxylic acid. Uptakes of these peptide analogues were examined by measuring intracellular accumulations into monolayers of the human intestinal epithelial cell line Caco-2 expressing the dipeptide transporter PEPT1. Kinetic analysis and effects of addition either of uncoupler (protonophore) or by Gly-Sar, one of the good substrates of PEPT1, revealed that fluorescent dipeptides were taken up by passive diffusion. In contrast, these analogues remarkably inhibited the Gly-Sar uptake by Caco-2 cells. Among the fluorescent analogues synthesized in this paper, Val-Lys(Flu) was the most potent competitive inhibitor against the Gly-Sar uptake with an inhibition constant of 5 microM. This value is the smallest among those ever reported: Val-Lys(Flu) has the highest affinity for PEPT1 among chemicals ever reported. The importance of the hydrophobic part of the substrate was pointed out.

A mutant of Mycobacterium smegmatis defective in dipeptide transport.

A mutant of Mycobacterium smegmatis unable to use the dipeptide carnosine (beta-alanyl-L-histidine) as a sole carbon or nitrogen source was isolated. Carnosinase activity and the ability to grow on beta-Ala and/or L-His were similar in the mutant and the wild type. However, the mutant showed significant impairment in the uptake of carnosine. This study is the first description of a peptide utilization mutant of a mycobacterium.

Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells.

The reabsorption of filtered di- and tripeptides as well as certain peptide mimetics from the tubular lumen into renal epithelial cells is mediated by an H+-coupled high-affinity transport process. Here we demonstrate for the first time H+-coupled uptake of dipeptides into the renal proximal tubule cell line LLC-PK1. Transport was assessed 1) by uptake studies using the radiolabeled dipeptide D-[3H]Phe-L-Ala, 2) by cellular accumulation of the fluorescent dipeptide D-Ala-Lys-AMCA, and 3) by measurement of intracellular pH (pHi) changes as a consequence of H+-coupled dipeptide transport. Uptake of D-Phe-L-Ala increased linearly over 11 days postconfluency and showed all the characteristics of the kidney cortex high-affinity peptide transporter, e.g., a pH optimum for transport of D-Phe-L-Ala of 6.0, an apparent Km value for influx of 25.8 +/- 3. 6 microM, and affinities of differently charged dipeptides or the beta-lactam antibiotic cefadroxil to the binding site in the range of 20-80 microM. pHi measurements established the peptide transporter to induce pronounced intracellular acidification in LLC-PK1 cells and confirm its postulated role as a cellular acid loader.

Membrane topology of the human dipeptide transporter, hPEPT1, determined by epitope insertions.

We have used epitope insertion to analyze the transmembrane topology of the human H+-dipeptide symporter hPEPT1. An epitope tag, EYMPME (EE), was inserted into different locations at amino acids 39, 78, 106, 412, and 708 of hPEPT1 by site-directed mutagenesis. The functional integrity of the tagged protein was tested by measuring its dipeptide transport activity in transfected Cos7 cells. Further, cells expressing hPEPT1 or EE-tagged hPEPT1 derivatives were labeled with an anti-EE-monoclonal antibody (anti-EE-mAb) or an antiserum raised against the carboxyl terminus of hPEPT1 (anti-hPEPT1) and examined by immunofluorescence confocal microscopy. EE106-, 412-, and 708-hPEPT1 transported the dipeptide tracer as well as wild-type hPEPT1. Tags at position 106 and 412 were shown to be extracellular because they were accessible to anti-epitope antibody in nonpermeabilized cells. In contrast, the carboxyl-terminal domain and EE708 were shown to be intracellular since they were only accessible to the antibodies in permeabilized cells. These results are consistent with a 12-transmembrane domain (TMD) topological model of PEPT1. Epitope insertions at regions linking the putative TMD1 and TMD2, and TMD2 and TMD3 (EE39- and EE78-hPEPT1), abolished the dipeptide transport into the cells. In transfected Cos7 cells, these tagged proteins remained largely intracellular rather than at the plasma membrane. These results suggest that the integrity of these regions is essential for transporter trafficking and/or function. Thus, the topology of the amino-terminal portion, including putative TMD1 and -2, remains to be clarified.

H+-Coupled Uphill Transport of the Dipeptide Glycylsarcosine by Bovine Intestinal Brush-Border Membrane Vesicles

In monogastric species, a considerable portion of amino acid nitrogen is absorbed across the brush-border membrane of the small intestine as small peptides (e.g., tripeptides and dipeptides). In ruminants, however, this process is less clear. Therefore, we investigated the uptake of radioactively labeled glycylsarcosine as a model dipeptide across the intestinal brush-border membrane using brush-border membrane vesicles prepared from the bovine small intestine. Uphill transport of glycylsarcosine was energized by a transmembrane H+ gradient and was further stimulated by an electrical potential difference across the membrane. Transport mediated by a carrier contributes to total glycylsarcosine transport across the brush-border membrane. Comparison of the apparent kinetic constants between brush-border membranes prepared from the proximal jejunum or ileum revealed similar half-saturating substrate concentrations (1.28 and 0.93 mmol/L for proximal jejunum and ileum, respectively), but maximal transport rates appeared to be somewhat higher in the proximal small intestine (2.15 and 1.20 nmol/mg of protein per 3s for proximal jejunum and ileum, respectively). Uptake of glycylsarcosine was strongly inhibited by other dipeptides, but the amino acids glycine and sarcosine did not affect transport. Inhibition of glycylsarcosine uptake by cephalexin indicated an affinity of the carrier for cephalosporin antibiotics. Transport of intact dipeptides across the brush-border membrane of the small intestine might be of physiological importance in ruminants because the microbial and dietary proteins resistant to rumen degradation are digested and absorbed in the small intestine.

The opt1 gene of Drosophila melanogaster encodes a proton-dependent dipeptide transporter.

We have cloned and characterized the opt1 gene of Drosophila melanogaster. This gene encodes a protein with significant similarity to the PTR family of oligopeptide transporters. The OPT1 protein is localized to the apical epithelial membrane domains of the midgut, rectum, and female reproductive tract. The opt1 message is maternally loaded into developing oocytes, and OPT1 is found in the alpha-yolk spheres of the developing embryo. It is also found throughout the neuropil of the central nervous system, with elevated expression within the alpha- and beta-lobes of the mushroom bodies. Transport activity was examined in HeLa cells transiently expressing OPT1. This protein is a high-affinity transporter of alanylalanine; the approximate Km constant is 48.8 microM for this substrate. OPT1 dipeptide transport activity is proton dependent. The ability of selected beta-lactams to inhibit alanylalanine transport suggests that OPT1 has a broad specificity in amino acid side chains and has a substrate requirement for an alpha-amino group. Together these data suggest an important role for OPT1 in regulating amino acid availability.

Molecular Identification of a Role for Tyrosine 167 in the Function of the Human Intestinal Proton- Coupled Dipeptide Transporter (hPepT1)

hPepT1 is a proton-coupled peptide transporter that mediates the absorption of di- and tripeptides. Here we show that tyrosine 167 (Y167) in transmembrane domain 5 (TMD5) of this 12-transmembrane spanning protein contributes to its transport function. We identified this particular amino acid by a computer model of the arrangement of the TMDs of hPepT1 and investigated its role by site-directed mutagenesis and dipeptide uptake studies. [3H]Gly-sar uptake in cells transiently transfected with Y167A-hPepT1 was abolished completely, even though the level of Y167A-hPepT1 expression by Western blot analysis and cell surface expression by immunofluorescence microscopy was similar to those of the wild type. Therefore, mutation affected transport function, but apparently not the steady-state protein level or trafficking of the transporter to the plasma membrane. Moreover, mutation of Y167 into phenylalanine, serine, or histidine all abolished gly-sar uptake in transfected HEK 293 cells. Taken together, these findings suggest that Y167 plays an essential role in hPepT1 function, perhaps due to the unique chemistry of its phenolic side chain.