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Functional characterization of a cloned pig intestinal peptide transporter (pPepT1)

^* Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061-0306

	Abstract

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Absorption of dietary protein can be mediated through the uptake of AA as free AA or small peptides. A H⁺-coupled, peptide transport protein, PepT1, is responsible for the absorption of small peptides arising from digestion of dietary proteins in the small intestine. The magnitude of peptide absorption and the nutritional significance of PepT1 are unknown for many food-producing animals; thus, the objective of this study was to clone and determine the functional characteristics of the pig PepT1 (pPepT1). Two cDNA-encoding pPepT1 were isolated, which contain alternative polyadenylation sites. The predicted pPepT1 is a 708-AA protein, which shows 82.8, 85.7, and 64.7% AA identity to human, sheep, and chicken PepT1, respectively. On northern blots, two pPepT1 mRNA of approximately 2.9 and 3.5 kb were detected in the duodenum, jejunum, and ileum of the small intestine and are presumed to result from alternative polyadenylation. Uptake of [³H]-Gly-Sar was measured in Chinese hamster ovary cells transiently transfected with a pPepT1 expression vector to study the functional expression of pPepT1. Peptide transport was H⁺-dependent, with an optimal pH of 6.0 to 6.5. The ability of pPepT1 to transport various peptides was assayed by calculating the concentration of unlabeled peptide that inhibited 50% of [³H]-Gly-Sar uptake (IC₅₀) in transfected cells. Eleven dipeptides and two tripeptides had IC₅₀ values that ranged from 0.004 to 0.53 mM. Three peptides, Lys-Lys, Arg-Lys, and Lys-Trp-Lys, had IC₅₀ values greater than 1. 38 mM and seem to be poor substrates for pPepT1. For all three tetrapeptides examined, uptake of Gly-Sar was too small to measure, even at a concentration of 10 mM tetrapeptide; therefore, IC₅₀ values could not be calculated. These results demonstrate that pPepT1 can transport a variety of dipeptides and tripeptides but not tetrapeptides.

Key Words: Intestinal • Peptide Transport • PepT1 • Pig

	Introduction

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A significant fraction of dietary amino nitrogen is absorbed as intact oligopeptides rather than free AA (Ganapathy et al., 1994). A number of mammalian peptide transporters have been cloned and constitute the proton-coupled oligopeptide transporter (POT) family (Herrera-Ruiz and Knipp, 2003). Two of these transporters, PepT1 and PepT2, are membrane proteins capable of transporting a broad array of neutral, acidic, and basic di- and tripeptides, as well as peptidomimetics (Daniel, 1996; Brodin et al., 2002). These transporters have been shown to play an essential role in the absorption of ß-lactam antibiotics, angiotensin-converting enzyme inhibitors, rennin inhibitors, and the antitumor drug, bestatin (Inui et al., 2000). The PepT1 plays an important role in transporting small peptides arising from digestion of dietary proteins in the small intestine (reviewed in Daniel, 2004). The PepT2 is specifically expressed in the kidney and functions to reabsorb filtered peptides, peptide-derived antibiotics, and peptides produced as a result of the action of luminal peptidases (Boll et al., 1996).

Expression of PepT1 mRNA has been observed in a variety of tissues. The PepT1 mRNA is detectable in the small intestine of sheep, cows, pigs, and chickens, and in the omasal and ruminal epithelium of sheep and dairy cows, with little expression in liver and kidney (Chen et al., 1999). The expression pattern of PepT1 in the rabbit small intestine differs depending on the sections of the small intestine as well as in different areas along the crypt-villus axis (Freeman et al., 1995). The PepT1 was highly expressed in the absorptive epithelial cells of the villi in the rat small intestine (Ogihara et al., 1999). The magnitude of peptide absorption and the nuritional and pharmaceutical significance of PepT1 are unknown for many food-producing animals; thus, the objective of this study was to clone the pig PepT1 cDNA and determine the substrate specificity of pig PepT1 (pPepT1).

	Materials and Methods

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Construction and Screening of a Pig Intestinal cDNA Library

A pig intestinal cDNA (>500 bp) library was constructed using the ZAP Express cDNA synthesis system (Stratagene, LaJolla, CA) starting with poly(A)⁺ RNA extracted from pig small intestine. Recombinant phage DNA were packaged with Gigapack III Gold packaging extract (Stratagene) and introduced into XL1-Blue MRF' Escherichia coli. Positive phages were identified by plaque hybridization using MSI Magna nylon transfer membranes (Osmonics, Inc., Westborough, MA) and a [³²P]-labeled, 446-bp fragment of sheep PepT1 cDNA as a probe (Chen et al., 1999). Hybridization was carried out for 16 h at 42°C in a solution containing 50% formamide, 5x Denhardt’s solution, 6x SSPE, 0.5% SDS, and 10 µg/mL denatured salmon sperm DNA. Posthybridization washing was done twice in 5x SSC, 0.5% SDS, at room temperature for 15 min, twice in 1x SSC, 0.5% SDS, at 37°C for 15 min, and twice in 0.1x SSC, 1% SDS, at 65°C for 15 min. In vivo excision was performed on the isolates to obtain the cDNA insert in the phagemid, pBK-CMV. Two clones, p17 and p18, were chosen for DNA sequence analysis. The nucleotide sequence for pig PepT1 was submitted to GenBank and has been assigned Accession No. AY157977.

Northern Blot Analysis

Tissue distribution of pPepT1 mRNA transcripts was determined by northern blot. Twenty micrograms of total intestinal RNA was size-fractionated on a 1% gel containing 2.2 M formaldehyde, and then transferred to a nylon membrane by capillary action. The membrane was probed with a [³²P]-labeled, 688-bp PCR fragment encoding for part of the pPepT1 cDNA located approximately 800 bp after the translation start codon. The blot was hybridized at 42°C as described for cDNA library screening and exposed to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY) with an intensifying screen.

Expression of PepT1 in Chinese Hamster Ovary Cells

Expression of pig Pep T1 (pPepT1) was assayed in transfected Chinese hamster ovary (CHO) cells. Pig PepT1 cDNA was subcloned into the eukaryotic expression vector pTargeT (Promega, Madison, WI). The PCR primers were designed with restriction enzyme sites such that each PCR product would contain a 5'BamHI site (5'ATC TAG GAT CCT CCA CAA TGG GAA TGT CC GTG CCA C3') and a 3'KpnI site (5'ATC TGG TAC CTG GCT CCT GTC TTG TTT AAC 3'). The PCR was carried out using PCR Master Mix (Promega) at 72°C for 5 min, 80°C for 15 min, 35 cycles of 95°C for 1 min, 56°C for 1 min, 70°C for 3 min, and a final step of 72°C for 15 min. The PCR products were digested with BamHI and KpnI and ligated into BamH1/KpnI digested pTargeT vector. The ligated vectors were electro-transformed into MaxEfficiency DH10B competent cells (Invitrogen, Gaithersburg, MD). The pPepT1 cDNA was reverified by DNA sequencing.

The CHO cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 1% nonessential AA, penicillin (100 U/mL) streptomycin (100 U/mL), and fungizone (250 µg/500 mL; Atlanta Biologicals, Norcross, GA) at 37°C in an atmosphere of 95% air:5% CO₂. The day before transfection, cells were trypsinized and plated on a standard 12-well plate (well diameter = 2.1 cm) at an approximate density of 2.0 x 10⁵ cells per well. Twenty-four hours later, transfection of the cells was performed at approximately 70% confluency. For each well, a mixture of 2.4 µL of lipofectamine (2 µg/µL, Invitrogen), 1.6 µg of pTargeT plasmid with and without insert, and 40 µL of OPTI-MEM (Invitrogen) was placed into a polystyrene tube and incubated for 30 min at room temperature. Then, 4.5 mL of OPTI-MEM was added to the lipid DNA complex, the tube was gently shaken, and 390 µL of the mixture was added to each well. One milliliter of OPTI-MEM was added to each well and incubated at 37°C for 5 h. At the end of 5 h, the medium was removed and 2 mL of Dulbecco’s modified Eagle’s medium plus 10% fetal bovine serum was added.

Measurement of Uptake of Peptides

Uptake measurements were performed 18 to 20 h after transfection with pPepT1. Growth medium was removed and the cells were washed with PBS. Room temperature uptake buffer containing 25 mM 2-morpholinoehtanesolfonate (pH 6.0), 5 mM glucose, 0.8 mM MgSO₄, 1.8 mM CaCl₂, 5.4 mM KCl, and 140 mM NaCl, pH 6.0, was used to dilute peptides and wash cells. A stock solution of [³H]-Gly-Sar (0.1 mCi/mL, 50 mCi/mmol; Moravek, Brea, CA) was prepared. Then, 2.5 µL of the Gly-Sar stock was added to 0.25 mL of uptake buffer per well (the final concentration of Gly-Sar was 20 µM). The mixture was added to the wells and incubated at room temperature for 25 min. The buffer was removed and the wells were washed three times with ice-cold uptake buffer to inhibit uptake. To lyse the cells, 500 µL of 0.1% SDS were added to each well for 20 min. Uptake of [³H] Gly-Sar was quantified by counting 450 µL of the cell lysate in a scintillation counter. The K_t (the concentration of substrate that yields half the maximal transport rate) and V_max (maximal transport rate), expressed as pmol/min, for Gly-Sar uptake were calculated by nonlinear regression using PRISM (GraphPad, San Diego, CA). Inhibition studies were performed in a similar manner as above but the [³H]-Gly-Sar was incubated with inhibitor peptides at concentrations ranging from 0.001 to 10 mM. The peptides chosen for the competition studies were Met-Met, Phe-Phe, Met-Leu, Met-Gly, Met-Glu, Lys-Met, Leu-Val, Lys-Lys, Lys-Phe, Trp-Phe, Val-Leu, Arg-Lys, Gly-Met, Met-Leu-Phe, Leu-Gly-Gly, Lys-Trp-Lys, Met-Gly-Met-Met, Val-Gly-Ser-Glu and Pro-Phe-Gly-Lys (Sigma, St. Louis, MO). All data are shown as means ±SD of three or four independent replicates from one transfection. Calculated IC₅₀ values (i.e., the concentration of the unlabeled compound necessary to inhibit 50% of [³H]-Gly-Sar uptake) were determined by nonlinear regression using PRISM.

	Results

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Sequence and Structure of pPepT1

Screening of the pig intestinal cDNA library generated two pPepT1 clones, p17 and p18, which were approximately 2.7 and 3.0 kb in length. The pPepT1 cDNA encodes a protein of 708 AA with a molecular weight of 78.9 kDa and an isoelectric point of 8.4. The predicted AA sequence of the pPepT1 protein is 82.8, 85.7, and 64.7% identical to human, sheep, and chicken PepT1, respectively (Figure 1). The two pPepT1 clones differ at their 3'end due to usage of alternative polyadenylation sites. In clone p17, a sequence (AGTAAA) similar to the consensus polyadenylation signal (AATAAA) precedes the poly (A) tail by 16 nucleotides. In contrast, clone p18 contains an additional 328 nucleotides of sequence after the polyadenylation site in clone p17. No consensus polyadenylation signal, however, was present immediately upstream of the poly (A) tail in clone p18. Clone p18 also contained a four-base deletion within the coding region, causing a frameshift in the PepT1 AA sequence; therefore, clone p18 was discarded and clone p17 was used for all subsequent analyses.

View larger version (67K): [in this window] [in a new window]   Figure 1. Comparison of the AA sequences for pig, sheep, human, and chicken PepT1 (GenBank Accession No. AY180903, AY027496, NM_005073, AY029615, respectively). Identical AA across species are indicated by an asterisk (*).

Two mRNA of approximately 2.9 and 3.5 kb were detected by northern blot analysis in the duodenum, jejunum, and ileum of the small intestine using a pPepT1 probe (Figure 2). These two pPepT1 mRNA are likely due to the alternative polyadenylation sites, which were identified during sequencing of the two pPepT1 cDNA. The difference in sizes of the cloned pPepT1 cDNA and the mRNA detected by northern blot may be due in part to the unknown lengths of the poly A tails. The 2.9-kb mRNA was the more abundant of the two transcripts, and both transcripts were detected strongly in the duodenum and jejunum and at lower levels in the ileum. There was no detectable hybridization with RNA isolated from liver tissue. There also was hybridization of a band at approximately 1.8 kb that may represent a degradation product. This lower band appears to be proportional to the pPepT1 mRNA in each tissue and is absent in liver tissue. These results are consistent with our previously reported observation that PepT1 is expressed predominantly in the small intestine and not expressed in the liver of sheep, dairy cows, pigs, and chickens (Chen et al., 1999).

View larger version (93K): [in this window] [in a new window]   Figure 2. Northern blot analysis of pPepT1 mRNA in different sections of the pig small intestine. Total RNA (20 µg) from the duodenum (duo), jejunum (jej), and ileum (ile) of the small intestine and from the liver (liv) were loaded in each lane. The northern blot was hybridized with a [32P]-labeled fragment of pPepT1 cDNA as a probe.

To study functional expression of pPepT1, uptake of [³H]-Gly-Sar was measured in transiently transfected CHO cells. In preliminary experiments, the optimal pH and time for [³H]-Gly-Sar uptake were determined. The optimal pH was 6.0 to 6.5, and all uptake studies subsequently used an uptake buffer at a pH of 6.0 (Figure 3A). Assays were run for 25 min because uptake was still observed to be linear at this time (Figure 3B). Uptake of [³H]-Gly-Sar in vector (no insert) transfected cells was only 370 cpm at 20 min, indicating that transport was carrier-mediated and not non-specific. To determine the transport kinetics of pig PepT1, uptake of [³H]-Gly-Sar was determined at various concentrations from 0.001 to 10 mM (Figure 3C). Nonlinear regression was used to derive the V_max and K_t for [³H]-Gly-Sar, which were determined to be 25.5 ± 1.0 pmol/min and 0.94 ± 0.14 mM, respectively (mean ± SE).

View larger version (19K): [in this window] [in a new window]   Figure 3. The pH, time, and substrate dependence of Gly-Sar uptake in transfected Chinese hamster ovary cells. The uptake of labeled Gly-Sar was determined at pH 5.0 to 7.0 (A), at pH 6.0 for 10 to 60 min (B) and at Gly-Sar concentrations ranging from 10 µM to 10 mM (C).

View larger version (19K): [in this window] [in a new window]   Figure 4. Transport of [3H]-glycylsarcosine (Gly-Sar) in pig peptide transporter transfected Chinese hamster ovary cells in the presence of unlabeled di-, tri-, and tetrapeptides (Met-Met, Lys-Lys, Leu-Gly-Gly, Pro-Phe-Gly-Lys) at concentrations ranging from 0.001 to 10 mM. Inhibition of Gly-Sar uptake was assayed at pH 6.0 for 25 min. The data represent the mean of four determinations.

	Discussion

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All cloned PepT1 have a conserved 12-transmembrane domain structure with a large extracellular loop located between transmembrane domains 9 and 10 and the amino and carboxyl termini on the cytoplasmic side of the membrane (Fei et al., 1994). Analysis of pPepT1 using the Transmembrane Hidden Markov Model program (version 2.0, http://www.cbs.dtu.dk/services/TMHMM-2.0) to predict the location of transmembrane helices and the location of intervening loop regions revealed 13 putative transmembrane domains rather than 12 transmembrane domains (Figure 5). In the 13 transmembrane domain model, the large hydrophilic loop of approximately 200 AA is located between transmembrane domains 10 and 11, rather than domains 9 and 10. The extra predicted transmembrane domain is located at the amino terminus of the protein, whereas the locations of the other 12 transmembrane domains are conserved. This model for the pPepT1 protein would place the amino terminus extracellular and the carboxy terminus intracellular. Hydrophobicity comparisons across species using the Transmembrane Hidden Markov Model program revealed that this thirteenth transmembrane domain may be present in other species, but the hydrophobicity scoring was too low to be considered a transmembrane domain. For example, human PepT1 has a hydrophobicity plot that most resembles the putative 13-transmembrane domain structure of pPepT1, but the calculated probability of the extra transmembrane domain is less than 60%. In contrast, the probability of the thirteenth transmembrane domain in pPepT1 is greater than 80%. This putative 13-transmembrane domain structure for pPepT1, however, remains to be experimentally verified.

View larger version (54K): [in this window] [in a new window]   Figure 5. The predicted transmembrane domains of pig, human, sheep, rabbit, mouse, rat, turkey, and chicken peptide transporter using the Transmembrane Hidden Markov Model (Version 2.0). The location (AA position) and probability (0 to 1.0) of each transmembrane domain is shown as a peak. At the top of each figure, the thick bar represents a putative transmembrane domain. A line connecting adjacent transmembrane domains at the top of the bar indicates that the intervening sequence is extracellular, whereas a line connecting transmembrane domains at the bottom of the bar represents intracellular regions.

Changes in the structural organization of the PepT1 at the amino terminus may have no effect on the transport properties of PepT1. Site-directed mutagenesis and the generation of chimeric proteins have been valuable techniques for mapping putative substrate binding regions. Analysis of the transport properties of chimeric PepT1/PepT2 proteins mapped the substrate-binding domain to transmembrane domains 7, 8, and 9 (Fei et al., 1998). Site-directed mutagenesis has also been used to map or identify a proposed binding region for the coupling H⁺ ion. The histidine residues at histidine-57 and histidine-121 are intimately involved in the binding of the coupling H⁺ ion and the recognition of transportable peptide substrates (Terada et al., 1996; Fei et al., 1997; Chen et al., 2000).

To evaluate the kinetics of peptide transport, our cloned pPepT1 was transfected into CHO cells and uptake and inhibition assays were performed. Similar to other characterized PepT1, pPepT1 transport was H⁺-dependent, with optimal uptake at a pH between 6.0 and 6.5. The transport of radiolabled Gly-Sar had a calculated K_t of 0.94 mM, which is similar to the calculated Michaelis constant (1.08 mM) for pPepT1 determined by Winckler et al. (1999). In the study by Winckler et al. (1999), the Ussing chamber technique was used to measure peptide transport in isolated pig jejunal tissue. Thus, our cloned pPepT1 expressed in CHO cells maintained the same transport properties as native pPepT1.

Inhibition studies in pPepT1 transfected CHO cells demonstrated that most dipeptides and tripeptides had a high affinity (low IC₅₀ values) for pPepT1. Exceptions were dipeptides and tripeptides that contained a C-terminal lysine such as Lys-Lys, Arg-Lys and Lys-Trp-Lys, which all had lower affinities for pPepT1 and high IC₅₀ values. These three peptides are also composed of at least two positively charged AA, and all three peptides are also very hydrophilic. This suggests that charge and/or hydrophobicity of a peptide may affect its affinity for the peptide transporter. Lister et al. (1997) previously showed that, at a pH of 6.8 compared with pH 7.4, the transport of negatively charged and neutral peptides increased in rat small intestinal tissue, whereas positively charged peptide transport decreased. The carboxy terminal lysine on each of the three peptides may have an effect on the uptake of the dipeptides because peptides with amino terminal Lys (Lys-Met and Lys-Phe) were readily transported. To determine whether the carboxy terminal lysine peptides in this study were inhibited by their charge or the presence of lysine, a number of peptides with varying combinations of positive charge and lysine position need to be tested. Chen et al. (2002b) examined peptide transport in sheep PepT1 transfected CHO cells and showed that the positively charged Met-Lys was transported readily (IC₅₀ = 0.123 mM) but that Thr-Ser-Lys was not (IC₅₀ = 3.0 mM). Similarly, Met-Lys was readily transported (IC₅₀ = 0.07 mM) in chicken PepT1 transfected CHO cells (Chen et al., 2002a).

When comparing the kinetics of peptide transport by pig, chicken, and sheep PepT1, there is a strong overall similarity (Table 2). In all three species, PepT1 is able to transport a wide variety of di- and tripeptides and shows lower affinity for peptides with a carboxy-terminal Lys. Interestingly, sheep PepT1 had a much higher affinity for Lys-Lys than did pig or chicken PepT1 (Chen et al., 2002a,b). The lower IC₅₀ of Lys-Lys in sheep may be an adaptation in ruminants to take up higher levels of lysine. Like sheep and chicken PepT1, pPepT1 had a high affinity for neutral peptides, although, pPepT1 demonstrated a lower affinity for Met-Glu and the tri-peptides (Met-Leu-Phe, Leu-Gly-Gly) than sheep or chicken PepT1. One important difference in pPepT1 compared with other species is that all three tetrapeptides studied for uptake did not show any significant inhibition of Gly-Sar, even at very high concentrations of 10 mM. Unlike sheep and chicken, which demonstrated transport of selective tetrapeptides in PepT1 transfected CHO cells, pPepT1 does not seem to transport tetrapeptides. The small amount of inhibition of Gly-Sar seen with high concentrations of tetrapeptides is probably due to contamination of di- and tripeptides in the tetrapeptide mix or from hydrolysis of the tetrapeptide in cell culture. Again, major functional differences between pPepT1 with a proposed 13-transmembrane domain structure and chicken or sheep PepT1 with a proposed 12-transmembrane structure are minimal, suggesting that the structure of the amino terminus does not affect transport.

	Footnotes

 
¹ This research was supported in part by the Virginia Agricultural Experiment Station Project No. 612990 and by a John Lee Pratt Animal Nutrition Graduate Fellowship awared to J. E. Klang.

² Current address: Trophogen Inc., Rockville, MD 20850.

³ Current address: Dept. of Biochemistry and Molecular Biology, Univ. of Florida, Gainesville 32610.

⁴ Correspondence: 3150 Litton Reaves (phone: 540-231-4737; fax: 540-231-3010; e-mail: ewong{at}vt.edu).

Received for publication July 21, 2004. Accepted for publication October 15, 2004.

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