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Nutrient-Gene Expression

Molecular Cloning and Functional Expression of a Chicken Intestinal Peptide Transporter (cPepT1) in Xenopus Oocytes and Chinese Hamster Ovary Cells^{1 ,2}

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

³To whom correspondence should be addressed. E-mail: webbk{at}vt.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
To study peptide absorption in chickens, an intestinal peptide transporter cDNA (cPepT1) was isolated from a chicken duodenal cDNA library. The cDNA was 2914 bp long and encoded a protein of 714 amino acid residues with an estimated molecular size of 79.3 kDa and an isoelectric point of 7.48. cPepT1 protein is 60% identical to PepT1 from rabbits, humans, mice, rats and sheep. Sixteen dipeptides, three tripeptides and four tetrapeptides that contained the essential amino acids Met, Lys and(or) Trp were used for functional analysis of cPepT1 in Xenopus oocytes and Chinese hamster ovary cells. For most di- and tripeptides tested, the substrate affinities were in the micromolar range, indicating that cPepT1 has high affinity for these peptides. Lys-Lys and Lys-Trp-Lys were exceptions, with substrate affinities in the millimolar range. Neither free amino acids nor tetrapeptides were transported by cPepT1. Northern blot analysis using a full-length cPepT1 cDNA as the probe demonstrated that cPepT1 is expressed strongly in the duodenum, jejunum and ileum, and at lower levels in kidney and ceca. The present study demonstrated for the first time the presence and functional characteristics of a peptide transport system from an avian species.

KEY WORDS: • peptide • transport • chickens • PepT1

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Protein is a costly nutrient in poultry diets. Optimizing or improving the efficiency of protein or amino acid utilization will result not only in a reduction in production costs but will also minimize excessive nitrogen pollution of the environment. Traditionally, it was believed that proteins had to be broken down to single amino acids to be absorbed into cells in animals. However, it is now known that peptide transport is an important physiologic process that occurs in tissues of animals (1 ). The cloning and characterization of two structurally similar peptide transporters, (PepT1)⁴ and PepT2, have provided valuable information about peptide transport in mammalian species (2 –6 ). These peptide transporters recognize di- and tripeptide substrates, as well as pharmacologically active compounds (7 ).

Although peptide transporters have been cloned from rabbits (2 ), humans (3 ), rats (4 ) and mice (8 ), nearly all functional analysis has focused on model peptide substrates or pharmacologically useful compounds. Less research has been conducted to examine the contributions of peptide transporters from a nutritional perspective. Recent reports from our laboratory demonstrate the existence and tissue distribution of a peptide transporter(s) in sheep, cows, pigs and chickens (9 –11 ). These results suggest that peptide absorption may be nutritionally important in all domestic animals. The cloning and expression of an ovine peptide transporter (oPepT1) in our laboratory provided information for the first time about the molecular structure and basic functions of a peptide transporter in food-producing animals (6 ). The purposes of this study were to analyze the peptide transport system in an avian species by cloning the chicken peptide transporter (cPepT1) and to characterize the function of this transporter in vitro using both Xenopus oocytes and Chinese hamster ovary (CHO) cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Materials.

All chemicals, substrates, and reagents were of either molecular biology or cell culture grade. The ZAP Express cDNA synthesis system and Gigapack III were purchased from Stratagene (La Jolla, CA). Magna nylon transfer membranes were purchased from Osmonics (Westborough, MA). Restriction enzymes were from New England BioLabs (Beverly, MA). The RNA transcription kit, mMESSAGE mMACHINE was obtained from Ambion (Austin, TX). Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, WI). Amino acids and peptides (dipeptides to tetrapeptides) were purchased from Sigma Chemical (St. Louis, MO). [³H]-Glycylsarcosine (Gly-Sar; specific radioactivity, 110 mCi/mmol) was purchased from Moravek Biochemical (Brea, CA).

Construction and screening of a chicken intestinal cDNA library.

Broiler chickens (n = 15; male and female) were killed by cervical dislocation and the small intestine was removed. Poly(A)⁺ RNA was isolated from a pool of duodenal tissues collected. All animal procedures were approved by Virginia Tech’s Animal Care Committee. A cDNA library was constructed using the ZAP Express cDNA synthesis system. Only cDNA of a size > 400 bp were used for library construction. Phage DNA containing cDNA were then packaged with Gigapack III Gold packaging extract and introduced into the XL1-Blue MRF' Escherichia coli cell line. The phage library was plated out immediately on a series of large, 150-mm NZY agar plates (50,000 plaques/plate) for screening.

Positive clones were identified by plaque hybridization of the cDNA library transferred to Magna nylon transfer membranes. The cDNA probe used for screening was a cloned oPepT1 (6 ). The probe was labeled with [-³²P]dATP (ICN Pharmaceutical, Costa Mesa, CA) by nick translation using DNA polymerase I/DNase I (Life Technologies, Gaithersburg, MD). Hybridization was carried out for 16 h at 42°C in a solution containing 50% formamide, 5X Denhardt’s solution, 6X SSPE (1 x SSPE = 0.15 mmol/L NaCl, 10 mmol/L NaH₂PO₄, and 1 mmol/L EDTA), 0.5% SDS and 10 mg/L yeast tRNA. Posthybridization washing was done under medium stringency conditions, which involved washing twice with 5X SSPE, 0.5% SDS at room temperature for 15 min and twice with 1X SSPE, 0.5% SDS at 42°C for 15 min. Positive clones were subjected to three more rounds of screening using the same conditions. After the quaternary screening, 100% of the plaques showed positive hybridization by autoradiography.

The positive plaques identified after screening of the cDNA library were used to generate the excised pBK-CMV phagemid containing the cDNA insert. Sequencing by the dideoxynucleotide chain termination method was performed manually using a Sequenase version 2.0 DNA sequencing kit (U.S. Biochemical, Cleveland, OH). Analysis of nucleotide and amino acid sequence was performed using the sequence analysis software Lasergene (DNAStar, Madison, WI).

Northern blot analysis.

Tissue distribution of cPepT1 mRNA transcripts was determined by Northern blot. Poly (A)⁺-enriched RNA samples (10 µg) from different tissues were denatured and size-fractionated on a 1% agarose gel containing 2.2 mol/L formaldehyde. The 18s rRNA was visualized by ethidium bromide staining and used as an estimate of the amount of RNA loaded per lane. The size-fractionated RNA was then transferred onto a nylon membrane and probed with the full-length cPepT1 cDNA. The probe was labeled with [-³²P]dATP (ICN Pharmaceutical) by nick translation using DNA polymerase I/DNase I (Life Technologies). Blots were hybridized overnight at 42°C for 16–18 h and posthybridization washing was done under medium stringency conditions. The blot was then exposed to Kodak XAR-5 film with an intensifying screen at -80°C.

Functional expression in Xenopus oocytes.

cRNA was synthesized using the RNA transcription kit mMESSAGE mMACHINE. For sense cRNA synthesis, phagemid containing the cDNA insert was transcribed in vitro by T₃ RNA polymerase in the presence of an RNA cap analog. For antisense cRNA synthesis, phagemid containing the cDNA insert was transcribed in vitro by T₇ RNA polymerase in the presence of an RNA cap analog. The resultant cRNA was purified and resuspended in nuclease-free water at a concentration of 1 g/L and stored frozen at -80°C in aliquots.

Healthy Xenopus oocytes at stage V were defolliculated and the resting membrane potential was sampled (12 ). Only oocytes with a resting membrane potential more negative than -30 mV 1 d after defolliculation were used for injection. Using a microinjection system, 25 ng of either sense cRNA or antisense cRNA were injected into the vegetal pole of each oocyte, near the polar interface. Antisense cRNA was used as a control. The injected oocytes were incubated in culture solution at 18°C for 1–7 d.

The two-electrode voltage-clamp technique was used to characterize the induced peptide transport activity in oocytes injected with sense cRNA or antisense cRNA. All responses were monitored by a two-electrode voltage-clamp amplifier (TEV-200, Dagan, Minneapolis, MN), and analyzed by a MacLab (AD Instruments, Milford, MA), which is an analog-digital converter and software system that uses an Apple Macintosh computer for performing data acquisition (13 ). Normally, electrophysiologic measurements in sense-cRNA- or antisense-cRNA–injected oocytes were carried out 3–6 d after injection. Only oocytes with a resting membrane potential more negative than -30 mV were used for recordings. An oocyte was perfused continuously with measurement buffer with or without peptide at a rate of 1.2 mL/min using a gravity feed perfusion system (Model BPS4, Ala Scientific Instruments, Westburg, NY). All peptide substrate solutions were prepared by dissolving the peptides in measurement buffer (96 mmol/L NaCl, 2 mmol/L KCl, 1 mmol/L MgCl₂, 1.8 mmol/L CaCl₂, 5 mmol/L HEPES, pH 6.0). All experiments were performed at room temperature (21°C).

Expression in CHO cells.

CHO cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 1% nonessential amino acids and penicillin (1 x 10⁵ U/L)/streptomycin (100 mg/L). All cells were maintained in an atmosphere of 5% CO₂ at a relative humidity of 90% at 37°C. One day before transfection, cells were trypsinized and plated onto 12-well plates at a density of 2.4 x 10⁵/well. For each well, 0.8 µg plasmid (with or without insert) was mixed with 2.4 µL Lipofectamine (2 g/L, Life Technologies) in 40 µL OPTI-MEM (Life Technologies) and incubated at room temperature for 30 min. The DNA-lipid complex was then added to each well and cells were transfected for 5 h at 37°C. For each assay, at least two transfections were performed.

Uptake measurements were done 16–18 h after transfection. The cells were washed three times with uptake buffer containing 25 mmol/L 2-(N-morpholino) ethanesulfonic acid (MES)/Tris, pH 6.0, 5 mmol/L glucose, 0.8 mmol/L MgSO₄, 1.8 mmol/L CaCl₂, 5.4 mmol/L KCl and 140 mmol/L NaCl. Gly-Sar solution was prepared at six concentrations (0.02–10 mmol/L, with 1 µCi/mL [³H]-Gly-Sar). Uptake solutions were added to each well and incubated for 20 min at room temperature. Uptake was stopped by washing cells with ice-cold uptake buffer. Cells were lysed by adding 0.5 mL 0.1% SDS and incubating at room temperature for 10 min. Uptake of [³H]-Gly-Sar was quantified by scintillation counting, and the amount of protein present in each well was quantified using Bio-Rad DC Protein kit (Bio-Rad, Hercules, CA). Inhibition studies were performed under the same conditions as described above, except that 20 µmol/L [³H]-Gly-Sar (50 mCi/mmol) was used for radiolabeled substrate and five concentrations (0.001–10 mmol/L) of inhibitor peptides were added for inhibition studies.

Calculations and statistics.

The kinetic parameters including the transport constant (K_t), the maximal current (I_max), the 50% inhibitory concentration (IC₅₀) and all other calculations (linear and nonlinear regression analysis) were performed using PRISM (GraphPad, San Diego, CA). The experiments were carried out with five or six replicates, and results are presented as the mean ± SEM. Data were evaluated using one-way ANOVA and the Least Significant Difference test was used for post-hoc comparisons (14 ).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Sequence and structure of the chicken intestinal PepT1 cDNA.

The screening of a chicken duodenal cDNA library using the cloned ovine intestinal peptide transporter cDNA as the probe resulted in the isolation of a chicken intestinal peptide transporter cDNA (cPepT1). The cPepT1 cDNA was 2914 bp long with an open reading frame of 2142 bp. There was a typical Kozak consensus sequence, GCCGCC(A/G)C[UNDERLN]ATG[/UNDERLN]G (15 ) flanking the initiation codon. A 69-bp 5' UTR and a 703-bp 3' UTR flanked the open reading frame. At the 3' end, the cDNA had a polyadenylation signal (AATAAA) 14 nt preceding the polyA tail. The encoded protein was predicted to have 714 amino acids with a molecular mass of 79.3 kDa and an isoelectric point of 7.48. Alignment results showed that the first fifteen amino acids of cPepT1 at the N-terminus were completely different from mammalian PepT1 (Fig. 1 ). The predicted amino acid sequence of cPepT1 protein is 62.4, 62.5, 63.8, 64.8 and 65.1% identical to PepT1 from rabbits, humans, mice, rats and sheep, respectively. Hydrophobicity analysis (16 ) indicated that cPepT1, like the mammalian PepT1, has 12 putative transmembrane domains with a large extracellular loop between transmembrane domains 9 and 10 (Fig. 2 ). The model also predicts that both the amino and the carboxyl termini are on the cytoplasmic side of the membrane. About 84% identity is found in the transmembrane domain regions, whereas the large extracellular loop shows only 21% identity with PepT1 from other species. Sequence analysis also showed the presence of the PTR2 family signatures, which are the signature sequences of members of the proton-dependent oligopeptide transporter (POT) superfamily (17 ). The PTR2 sequences in the cPepT1 are the sequences spanning the second and the third transmembrane domains (GALIADSWLGKFKTIVSLSIVYTI) and the sequences in the core of the fifth transmembrane domain (FSIFYLAINAGSL, Fig. 1 ).

View larger version (59K): [in this window] [in a new window]   Figure 1. Alignment of predicted amino acid sequences of human, rat, mouse, rabbit, sheep and chicken peptide transporter 1 (cPepT1). Amino acids identical to the human sequence are indicated by dashes (-). The underlined sequences represent the PTR2 signature sequences of the proton-dependent oligopeptide transporter (POT) superfamily. The cPepT1 nucleotide and amino acid sequences reported in this paper have been submitted to GenBank under accession number AY029615.

View larger version (24K): [in this window] [in a new window]   Figure 2. Putative membrane-spanning model of chicken peptide transporter 1 (cPepT1). Hydrophobicity analysis indicated that cPepT1 has 12 putative transmembrane domains with a large extracellular loop between transmembrane domains 9 and 10. Potential N-linked glycosylation sites are indicated by the symbol (). Potential protein kinase C (PKC) phosphorylation sites and protein kinase A (PKA) phosphorylation sites are also indicated.

Our screening identified two cPepT1 cDNA variants from the same cDNA library. The cDNA differed by a single amino acid alteration from leucine to serine at amino acid 703 (TTGTCG). These cPepT1 were designated as cPepT1-Leu703 and cPepT1-Ser703. To verify the presence of these variants, total RNA from a pool of small intestinal tissues of 15 broilers as well as RNA from 10 individual broilers were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) using cPepT1-specific primers. DNA sequencing of the RT-PCR products showed that both TTG (Leu703) and TCG (Ser703) polymorphisms exist naturally in our broiler flock (data not shown).

Tissue distribution of cPepT1 mRNA.

A 2.9-kb mRNA was detected in the duodenum, jejunum, ileum, kidney and cecum under medium stringency hybridization conditions (Fig. 3 ). mRNA from duodenal, jejunal and ileal tissues showed the strongest hybridization, whereas the kidney and cecum mRNA showed less hybridization. Poly(A)⁺ RNA from other tissues, including the liver, crop, proventriculus, pectoralis and fibularis longus muscles, showed no detectable hybridization.

View larger version (52K): [in this window] [in a new window]   Figure 3. Northern blot analysis of chicken peptide transporter 1 (cPepT1) mRNA in chicken tissues. Poly (A)+ enriched RNA (10 µg) was loaded per lane and the blot was hybridized with a full-length cPepT1 cDNA as the probe. The 18s rRNA was used as an internal control for loading. Lanes represent the pectoralis muscle (PM), fibularis longus muscle (LM), kidney (K), liver (L), crop (Cr), proventriculus (P), cecum (Ce), duodenum (D), jejunum (J) and ileum (I).

Two systems were used to investigate the function of cPepT1, the Xenopus oocyte expression system and a mammalian cell culture system. Two-electrode voltage-clamp analysis demonstrated that control oocytes (antisense cRNA injected) did not evoke any current with the substrates in all experiments. Inward currents are indicative of peptide transport into oocytes. In standard recording buffer, inward currents were detected in sense cPepT1 cRNA-injected oocytes after perfusion of 1 mmol/L Gly-Sar. Currents were greater (P < 0.05) at pH 6.0 and 6.5 than at pH 5.0, 5.5 and 7.0 (Fig. 4A ). Therefore, the transport process was pH dependent with an optimal pH of 6.0 to 6.5. Substitution of Na⁺, K⁺ or Ca²⁺ did not have any effect on peptide transport activity of cPepT1 at any pH tested (Fig. 4 A). The inward currents in the standard measurement buffer induced by cPepT1 at all pH levels were similar to the values obtained in the absence of Na⁺, K⁺ or Ca²⁺.

View larger version (50K): [in this window] [in a new window]   Figure 4. pH and ion-dependency of chicken peptide transporter 1 (cPepT1) in cRNA-injected Xenopus oocytes and cPepT1-transfected Chinese hamster ovary (CHO) cells. (A) Oocytes were clamped at -60 mV and perfused with 1 mmol/L dipeptide glycylsarcosine (Gly-Sar) in standard measurement buffer at pH 5.0, 5.5, 6.0, 6.5 and 7.0. To determine whether this transport process was dependent upon the presence of Na+, K+ or Ca2+ ions, transport was measured in Na+-free, K+-free and Ca2+-free buffers. Na+-free and K+-free buffers were prepared by replacing NaCl or KCl with choline chloride. Ca2+-free buffer was prepared by replacing CaCl2 with choline chloride in standard measurement buffer. Values are means ± SEM, n = 5. Means without a common letter differ, P < 0.05. (B) CHO cells were transfected with cPepT1 cDNA using Lipofectamine. Transfected cells were then incubated with uptake buffer containing [3H]-Gly-Sar at pH 5.0–7.5. Values are the means of maximum uptake ± SEM, n = 6 total wells in two transfections.

Transport kinetics of cPepT1 were measured using the hydrolysis-resistant dipeptide Gly-Sar as a model peptide. Currents evoked when Gly-Sar was perfused at concentrations ranging from 0.01 to 10 mmol/L demonstrated that transport of Gly-Sar was saturable (Fig. 5A ). The K_t for Gly-Sar was 0.47 mmol/L. Uptake of Gly-Sar by cPepT1-transfected CHO cells showed a K_t of 2.6 ± 0.3 mmol/L and a V_max of 34.6 ± 1.2 nmol/(mg protein · 20 min) (Fig. 5 B). Therefore, the uptake of Gly-Sar in oocytes and CHO cells expressing cPepT1 was concentration dependent and saturable. Transformation of the data from uptake of Gly-Sar resulted in a straight line in the Eadie-Hofstee plot (r = 0.98; Fig. 5 , insets), which indicated the presence of a single transport system responsible for the uptake of Gly-Sar in both cPepT1-injected oocytes and cPepT1-transfected CHO cells.

View larger version (17K): [in this window] [in a new window]   Figure 5. Kinetic parameters of glycylsarcosine (Gly-Sar) uptake by chicken peptide transporter 1 (cPepT1) measured by recording current amplitudes in Xenopus oocytes injected with cPepT1 cRNA and [3H]-Gly-Sar uptake in Chinese hamster ovary (CHO) cells transfected with cPepT1 cDNA. (A) Oocytes were clamped at -60 mV and perfused with 1.2 mL/min at Gly-Sar concentrations ranging from 10 µmol/L to 10 mmol/L in standard measurement buffer at pH 6.0. Values are means ± SEM, n = 5. Inset: Eadie-Hofstee plot of Gly-Sar uptake. (B) cPepT1-transfected CHO cells were incubated with six Gly-Sar concentrations (0.02–10 mmol/L, with 1 µCi/mL [3H]-Gly-Sar). Vector pBK-CMV without insert was transfected into CHO cells as a control. Values are means ± SEM, n = 6 total wells in two transfections. Inset: Eadie-Hofstee plot of Gly-Sar uptake in cPepT1-transfected CHO cells.

Both cPepT1-Leu703 and cPepT1-Ser 703 were present in our chicken RNA pools. A comparison of kinetic features between the two cPepT1 is shown in Table 2 . Changing this amino acid of cPepT1 had no significant effect on transport ability of the cPepT1 to the substrates we tested, with the possible exceptions of Trp-Ala and Trp-Leu.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The cPepT1 mRNA transcript appeared to be expressed predominantly in the small intestine. The expression pattern of poly (A)⁺ RNA in these tissues was consistent with those from a previous study in our laboratory (11 ), which indicated that the small intestine appeared to be the primary site of expression of cPepT1 mRNA transcripts. However, the present study using the cloned cPepT1 as the probe detected a 2.9-kb mRNA, whereas our previous study detected only the 1.9-kb mRNA using a short sheep PepT1 probe (11 ). When low stringency washing was used and exposure to Kodak XAR-5 film was markedly extended, a small amount of hybridization was detected to a 2.9-kb transcript when the sheep probe was used. However, when similar hybridization conditions were used with the cloned cPepT1 as the probe, no hybridization was detected to a 1.9-kb transcript. The difference in mRNA size indicates the presence of a 1.9-kb transcript in chickens that has structural homology to the sheep peptide transporter. What the function of the protein that is encoded by this transcript might be is yet to be determined.

Although previous reports from our laboratory showed that rat myogenic cells (C₂C₁₂) and ovine myogenic satellite cells could utilize exogenous peptides as sources for protein synthesis (23 ,24 ), we did not detect any hybridization in mRNA from pectoralis or fibularis longus muscle. Other studies reported the presence of PepT1 mRNA in the liver and kidney of rabbits, rats and humans (2 ,3 ,5 ). In the present study, cPepT1 mRNA was barely detectable in kidney and cecum, but not detectable in liver. Therefore, the expression pattern of cPepT1 mRNA is slightly different from other PepT1.

The influence of different ions on peptide transport in cPepT1-injected oocytes agrees with other reports that the proton is critical for the transport process, but Na⁺, Cl^- and K⁺ are not (2 ,25 ). However, the transport activity of a PepT1-like transporter in the canine renal cell line (MDCK cells) was affected by Ca²⁺ and a calmodulin-dependent pathway (26 ). Transport of dipeptides by PepT2 in the porcine cell line (LLC-PK1 cells) was also shown to be altered by Ca²⁺, but not through a calmodulin-specific pathway (27 ). In the present study, peptide transport activity of cPepT1 in Xenopus oocytes was independent of Na⁺, K⁺ or Ca²⁺. Overall, in both the oocyte and mammalian cell systems, cPepT1-mediated Gly-Sar uptake was pH dependent with an optimal pH between 6.0 and 6.5.

Most efforts to date that have characterized PepT1 and PepT2 in other species (rabbits, humans, rats and mice) have focused on model peptides and drugs. There has been less attention directed toward the nutritional implications of this process although a few peptides have been examined. We evaluated the transport characteristics of cPepT1 using peptides containing essential amino acids (mainly methionine and lysine). The maximum transport rate and transport affinity of different peptides varied. The nutritional implications of our data are not clear at the moment; however, the wide range of peptides transported by cPepT1 indicates that peptide transport in chickens could be quite important. The very large K_t and IC₅₀ of Lys-Lys and Lys-Trp-Lys represent two peptides with low affinity for cPepT1. This indicates that either these two peptides are less favorable substrates for cPepT1, or they are able to be transported because the physiologic concentrations of these peptides are higher than those of other peptides.

Comparison of the two expression systems showed similar results for substrate affinity. The CHO cell is a suitable system with which to study competition between substrates, whereas the Xenopus oocyte system monitors the cotransport of protons with the peptides. In the present study, for example, tetrapeptides showed weak inhibition of Gly-Sar uptake in cPepT1-expressing CHO cells. In the Xenopus oocyte system, these tetrapeptides did not evoke any detectable inward current at all. Therefore, we hypothesize that in the CHO cell system, these tetrapeptides may be degraded by membrane peptidases and compete for Gly-Sar uptake as smaller di- or tripeptides. Alternatively, they might interact with the transporter itself without actually being transported, causing a change in the transporter conformation that indirectly affects Gly-Sar uptake. Therefore, it seems that cPepT1 uses mainly di- and tripeptides as its substrates.

In summary, this paper describes the cloning and characterization of a novel member of the POT family, cPepT1, with the ability to transport oligopeptides. The cPepT1 has lower amino acid identity compared with PepT1 from other species. Northern blot analysis demonstrated that cPepT1 is strongly expressed in the small intestine, and at lower levels in cecum and kidney, but not in liver, crop, proventriculus, or pectoralis and fibularis longus muscles. The transport function of cPepT1 in Xenopus oocytes and CHO cells indicated that cPepT1 has high affinity for most of the dipeptides and tripeptides tested. The transport process is electrogenic and independent of Na⁺, K⁺ and Ca²⁺. Neither tetrapeptides nor free amino acids are substrates for cPepT1. Our studies have characterized the substrate specificities of cPepT1, which provides new information for better understanding of protein absorption in chickens and ultimately may lead to the formulation of new chicken diets to enhance growth performance.

	FOOTNOTES

 
¹ Supported in part by the Animal Growth, Development and Nutrient Utilization Program of the Animals Division of the National Research Initiative Competitive Grants Program of the U.S. Department of Agriculture project no. 99–03265; Virginia Agricultural Council project no. 358; Virginia Agricultural Experiment Station project no.6129990: and the John Lee Pratt Animal Nutrition Program at Virginia Polytechnic Institute and State University and the Virginia Agriculture Council.

² The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EBI Data Bank under accession number AY029615.

⁴ Abbreviations used: CHO cells, Chinese hamster ovary cells; cPepT1, chicken peptide transporter 1; Gly-Sar, glycylsarcosine; I_max, maximum current; IC₅₀, 50% inhibitory concentration; K_t, affinity constant; oPepT1, ovine peptide transporter 1; PepT, peptide transporter; POT, proton-dependent oligopeptide transporter; RT-PCR, reverse transcriptase-polymerase chain reaction; V_max, maximum velocity.

Manuscript received 10 September 2001. Initial review completed 6 October 2001. Revision accepted 22 December 2001.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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