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Articles

Expression of a Cloned Ovine Gastrointestinal Peptide Transporter (oPepT1) in Xenopus Oocytes Induces Uptake of Oligopeptides in Vitro¹

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

²To whom correspondence should be addressed. E-mail: webbk{at}vt.edu.

	ABSTRACT

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We determined the primary structure, tissue distribution and in vitro functional characterization of a peptide transporter, oPepT1, from ovine intestine. Ovine PepT1 (oPepT1) cDNA was 2829-bp long, encoding a protein of 707 amino acid residues with an estimated molecular size of 78 kDa and an isoelectric point (pI) of 6.57. Transport function of oPepT1 was assessed by expressing oPepT1 in Xenopus oocytes using a two-electrode voltage-clamp technique. The transport process was electrogenic and pH dependent, but independent of Na⁺, Cl^- and Ca²⁺. The oPepT1 displayed a broad substrate specificity for transport of neutral and charged dipeptides and tripeptides. All dipeptides and tripeptides examined evoked inward currents in a saturable manner, with an affinity constant (K_t) ranging from 27 µmol/L to 3.0 mmol/L. No responses were detected from tetrapeptides or free amino acids. Northern blot analysis demonstrated that oPepT1 was expressed in the small intestine, omasum and rumen, but was not expressed in liver and kidney. The presence of the peptide transporter in the forestomach at such levels could provide nutritionally important amino acid nitrogen to ruminants.

KEY WORDS: • ovine • peptide • transport • molecular cloning • Xenopus

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The cloning and characterization of peptide transporters (PepT)³ has provided valuable information about peptide transport in mammalian species (Fei et al. 1994 , Liang et al. 1995 , Miyamoto et al. 1996 , Saito et al. 1995 ). Two peptide transporters, PepT1 and PepT2, with similar structural and functional features, have been cloned. Both PepT1 and PepT2 are membrane proteins with 12 transmembrane domains and a large extracellular loop between transmembrane domains 9 and 10. These peptide transporters recognize di- and tripeptide substrates, as well as pharmacologically active compounds, including ß-lactam antibiotics, angiotensin-converting enzyme inhibitors and the antitumor agent, bestatin (Leibach and Ganapathy 1996 ). The peptide transporter, PepT1, is expressed mainly in the small intestine with little expression occurring in liver and kidney (Fei et al. 1994 , Liang et al. 1995 , Miyamoto et al. 1996 ), whereas PepT2 is expressed mainly in kidney (Saito et al. 1996 ).

Peptide transport is an important physiologic process that occurs in tissues of animals (Matthews 1991 ). However, little research has been conducted to identify the system(s) responsible for the absorption of peptides in domestic animals. Early studies from our laboratory indicated that peptides might be absorbed from the ruminant gastrointestinal tract (Koeln and Webb 1982 ), and recent reports demonstrate the existence and tissue distribution of a peptide transporter(s) in sheep, cows, pigs and chickens (Chen et al. 1999 , Matthews et al. 1996 , Pan et al. 1997 ). Although the size of the mRNA varied among species, the mRNA was present in the small intestine of all animals examined and the omasal and ruminal epithelium of sheep and cows. Expression of mRNA extracted from sheep omasal epithelial tissue was capable of transporting di- to tetrapeptides in Xenopus oocytes. These results suggest that peptide absorption may be nutritionally important in the ruminant as well as other domestic animals. The purpose of this study was to clone and express the ovine peptide transporter (oPepT1) and to characterize the function of this transporter in vitro by expression in Xenopus oocytes.

	MATERIALS AND METHODS

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Materials.

All chemicals, substrates, and reagents were of either molecular biology or cell culture tested chemical grades. The ZAP Express cDNA synthesis system and ZAP Express cDNA Gigapack III cloning kit were purchased from Stratagene (La Jolla, CA). Restriction enzymes were from New England BioLabs (Beverly, MA). Magna nylon transfer membranes were purchased from Micron Separation (Westboro, MA). The RNA transcription kit, mMESSAGE mMACHINE, for synthesis of cRNA was obtained from Ambion (Austin, TX). Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, WI) or Xenopus One (Ann Arbor, MI). Collagenase A was purchased from Boehringer Mannheim (Indianapolis, IN). Streptomycin, penicillin, diethyl pyrocarbonate and eighteen peptides (dipeptides to tetrapeptides) were purchased from Sigma Chemical (St. Louis, MO).

Construction of a directional ovine cDNA library.

The method of Puissant and Houdebine (1990) was used to extract total RNA from tissues of crossbred sheep (average body weight, 60 kg). Poly(A)⁺ RNA was purified from total RNA on oligo(dT) cellulose following established procedures (Sambrook et al. 1989 ). A cDNA library was constructed using poly(A)⁺ RNA isolated from sheep jejunal tissue according to the manufacturer’s protocol with minor modifications. The ZAP Express cDNA synthesis system was employed for this purpose. Before ligation to the ZAP Express vector, cDNA were size-fractionated by gel filtration chromatography (spin column with Sepharose CL-4B, Sigma Chemical). Only the fractions containing cDNA >0.4 kb in size were used for library construction. Vector-ligated cDNA were packaged with Gigapack III Gold packaging extract according to the manufacturer’s packaging protocol. The phage were titered and then introduced into the XL1-Blue MRF' Escherichia coli cell line. The primary library was very unstable; therefore, the library was plated out immediately on a series of large 150-mm NZY agar plates (5000 plaques/plate) to perform plaque lifts for screening of the library.

Screening of the cDNA Library.

Positive clones were identified by plaque hybridization of the cDNA library transferred to Magna nylon transfer membranes. For primary screening, the library was divided into 20 pools of 5000 plaques each. The cDNA probe used for screening was a 446-bp fragment cloned from sheep omasal epithelial total RNA by reverse transcriptase-polymerase chain reaction (Chen et al. 1999 ). 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) and purified by Sephadex G-25 (Sigma, St. Louis, MO) spin column chromatography. Hybridization was carried out for 16 h at 42°C in a solution containing 50% formamide, 5X Denhardt’s solution, 6X SSPE (1X 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 high stringency conditions, which involved washing twice in 5X standard saline citrate (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. Positive clones, identified after primary screening, were subjected to three more rounds of screening under the same conditions. After the quaternary screen, 100% of the plaques showed positive hybridization by autoradiography, which confirmed the purity of the phage.

Sequencing of the full-length cDNA insert.

Both sense and antisense strands of the cDNA were sequenced by primer walking. Sequencing by the dideoxynucleotide chain termination method was performed manually using a Sequenase version 2.0 DNA sequencing kit (U.S. Biochemical, Cleveland, OH). Internal regions of the cDNA were sequenced using 17- to 19-mer oPepT1-specific primers. Sequencing reactions were run on 7% polyacrylamide gels. Analysis of nucleotide and amino acid sequence was performed using the sequence analysis software Lasergene (DNAStar, Madison, WI). Database searches were done using the GenBank Program BLAST.

Northern analysis.

Tissue distribution of oPepT1 mRNA transcripts was determined by Northern blot. Poly(A)⁺ RNA was isolated and purified as described above from sheep tissues. Poly(A)⁺ RNA samples (10 µg) from these tissues were denatured and size-fractionated on a 1% agarose gel containing 2.2 mol/L formaldehyde. As an internal control to ensure equal RNA loading, 18s rRNA was monitored by ethidium bromide staining. The size-fractionated RNA was then transferred onto a nylon membrane and probed with the full-length oPepT1 cDNA. The probe was labeled with [-³²P]dATP (ICN Pharmaceutical) by nick translation using DNA polymerase I/DNase I (Life Technologies) and purified by Sephadex G-25 spin column chromatography. The blot was hybridized overnight at 42°C for 16–18 h and washed under high stringency conditions according to the manufacturer’s protocol (Micron Separation). The blot was exposed to Kodak XAR-5 film with an intensifying screen at -80°C.

In vitro transcription of cRNA.

cRNA was synthesized using the RNA transcription kit mMESSAGE mMACHINE (Ambion) according to the manufacturer’s protocol. For sense cRNA synthesis, phagemid containing the cDNA insert was linearized using Sma I and 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 linearized using Sal I and transcribed in vitro by T₇ RNA polymerase in the presence of an RNA cap analog. The resultant cRNA was purified by multiple extractions with phenol/chloroform and precipitated with ethanol. The cRNA was recovered by centrifugation at 12,000 x g at 4°C for 30 min, resuspended in nuclease-free water at a concentration of 1 g/L and stored frozen at -80°C in aliquots. Concentration was determined by UV spectrophotometry, and the integrity of the cRNA was verified by denaturing 1% agarose-formaldehyde gel electrophoresis and visualization using ethidium bromide staining.

Electrophysiology.

Stage V oocytes were collected as described by Goldin (1992) . Healthy oocytes at stage V were sampled and only batches with a resting membrane potential (V_m) more negative than -30 mV 1 d after removal of follicular tissue were used for injection. Using a microinjection system, 50 ng of either sense cRNA or antisense cRNA was injected into each oocyte in the vegetal pole, near the polar interface. Antisense cRNA solution was used as a control. The injected oocytes were returned to the culture solution and incubated at 18°C for 1–7 d. The culture solution was changed daily and damaged oocytes, as indicated by misshapen and ruptured oocytes, were discarded.

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 (Soderlund et al. 1989 ). Normally, electrophysiologic measurements in sense-cRNA– or antisense-cRNA–injected oocytes were carried out 4–7 d after injection. A single oocyte was placed in a recording chamber (200 µL) in the presence of standard measurement buffer (in mmol/L: 96 NaCl, 2 KCl, 1 MgCl₂, 1.8 CaCl_2, 5 HEPES). Oocytes were maintained in the buffer for at least 10 min before impalement. Oocytes were always impaled to enable measurement of the resting V_m before the current electrode was inserted. A subsequent 10-min period for stabilization before the start of the experiment was included. In all cases, the electrodes were inserted into the dark, animal pole for better visualization of the two electrodes. Only oocytes with a resting V_m more negative than -30 mV were used for recordings.

All peptide substrate solutions were prepared by dissolving the peptides in the appropriate measurement buffer and adjusting the pH with NaOH or HCl. 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). The oocytes were thoroughly washed with fresh buffer before exposure to the next testing substrate, and several consecutive treatments were applied to the same oocyte. The experiments were repeated in at least five independent oocytes for data analysis. All experiments were performed at room temperature (21°C).

Computational analysis.

A hydrophobicity plot of oPepT1 was constructed according to the Kyte and Doolittle (1982) hydropathy analysis using a window of 21 amino acid residues via the TMpred service program from the European Molecular Biology Network. The TMpred program makes a prediction of membrane-spanning regions and their orientation. The algorithm is based on the statistical analysis of TMbase, a database of naturally occurring transmembrane proteins (Hofmann and Stoffel 1993 ).

Protein phosphorylation/dephosphorylation sites of oPepT1 were predicted on the basis of consensus amino acid sequences as substrate specificity determinants for protein kinases and phosphatases (Kennelly and Krebs 1991 ). The consensus amino acid sequences for cAMP-dependent protein kinase (PKA) site prediction are R-R/K-X-S/T, R-X₂-S/T or R-X-S/T. The consensus amino acid sequences for protein kinase C (PKC) site prediction are (R/K_1–3, X_2–0)-S/T-(X_2–0, R/K_1–3), S/T-(X_2–0, R/K_1–3) or (R/K_1–3, X_2–0)-S/T. The consensus amino acid sequence for N-linked glycosylation site prediction is N-X-T/S (X = any amino acid).

Calculations and statistics.

The kinetic parameters, the Michaelis-Menten constant, K_t, and the maximal velocity, I_max, and all other calculations (linear as well as nonlinear regression analysis) were performed using PRISM (GraphPad, San Diego, CA). The experiments were generally carried out with 5–6 oocytes from at least two different batches, and results are presented as means ± SEM. Comparative analysis of transport parameters for the different substrates was performed in the same batch of oocytes. Data were evaluated using one-way ANOVA. The least significant difference test was used for post-hoc comparisons. A P-value of < 0.05 was considered to indicate significant difference. Data were also analyzed by regression analysis to investigate the form of the relationship between transport affinity constant and peptide characteristics. The REG procedure of SAS (1989) was used in this study for regression analysis.

	RESULTS

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Structural features of the ovine intestinal PepT1 cDNA.

The ovine intestinal oPepT1 cDNA was 2829-bp long with an open reading frame of 2121 bp. A 79-bp 5'UTR and a 629-bp 3' UTR flanked the open reading frame. The initiation codon was consistent with the Kozak consensus sequence, GCCGCC(A/G)CATGG (Kozak 1987 ). At the 3' end, the cDNA had a polyadenylation signal (AATAAA) 12 nt preceding the polyA tail. The encoded protein was predicted to have 707 amino acids with a molecular mass of 78 kDa and an isoelectric point (pI) of 6.57. Alignment results showed that the predicted amino acid sequence of oPepT1 was 83, 81 and 78% identical to human, rat and rabbit PepT1, respectively (Fig. 1 ).

View larger version (45K): [in this window] [in a new window]   Figure 1. Predicted amino acid sequence of ovine gastrointestinal peptide transporter (oPepT1). The alignment of deduced amino acid sequences of human, rat, rabbit and sheep PepT1. Amino acids identical to the human sequence are indicated by dashes (-). The nucleotide sequence of oPepT1 cDNA has been deposited in the GenBank (accession number AY027496).

View larger version (18K): [in this window] [in a new window]   Figure 2. Membrane model of ovine gastrointestinal peptide transporter (oPepT1). 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.

A 2.8-kb poly(A)⁺ RNA was detected from rumen, omasum and small intestine tissues (Fig. 3 ). The poly(A)⁺ RNA from other tissues, including the liver, kidney, semitendinosus muscle, longissimus muscle, abomasum, cecum and colon showed no detectable hybridization. The ethidium bromide staining of the residual 18s rRNA was relatively constant among lanes, indicating that approximately equal amounts of total RNA were loaded in each lane (Fig. 3) . Therefore, relative levels of oPepT1 expression as a proportion of poly(A)⁺ RNA isolated were higher in the small intestine than in the stomach.

View larger version (40K): [in this window] [in a new window]   Figure 3. Northern blot analysis of ovine gastrointestinal peptide transporter (oPepT1) mRNA in sheep tissues. Poly(A)+–enriched RNA (10 µg) was loaded per lane and the blot was hybridized with a full-length oPepT1 cDNA as the probe. Ethidium bromide staining of 18s rRNA was used as an internal control to ensure equal RNA loading. Lanes represent the liver (L), kidney (K), semitendinosus muscle (SM), longissimus muscle (LM), omasum (O), rumen (R), abomasum (A), duodenum (D), jejunum (J), ileum (I), cecum (Ce) and colon (Co).

Control oocytes (antisense cRNA injected) did not show any response to perfusion with the substrate in this and all subsequent experiments. Inward currents of 35 ± 3 nA at pH 5.0, 43 ± 4 nA at pH 5.5, 42 ± 3 nA at pH 6.0, 30 ± 4 nA at pH 6.5 and 20 ± 5 nA at pH 7.0 were detected in sense oPepT1 cRNA-injected oocytes after perfusion of 1 mmol/L glycyl-sarcosine (Gly-Sar; Fig. 4 ). Therefore, inward currents, which are indicative of peptide transport in oocytes (Matthews et al. 1996 ), were greater (P < 0.05) at pH 5.5 and 6.0 compared with pH 5.0, 6.5 and 7. Substitution of ions did not have any effect on peptide transport activity of oPepT1 at all pH levels tested (Fig. 4) . The inward currents in the standard measurement buffer induced by oPepT1 at pH 5.5–6.0 (42 ± 4 nA) were similar to the values obtained in the absence of Na⁺ (42 ± 4 nA), Cl^- (41 ± 5 nA) or Ca²⁺ (42 ± 3 nA). Therefore, the data indicated that peptide transport activity of oPepT1 was driven by an inwardly directed H⁺ gradient and was independent of Na⁺, Cl^- or Ca²⁺.

View larger version (21K): [in this window] [in a new window]   Figure 4. Influence of ions on current flow in oocytes injected with ovine gastrointestinal peptide transporter (oPepT1) cRNA. Oocytes were clamped at -60 mV and perfused with 1 mmol/L dipeptide glygyl-sarcosine (Gly-Sar) in standard measurement buffer at pH 5.0, 5.5, 6.0, 6.5 and 7.0. Transport was measured in standard, Na+-free, Cl--free, and Ca2+-free buffers. Na+-free and Cl--free buffers were prepared by replacing NaCl with choline chloride or sodium gluconate, respectively. 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.

Currents generated in sense cRNA–injected oocytes by 5 mmol/L zwitterionic and anionic substrates Gly-Sar, Met-Met and Glu-Glu increased as buffer pH decreased from 7.0 (Gly-Sar, 18 ± 5 nA; Glu-Glu, 20 ± 4 nA; Met-Met, 15 ± 6 nA;) to 5.5 (Gly-Sar, 41 ± 4 nA; Glu-Glu, 41 ± 6 nA; Met-Met, 36 ± 5 nA) (Fig. 5 ). The cationic peptide, Lys-Lys, showed the opposite effect, with high inward currents at pH 7.0 (33 ± 5 nA) and low at pH 5.0 (18 ± 4 nA). Thus, not all peptides were transported with the same pH dependency.

View larger version (13K): [in this window] [in a new window]   Figure 5. Influence of net charge of substrates on current responses in oocytes injected with ovine gastrointestinal peptide transporter (oPepT1) cRNA. The effect of peptide charge was examined in oocytes clamped at -60 mV and perfused with 5 mmol/L (saturating substrate concentrations) dipeptides [glygyl-sarcosine (Gly-Sar), Met-Met, Glu-Glu, or Lys-Lys] in standard measurement buffer at pH 5.0, 5.5, 6.0, 6.5 and 7.0. Values are means ± SEM, n = 5.

Perfusion of the three peptides (5 mmol/L dipeptides Met-Met, Glu-Glu, and Lys-Lys) alone, or in combination simultaneously or sequentially did not cause any additional responses. For instance, evoked current by perfusion of 5 mmol/L Met-Met was not affected when either Glu-Glu or Lys-Lys or both were added in the perfusion (Fig. 6 ). Therefore, all peptides appeared to interact at the same binding site on oPepT1.

View larger version (19K): [in this window] [in a new window]   Figure 6. The effects of combined dipeptide perfusion to the current responses determined in cRNA-injected oocytes recorded using the two-electrode voltage clamp. To determine whether peptide-evoked inward currents were generated by the same substrate binding site on ovine gastrointestinal peptide transporter (oPepT1), oocytes were clamped at -60 mV and perfused with 5 mmol/L dipeptides Met-Met, Glu-Glu and Lys-Lys in standard measurement buffer at pH 5.5.

Perfusion of 1 mmol/L of the amino acids, Gly, Met, Glu, or Lys, did not have any effect. All di- and tripeptides examined (concentration ranging from 0.01 to 10 mmol/L) evoked inward currents in a saturable manner, resulting in an affinity constant (K_t) range of 27 µmol/L to 3.0 mmol/L. No responses were detected from tetrapeptides in this perfusion study (Table 1 ). The peptides examined constitute a variety of substrates, which differ in their molecular weight, electrical charge and hydrophobicity. However, for all peptides, no correlation was found between affinity constant and molecular weight or net charge or hydrophobicity. Therefore, oPepT1 seemed to be able to transport peptides regardless of their molecular weight, net charge or hydrophobicity. For five N-terminal methionine-containing dipeptides, no correlation was found between K_t and the nature of the C-terminal amino acid. The dipeptides, Leu-Val and Val-Leu, had similar K_t, suggesting that they were recognized by oPepT1 in a similar manner.

	DISCUSSION

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Structural characteristics of oPepT1.

Despite the similarity of oPepT1 to other PepT1, there were some noticeable differences. First, the calculated pI of oPepT1 (6.57) was lower than that of others (rat PepT1, pI 7.39; rabbit PepT1, pI 7.47; human PepT1, pI 8.58). There may be a physiologic reason for this difference. Bicarbonate is the principle buffer that is secreted by mucosal cells throughout the small and large intestinal tract and is also provided in pancreatic and biliary secretions. The bicarbonate provided in pancreatic and biliary secretions is used to neutralize stomach acid and to provide a more favorable intestinal pH for enzymatic breakdown of feed components and absorption of nutrients. The pH of the unstirred water layer in the intestinal lumen of monogastric animals is 5.5–6.0 (Ganapathy and Leibach 1983 ). In ruminants, pancreatic and biliary secretions appear inadequate to handle the acid load delivered from the stomach to the small intestine. The pH of the duodenum and upper jejunum in sheep is rather acidic, ranging from 2.5 to 4.5, and it is not until the lower jejunum that the intestinal pH approaches 6 (Ben Ghedalia et al. 1974 ). Thus, a PepT1 protein with a lower pI might be expected in a ruminant.

Second, significant differences exist in the putative phosphorylation sites for protein kinases. Rabbit PepT1 has only one PKC phosphorylation site and one PKA phosphorylation site (Fei et al. 1994 ). Human PepT1 has two PKC phosphorylation sites, but no site for PKA phosphorylation (Liang et al. 1995 ). Rat PepT1 has one PKC phosphorylation site and one PKA phosphorylation site (Miyamoto et al. 1996 ). Results from a study of Brandsch et al. (1994) showed that a H⁺-dependent peptide transport system expressed in human Caco-2 cells was regulated by PKC. Therefore, it seems reasonable to speculate at this juncture that oPepT1 may be regulated by protein kinases. However, the functional importance of these different potential phosphorylation sites remains to be determined.

Analysis of charge distribution shows that oPepT1 has a negatively charged cluster from 672 to 691. Compared with PepT1 from other species, oPepT1 has more negatively charged amino acids in this cluster. No information is available at the moment concerning how the negatively charged cluster at the C-terminal end of the peptide transporter affects the function of the transport system. Functional analysis of a chimeric peptide transporter derived from PepT1 and PepT2 showed that the large extracellular loop and the C-terminal end were not responsible for the kinetic characteristics of the transport system (Doring et al. 1997 ). It is likely that this region is important for peptide transporter protein trafficking inside the cells or for regulating transport function because of the potential PKC site located at the C-terminus. Thus, the negatively charged C-terminal end may also affect oPepT1 transport activity by altering the rate of oPepT1 protein insertion into the plasma membrane.

Expression of oPepT1 mRNA.

The oPepT1 transcript appeared to be expressed in the small intestine, omasum and rumen as determined by Northern blot analysis. The expression pattern of poly(A)⁺ RNA in these tissues was consistent with that from a previous study in our laboratory (Chen et al. 1999 ). The small intestine appeared to be the primary site of expression of oPepT1 mRNA transcripts. This pattern is in agreement with previous studies that the major site of absorption of protein digestion products (e.g., amino acids) is the small intestine in ruminant animals (Phillips et al. 1979 , Wilson and Webb, 1990 ). In addition to the small intestine, the rumen and omasum are recognized for their absorptive activity of large quantities of volatile fatty acids and ammonia. The presence of oPepT1 mRNA in the omasum and rumen indicates that the oPepT1 protein could contribute to the absorption of small peptides in the forestomach of sheep. Previous reports from our laboratory showed that rat myogenic cells (C₂C₁₂) and ovine myogenic satellite cells can utilize exogenous methionine-containing peptides as sources of methionine for protein synthesis and, in some cases, the peptides were more extensively used than methionine (Pan et al. 1996 , Pan and Webb, 1998 ). We were unable to detect any hybridization to mRNA from longissimus muscle or semitendinosus muscle. The possible presence of one or more peptide transport protein(s) in muscle cells other than oPepT1 or the utilization of peptides by cultured cells via mechanisms other than peptide transporters cannot be discounted. Other studies reported the presence of PepT1 mRNA in the liver and kidney of rabbits, rats and humans (Fei et al. 1994 , Liang et al. 1995 , Miyamoto et al. 1996 ). In the present study, oPepT1 mRNA was not detectable in liver or kidney. This may indicate a different pattern of expression for the peptide transporter mRNA in ruminant animals.

Functional characteristics of oPepT1.

The influence of different ions on peptide transport in oPepT1-injected oocytes indicated that peptide transport activity of oPepT1 was driven by an inwardly directed H⁺ gradient and was independent of Na⁺, Cl^- or Ca²⁺. These data agree with other reports that a H⁺ gradient is critical for the transport process but that Na⁺, Cl^- and K⁺ are not (Fei et al. 1994 , Mackenzie et al. 1996 ). Unlike oPepT1, the canine renal cell line (MDCK cells) has transport activity of a PepT1-like transporter that was affected by Ca²⁺ and calmodulin-dependent processes (Brandsch et al. 1995 ). Transport of dipeptides in the porcine cell line (LLC-PK1 cells) by PepT2 was also shown to be altered by Ca²⁺, but not through a calmodulin-specific pathway (Wenzel et al. 1999 ).

The pH had a dramatic effect on the transport process of charged peptides. Zwitterionic and anionic substrates (Gly-Sar, Met-Met and Glu-Glu) were transported via oPepT1 with a pH optimum of 5.0, whereas for the cationic substrate, Lys-Lys, the pH optimum was 7.0. However, at pH 5.5, Lys-Lys still caused inward currents. This observation indicates that, at the physiologic pH in the intestine, which is pH 4.5–5.0 for ruminants, oPepT1 can transport all peptide substrates, regardless of their charges. But zwitterionic and anionic peptides are probably transported more quickly than cationic peptides. These data agree with a published report indicating that at physiologic pH (5.5–6.0), rabbit and human PepT1 (Amasheh et al. 1997 , Steel et al. 1997 ) preferred neutral and acidic peptides as their substrates.

Much effort has been expended to characterize PepT1 and PepT2 in humans, rats and rabbits. Throughout all of this, the major focus has been on the characterization of peptidomimetic drugs. Although a few peptides have been evaluated, there has been essentially no attention given to the implications this process may have with regard to the nutrition of the animal. Given the large amount of protein that is synthesized by food-producing animals in a very short time, it seems reasonable to assume that processes such as peptide absorption could be quite important.

The transport of 14 dipeptides and tripeptides by oocytes expressing oPepT1 in the present study obeyed Michaelis-Menten-type kinetics and a K_t range of 0.027 to 0.61 mmol/L for dipeptides and 0.15 to 3.0 mmol/L for tripeptides was observed. Even though the peptides evaluated in this study constitute only a sample of the possible di- and tripeptides present as a result of protein hydrolysis, there appears to be a considerable range of affinity for these substrates by oPepT1. What nutritional implication this may have is not clear. Certainly, proteins differ in their amino acid composition and thus also in the composition of peptides produced upon hydrolysis. Whether these peptides and their subsequent absorption are in any way related to protein quality is yet to be determined, but the possibility cannot be discounted. Much remains to be learned about the absorption of peptides and the relative rate of peptide amino acid compared with free amino acid absorption. With the additional information that will come with continued investigation of peptide transport may come the insight that will allow nutritionists to formulate diets that are more efficiently used and are more nutritionally adequate.

In summary, we have cloned an ovine intestinal peptide transporter, oPepT1, from sheep intestine. The oPepT1 has high homology with PepT1 from other species. High stringency Northern blot analysis demonstrated that oPepT1 is strongly expressed in the small intestine, at lower levels in omasum and much lower in rumen, but not in liver, kidney or muscle. Our studies demonstrate that oPepT1 is functionally expressed in Xenopus oocytes. The transport process is electrogenic and independent of Na⁺, Cl^- and Ca²⁺. Characterization of oPepT1 shows that oPepT1 transports a variety of dipeptides and tripeptides as its substrates, independent of their physicochemical characteristics, but not tetrapeptides or free amino acids.

	FOOTNOTES

 
¹ Supported in part by the Virginia Agricultural Experimental Station under project no. 6129990. Support was also provided by the John Lee Pratt Animal Nutrition Program at Virginia Polytechnic Institute and State University.

³ Abbreviations used: Gly-Sar, glycyl-sarcosine; K_t, affinity constant; oPepT, ovine gastrointestinal peptide transporter; PepT, peptide transporter; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; SSC, standard saline citrate; V_m, resting membrane potential.

Manuscript received June 19, 2000. Initial review completed August 10, 2000. Revision accepted January 16, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Amasheh S., Wenzel U., Boll M., Dorn D., Weber W.-M., Clauss W., Daniel H. Transport of charged dipeptides by the intestinal H+/peptide symporter PepT1 expressed in Xenopus laevis oocytes. J. Membr. Biol. 1997;155:247-256[Medline]

2. Ben-Ghedalia D., Tagari H., Bondi A. Protein digestion in the intestine of sheep. Br. J. Nutr. 1974;31:125-130[Medline]

3. Boll M., Markovich D., Weber W.-M., Korte H., Daniel H., Murer H. Expression cloning of a cDNA from rabbit small intestine related to proton-coupled transport of peptides, ß-lactam antibiotics and ACE-inhibitors. Eur. J. Physiol. 1994;429:146-149[Medline]

4. Brandsch M., Ganapathy V., Leibach F. H. H⁺-peptide cotransport in Madin-Darby canine kidney cells: expression and calmodulin-dependent regulation. Am. J. Physiol. 1995;368:F391-F398

5. Brandsch M., Miyamoto Y., Ganapathy V., Leibach F. H. Expression and protein kinase C-dependent regulation of peptide/H⁺ co-transport system in the Caco-2 human colon carcinoma cell line. Biochem. J. 1994;299:253-260

6. Chen H., Wong E. A., Webb K. E., Jr Tissue distribution of a peptide transporter mRNA in sheep, dairy cows, pigs, and chickens. J. Anim. Sci. 1999;77:1277-1283[Abstract/Free Full Text]

7. Doring F., Dorn D., Bachfischer U., Amasheh S., Herget M., Daniel H. Functional analysis of a chimeric mammalian peptide transporter derived from the intestinal and renal isoforms. J. Physiol. 1997;497:773-779[Medline]

8. Fei Y.-J., Kanal Y., Nussberger S., Ganapathy V., Leibach F. H., Romero M. F., Singh S. K., Boron W. F., Hediger M. A. Expression cloning of a mammalian proton-coupled oligopeptide transporter. Nature (Lond.) 1994;368:563-566[Medline]

9. Fei Y.-J., Liu W., Prasad P. D., Kekuda R., Oblak T. G., Ganapathy V., Leibach F. H. Identification of the histidyl residue obligatory for the catalytic activity of the human H⁺/peptide cotransporters PepT1 and PepT2. Biochemistry 1997;36:452-460[Medline]

10. Ganapathy V., Leibach F. H. Role of pH gradient and membrane potential in dipeptide transport in intestinal and renal brush-border membrane vesicles from the rabbit. Studies with L-carnosine and glycyl-L-proline. J. Biol. Chem. 1983;258:14189-14192[Abstract/Free Full Text]

11. Goldin A. L. Maintenance of Xenopus laevis and oocyte injection. Methods Enzymol 1992;207:266-279[Medline]

12. Hofmann K., Stoffel W. TMbase—a database of membrane spanning proteins segments. Biol. Chem. 1993;347:166-180

13. Kennelly P. J., Krebs E. G. Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. J. Biol. Chem. 1991;266:15555-15558[Free Full Text]

14. Koeln L. L., Webb K. E., Jr Peptide, erythrocyte and plasma amino acid transport across the gastrointestinal tract and liver of calves. Fed. Proc. 1982;41:948-953

15. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 1987;15:8125-8148[Abstract/Free Full Text]

16. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 1982;157:105-132[Medline]

17. Leibach F. H., Ganapathy V. Peptide transporters in the intestine and the kidney. Annu. Rev. Nutr. 1996;16:99-119[Medline]

18. Liang R., Fei Y.-J., Prasad P. D., Ramamoorthy S., Han H., Yang-Feng T. L., Hediger M. A., Ganapathy V., Leibach F. H. Human intestinal H⁺/peptide cotransporter cloning, functional expression, and chromosomal localization. J. Biol. Chem. 1995;270:6456-6463[Abstract/Free Full Text]

19. Liu W., Liang R., Ramamoorthy S., Fei Y.-J., Ganapathy M. E., Hediger M. A., Ganapathy V., Leibach F. H. Molecular cloning of PEPT 2, a new member of the H⁺/peptide cotransporter family, from human kidney. Biochim. Biophys. Acta 1995;1235:461-466[Medline]

20. Mackenzie B., Fei Y. J., Ganapathy V., Leibach F. H. The human intestinal H⁺/oligopeptide cotransporter hPEPT1 transports differently-charged dipeptides with identical electrogenic properties. Biochim. Biophys. Acta. 1996;1284:125-128[Medline]

21. Matthews D. M. Protein Absorption: Development and Present State of the Subject 1991 Wiley-Liss New York, NY.

22. Matthews J. C., Wong E. A., Bender P. K., Bloomquist J. R., Webb K. E., Jr Demonstration and characterization of dipeptide transport system activity in sheep omasal epithelium by expression of mRNA in Xenopus laevis oocytes. J. Anim. Sci. 1996;74:1720-1727[Abstract]

23. Miyamoto K.-I., Shiraga T., Morita K., Yamamoto H., Haga H., Taketani Y., Tamai I., Sai Y., Tsuji A., Takeda E. Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide transporter. Biochim. Biophys. Acta 1996;1305:34-38[Medline]

24. Pan Y., Bender P. K., Akers R. M., Webb K. E., Jr Methionine-containing peptides can be used as methionine sources for protein accretion in cultured C₂C₁₂ and MAC-T cells. J. Nutr. 1996;126:232-241

25. Pan Y., Webb K. E., Jr Peptide-bound methionine as methionine sources for protein accretion and cell proliferation in primary cultures of ovine skeletal muscle. J. Nutr. 1998;128:251-256[Abstract/Free Full Text]

26. Pan Y.-X., Wong E. A., Bloomquist J. R., Webb K. E., Jr Poly(A)⁺ RNA from sheep omasal epithelium induces expression of a peptide transport protein(s) in Xenopus laevis oocytes. J. Anim. Sci. 1997;75:3323-3330[Abstract/Free Full Text]

27. Phillips W. A., Webb K. E., Jr, Fontenot J. P. Characteristics of threonine, valine, and methionine absorption in the jejunum and ileum of sheep. J. Anim. Sci. 1979;48:926-933

28. Puissant C., Houdebine L.-M. An improvement of the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. BioTechniques 1990;8:148-149[Medline]

29. Saito H., Okuda M., Terada T., Sasaki S., Inui K.-I. Cloning and characterization of a rat H⁺/peptide cotransporter mediating absorption of beta-lactam antibiotics in the intestine and kidney. J. Pharmacol. Exp. Ther. 1995;275:1631-1637[Abstract/Free Full Text]

30. Saito H., Terada T., Okuda M., Sasaki S., Inui K.-I. Molecular cloning and tissue distribution of rat peptide transporter PepT2. Biochim. Biophys. Acta 1996;1280:173-177[Medline]

31. Sambrook J., Fritsch E. F., Maniatis T. Molecular Cloning: A Laboratory Manual 2nd ed. 1989 Cold Spring Harbor NY.

32. SAS Institute Inc SAS User’s Guide: Statistics 1989 SAS Institute Cary, NC.

33. Soderlund D. M., Bloomquist J. R., Wong F., Payne L. L., Knipple D. C. Molecular neurobiology: implications for insecticide action and resistance. Pestic. Sci. 1989;26:359-374

34. Steel A., Nussberger S., Romero M. F., Boron W. F., Boyd C.A.R., Hediger M. A. Stoichiometry and pH dependence of the rabbit proton-dependent oligopeptide transporter PepT1. J. Physiol. 1997;498:563-569[Medline]

35. Wenzel U., Diehl D., Herget M., Kuntz S., Daniel H. Regulation of the high-affinity H+/peptide cotransporter in renal LLC-PK1 cells. J. Cell. Physiol. 1999;178:341-348[Medline]

36. Wilson J. W., Webb K. E., Jr Lysine and methionine transport by bovine jejunal and ileal brush border membrane vesicles. J. Anim. Sci. 1990;68:504-514[Abstract]

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