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Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg VA 24061
2To whom correspondence should be addressed. E-mail: webbk{at}vt.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • cloning • protein kinase • sequence • inhibitor • activator
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The cloning and characterization of peptide transporters, PepT1 and PepT2, has provided valuable information about peptide transport in mammalian species (2
–5
). 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 (6
). The peptide transporter, PepT1, is mainly expressed in the small intestine with little expression occurring in liver and kidney (2
,7
), whereas PepT2 is mainly expressed in kidney (8
–10
).
However, little research has been conducted to identify the system(s) responsible for the absorption of peptides in food-producing animals. The cloning and expression of an ovine peptide transporter (oPepT1) in Xenopus oocytes provided information for the first time on the molecular structure and basic functions of a peptide transporter in food-producing animals (11
). To investigate the function of oPepT1 in mammalian cells, oPepT1 was transiently transfected in Chinese hamster ovary (CHO)3
cells and expression of oPepT1 was studied by measuring the uptake of [3H]-Gly-Sar into transfected CHO cells. Our results show that oPepT1 can efficiently transport di- and tripeptides and that activity of oPepT1 can be altered by protein kinase C (PKC) activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All chemicals, substrates and reagents were of either molecular biology or cell culture-tested chemical grades. [3H]-Gly-Sar (specific radioactivity, 110 mCi/mmol) was purchased from Moravek Biochemical (Brea, CA). Media, nonessential amino acids and trypsin were purchased from Fisher Scientific (Pittsburgh, PA). Lipofectamine was purchased from Life Technologies (Gaithersburg, MD). CHO cells were supplied by American Type Culture Collection (Rockville, MD). Penicillin, streptomycin, staurosporine, phorbol 12-myristate-13-acetate (PMA), cycloheximide and unlabeled peptides (dipeptides to tetrapeptides) were purchased from Sigma (St. Louis, MO).
Cell culture.
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 g/L). All cells were cultured in an atmosphere of 5% CO2 and 90% relative humidity at 37°C. Culture medium was changed daily. One day before transfection, cells were trypsinized and plated onto 12-well plates at a density of 2.4 x 105/well.
Transfection.
The construct used in all expression studies contains the oPepT1 cDNA (11
) cloned into the pBK-CMV expression vector (Stratagene, La Jolla, CA) under the control of the CMV promotor. CHO cells were transfected with the pBK-oPepT1 expression vector or just the pBK-CMV vector as a control of measuring endogenous transport activity. Transfection was done according to the manufacturer’s protocol. Briefly, for each well, 0.8 µg of plasmid (with or without insert) were mixed with 2.4 µL of lipofectamine (2 g/L) in 40 µL of OPTI-MEM reduced serum medium 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 in a 5% CO2, 90% relative humidity incubator. For each assay, at least two transfections were performed.
Transport assay.
Transport activity of CHO cells transiently transfected with oPepT1 was examined with [3H]-Gly-Sar, a known hydrolysis-resistant peptide substrate. Transfected cells were washed three times with pH 6.0 uptake buffer containing 25 mmol/L MES/Tris, 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 mCi/L [3H]-Gly-Sar). Uptake solutions of varying pH were added to each well and incubated for 40 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 followed by incubation at room temperature for 10 min. The 3H content of the cell lysate was quantified by liquid scintillation counting (LS 500TA scintillation counter; Beckman Instrument, Fullerton, CA) and the protein amount of each cell extract was measured by a modified Lowry assay using the Bio-Rad DC Protein kit (Bio-Rad, Hercules, CA).
Inhibition studies were performed under the same condition as described above, except that 20 µmol/L [3H]-Gly-Sar (50 mCi/mmol) was used for the radiolabeled substrate and five concentrations (0.001 to 10 mmol/L) of inhibitor substrates were added to the reaction to measure inhibition of [3H]-Gly-Sar uptake. The concentration of competitive peptide that caused 50% inhibition of Gly-Sar uptake (IC50) was calculated by PRISM (GraphPad, San Diego, CA).
In vitro regulation assay.
For studies of PKC inhibition or activation on peptide transport activity, transfected CHO cells were preincubated in the presence of 0.1 and 1 µmol/L staurosporine or 1 and 10 µmol/L PMA. Solutions of staurosporine, PMA and cycloheximide were prepared in dimethyl sulfoxide. The effects of pretreatment time and concentration of effectors on Gly-Sar uptake were studied at 30 and 60 min. After treatment, cells were washed twice with uptake buffer before initiation of uptake measurements in the absence of effectors. Gly-Sar solution was prepared at 0.02 mmol/L with 1 mCi/L [3H]-Gly-Sar. Uptake solutions were added to each well and incubated for 40 min at room temperature. Uptake was stopped by washing cells with ice-cold uptake buffer. [3H]-Gly-Sar uptake was determined as described above.
Calculations and statistics.
Determination of kinetic parameters and all other calculations (linear as well as nonlinear regression analysis) were performed using PRISM. Results are presented as the mean ± SEM. Data were evaluated using one-way ANOVA. The least significant difference test was used for posthoc comparisons. Differences with P < 0.05 were considered significant. 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 (12
) was used in this study for regression analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The time course for expression of oPepT1 in transfected CHO cells was examined by measuring the uptake of [3H]-Gly-Sar. Expression of oPepT1 was detectable at 8 h post-transfection and plateaued between 16 and 21 h (Fig. 1
A). In all subsequent experiments, transport was measured 16 h after transfection. Uptake of [3H]Gly-Sar by CHO cells transfected with the pBK-CMV vector alone was only 
1% of that observed in pBK-oPepT1-transfected CHO cells. Thus, there was negligible endogenous Gly-Sar transport activity in CHO cells. Therefore, the uptake values measured in pBK-oPepT1-transfected cells were analyzed directly without being adjusted for the values obtained in pBK-CMV-transfected CHO cells.
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B). Uptake of Gly-Sar by pBK-oPepT1-transfected CHO cells increased as incubation time with [3H]-Gly-Sar increased. The uptake rate plateaued between 40 and 80 min. There was no increase of the uptake of [3H]-Gly-Sar in pBK-CMV-transfected CHO cells. Therefore, in all subsequent experiments, the time of incubation for transport measurements was 40 min. pH and concentration effects on Gly-Sar uptake.
The effect of extracellular pH on the uptake of [3H]-Gly-Sar was studied in transfected CHO cells. In pBK-oPepT1-transfected CHO cells, uptake was greater at pH 5.5 and 6.0 than at 5.0, 6.5, 7.0 and 7.5 (Fig. 2
). Thus, optimum uptake was at pH 5.5 and 6.0. Control CHO cells transfected with only the pBK-CMV vector showed negligible transport when measured (data not shown). Therefore, an extracellular pH of 6.0 was used in all subsequent experiments with oPepT1-transfected cells.
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). The transport constant (Kt) and maximum velocity for Gly-Sar uptake were determined to be 1.0 ± 0.1 mmol/L and 14.3 ± 0.4 nmol/(mg protein · 40 min), respectively. When the data from uptake of Gly-Sar were plotted as an Eadie-Hofstee graph (uptake rate/substrate concentration versus uptake rate), a straight line was observed (r = 0.98; Fig. 4
, inset). This is indicative of the presence of a single transporter system.
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The transport of different peptides in transfected CHO cells was determined by inhibition studies. In oPepT1-transfected CHO cells, inhibition of [3H]-Gly-Sar uptake by seven dipeptides, six tripeptides, three tetrapeptides and two amino acids was examined to calculate the IC50 values of the competitive substrates. Among 18 peptides used in the inhibition assays, the IC50 of six dipeptides and three tripeptides for inhibition of Gly-Sar uptake ranged from 13.6 to 126.7 µmol/L (Table 1
). IC50 was defined as the concentration of competitive peptide that showed 50% inhibition of Gly-Sar uptake. Lower IC50 values indicated higher binding affinity of the competitive peptide to the peptide transporter. Some C-terminal lys-containing peptides, such as Lys-Lys, Lys-Trp-Lys, Lys-Tyr-Lys and Thr-Ser-Lys showed weak inhibition of Gly-Sar uptake (IC50 from 739.8 to 3732.0 µmol/L). No inhibition of Gly-Sar uptake by free amino acids (Met and Lys) was observed (data not shown). Three tetrapeptides were tested and each had large IC50 values in pBK-oPepT1-transfected CHO cells. Because there was no transport of these tetrapeptides in Xenopus oocytes (11
), the large IC50 observed in transfected CHO cells may be due to membrane peptidases that cleave the tetrapeptides to di- and/or tripeptides, which then compete for transport of Gly-Sar. A summary of the overall inhibition pattern of Gly-Sar uptake by different classes of peptides is shown in Figure 4
. Di- and tripeptides are clearly preferred substrates for oPepT1. The peptides examined constitute a variety of substrates differing in their molecular weight, net electrical charge, hydrophobicity and isoelectric point. However, for all peptides, no correlation was found among IC50 and molecular weight, net charge, hydrophobicity and isoelectric point. Therefore, oPepT1 transported peptides independent of these physical characteristics.
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The effects of a PKC inhibitor or activator on oPepT1 transport activity were investigated. Effect of pretreatment of the CHO cells with uptake buffer (MES) only as the control, 0.1 and 1 µmol/L of staurosporine (an inhibitor of PKC), and 1 and 10 µmol/L of PMA (an activator of PKC) for 30 and 60 min is shown in Figure 5
A. A significant increase (35%) of the transport system occurred when transfected CHO cells were incubated with 1 µmol/L of staurosporine for 30 min before uptake measurement. A significant decrease (28%) of the transport system occurred when transfected CHO cells were incubated with 10 µmol/L of PMA for 30 min before uptake measurement. Treatment of the cells with MES buffer only, staurosporine or PMA for 60 min before uptake measurement resulted in lower transport activity in all treatment groups compared with the cells treated only 30 min (P < 0.05). Therefore, prolonged incubation before uptake measurement was harmful to transfected cells.
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B). The increase of transport activity by staurosporine was specific and could be blocked if treatment was done in the presence of PMA. To evaluate whether the increase or decrease of transport activity by staurosporine or PMA was due to changes in de novo protein synthesis, the transfected cells were cotreated with staurosporine or PMA in the presence of cycloheximide, an inhibitor of cellular protein synthesis, before the uptake measurement. The staurosporine- or PMA induced changes of the peptide-transport system were not affected by cotreatment with cycloheximide (Fig. 5
B). Together these data suggest that PKC phosphorylation plays a regulatory role in oPepT1 function. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Xenopus oocytes are a robust system for the expression of many different proteins of animal or plant origin (13
). However, one of the unresolved issues regarding evaluation of peptide transport is whether a difference in substrate affinity and transport might exist for PepT1 expressed in Xenopus oocytes and mammalian cells. The present study was designed to characterize the activity of oPepT1 expressed in a mammalian cell line, CHO cells.
The present observations with oPepT1-transfected CHO cells are in agreement with previous reports that protons are critical for the transport process with an optimal pH of 5.5 and 6.0 (11
,14
). Transport kinetics of Gly-Sar uptake determined in oPepT1-transfected CHO cells were also comparable to those obtained previously (11
). In general, functional expression in CHO cells indicated that oPepT1 has high affinity (low IC50 values) for most of the dipeptides and tripeptides examined. A low affinity (high IC50 values) was observed for dipeptides and tripeptides containing a C-terminal lysine. The nutritional implications of our data are not clear at the moment. Either these peptides with low affinity are less favorable substrates for oPepT1 or the physiological concentrations of these peptides are higher than other peptides so that they can still be transported by oPepT1.
We evaluated the transport characteristics of oPepT1 using peptides containing essential amino acids (mainly methionine and lysine). A comparison of the IC50 data of CHO cells in the present study with the Kt data obtained previously (11
) revealed that the relationship between IC50 and Kt is high (r = 0.81; P < 0.002). Thus, the two in vitro expression systems predict similar transport characteristics for oPepT1. This is also confirmed by other reports using multiple expression systems (3
,15
). The CHO cell is a suitable system to study competition between substrates, whereas the Xenopus oocyte system monitors the cotransport of protons with the peptides. Because of the nature of inhibition studies, false transport results might be observed when the competitor binds to the transporter without actually being transported inside the cell. Likewise, membrane peptidases, if present, could result in the hydrolysis of large peptides followed by uptake of the smaller peptides. For example, in the present study, tetrapeptides showed inhibition of Gly-Sar uptake in oPepT1-expressing CHO cells. In the Xenopus oocyte system, these tetrapeptides did not evoke any detectable inward current (11
). Therefore, we conclude that in the CHO cell system, these tetrapeptides may be degraded by membrane peptidases and absorbed as smaller di- or tripeptides, which then compete for Gly-Sar uptake. Alternatively, these tetrapeptides might just bind to the transporter itself without being actually transported inside, causing a change in the transporter conformation that indirectly affects Gly-Sar uptake.
The translocation of the PepT1 protein from intracellular storage sites to the cell surface and the signaling pathways that link the regulators to PepT1 translocation remain poorly understood. Results from our previous study have predicted the existence of consensus amino acid sequences for PKC phosphorylation of oPepT1 (11
). In the present study, the function of oPepT1 was affected by PKC activation or repression, which was also observed in other studies (16
,17
). However, whether the functional regulation of oPepT1 by PKC is due to direct phosphorylation of oPepT1 protein or is achieved in an indirect manner, such as phosphorylation of a mediator protein, remains unknown. Further study using a PKC null mutant, where all putative PKC phosphorylation sites are eliminated by replacement of specific serine/threonine residues, will more directly address this question.
In summary, this article describes the functional characterization of a peptide transporter oPepT1 in transfected mammalian cells. The transport function of oPepT1 in CHO cells indicated that oPepT1 is capable of transporting all dipeptides and tripeptides examined in a proton dependent manner. Neither tetrapeptides nor free amino acids are proper substrates for oPepT1. The transport of peptide substrates by oPepT1 in transfected mammalian cells is similar to that in microinjected Xenopus oocytes. In vitro treatment of CHO cells with PKC effectors indicated that PKC phosphorylation plays a regulatory role in oPepT1 function. Our studies not only demonstrated that the transport of peptide substrates by oPepT1 is independent of the expression system but also lay the foundation for additional in vivo studies to better understand the mechanisms of protein absorption.
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FOOTNOTES |
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3 Abbreviations used: CHO, Chinese hamster ovary; IC50, 50% inhibition of Gly-Sar uptake; Kt, transport constant; PKC, protein kinase C; PMA, phorbol 12-myristate-13-acetate.
Manuscript received 2 July 2001. Initial review completed 2 August 2001. Revision accepted 27 September 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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15. Han, H. K., Rhie, J. K., Oh, D. M., Saito, G., Hsu, C. P., Stewart, B. H. & Amidon, G. L. (1999) CHO/hPEPT1 cells overexpressing the human peptide transporter (hPEPT1) as an alternative in vitro model for peptidomimetic drugs. J. Pharm. Sci. 88:347-350.[Medline]
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