©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Intestinal H/Peptide Cotransporter
CLONING, FUNCTIONAL EXPRESSION, AND CHROMOSOMAL LOCALIZATION (*)

(Received for publication, July 18, 1994; and in revised form, December 22, 1994)

Rong Liang (1) You-Jun Fei (1) Puttur D. Prasad (1) Sammanda Ramamoorthy (1) Hong Han (2) Teresa L. Yang-Feng (2) Matthias A. Hediger (3) Vadivel Ganapathy (1) Frederick H. Leibach (1)(§)

From the  (1)Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912-2100, the (2)Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, and the (3)Departments of Medicine and Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In mammalian small intestine, a H-coupled peptide transporter is responsible for the absorption of small peptides arising from digestion of dietary proteins. Recently a cDNA clone encoding a H/peptide cotransporter has been isolated from a rabbit intestinal cDNA library (Fei, Y. J., Kanai, Y., Nussberger, S., Ganapathy, V., Leibach, F. H., Romero, M. F., Singh, S. K., Boron, W. F., and Hediger, M. A.(1994) Nature 368, 563-566). Screening of a human intestinal cDNA library with a probe derived from the rabbit H/peptide cotransporter cDNA resulted in the identification of a cDNA which when expressed in HeLa cells or in Xenopus laevis oocytes induced H-dependent peptide transport activity. The predicted protein consists of 708 amino acids with 12 membrane-spanning domains and two putative sites for protein kinase C-dependent phosphorylation. The cDNA-induced transport process accepts dipeptides, tripeptides, and amino beta-lactam antibiotics but not free amino acids as substrates. The human H/peptide cotransporter exhibits a high degree of homology (81% identity and 92% similarity) to the rabbit H/peptide cotransporter. But surprisingly these transporters show only a weak homology to the H-coupled peptide transport proteins present in bacteria and yeast. Chromosomal assignment studies with somatic cell hybrid analysis and in situ hybridization have located the gene encoding the cloned human H/peptide cotransporter to chromosome 13 q33q34.


INTRODUCTION

Mammalian small intestine expresses a transport system that is specific for small peptides consisting of 2-4 amino acids(1) . Free amino acids do not serve as substrates for this system. The physiological role of the intestinal peptide transporter is to absorb small peptides arising from digestion of dietary proteins. The pharmacological relevance of this transporter has become evident in recent years because intestinal absorption of orally active amino beta-lactam antibiotics and other peptide-like drugs is mediated by this transporter(1, 2, 3) . It has been proposed that the transporter has the potential to become an important drug delivery system(3) . Studies on the energetic aspects of the transport system have demonstrated that the driving force for the transporter is an electrochemical H gradient rather than an electrochemical Na gradient(1, 4, 5, 6) . A H-dependent peptide transport system is also expressed in the mammalian kidney(7, 8, 9) , but it is not known whether the renal transporter is identical to or distinct from the intestinal transporter.

Microinjection of rabbit intestinal mRNA into Xenopus laevis oocytes leads to functional expression of a H-dependent peptide transport system with characteristics similar to those of the transporter in the native tissue(10) . Recently we have isolated a cDNA encoding a H-dependent peptide transporter from a rabbit small intestinal cDNA library using this Xenopus oocyte expression system as the screening procedure(11) . We report in this paper the cloning of a human intestinal H/peptide cotransporter. The cloned transporter has been characterized by functionally expressing the transporter in HeLa cells as well as in X. laevis oocytes. Chromosomal localization studies have revealed that the gene for the transporter is present on chromosome 13 q33q34.


MATERIALS AND METHODS

Screening of the cDNA Library

The human intestinal cDNA library (gt10) used here was prepared with mRNA from the ileum(12) . The library was screened by plaque hybridization using VCS 257 cells. The probe was a 0.6-kb (^1)fragment arising from the 5` end of the rabbit intestinal H/peptide cotransporter cDNA(11) . The fragment was released from the full-length cDNA by EcoRI digestion and radiolabeled with [alpha-P]dCTP using the oligolabeling kit from Pharmacia LKB Biotechnol. Hybridization was carried out at 42 °C in a solution containing 50% formamide, 6 times SSC, 5 times Denhardt's solution, 0.1% SDS, and 100 µg/ml of salmon sperm DNA. Washing was done under medium stringency conditions, two times in 3 times SSC, 0.5% SDS at 55 °C for 30 min and one time in 1 times SSC, 0.1% SDS at 60 °C for 30 min. Positive clones were identified and plaque purified by secondary and tertiary screening. On the tertiary autoradiographs, 100% of the plaques showed positive hybridization signal, confirming the purity of the clones.

Isolation of the cDNA Insert

Phage DNA was prepared using the Wizard Lambda Prep DNA Purification System (Promega). Digestion of the DNA with EcoRI yielded three DNA fragments, 1.2, 0.6 and 0.4 kb in size, in addition to the two fragments (11 and 40 kb in size) arising from the vector. All three fragments of the cDNA insert hybridized to the full-length (2.7 kb) rabbit intestinal H/peptide cotransporter cDNA probe. The digestion pattern indicated that there are two internal sites for EcoRI in the insert and that the size of the full-length insert is approximately 2.2 kb. In order to obtain the full-length cDNA for functional expression, partial digestion with EcoRI was used. The phage DNA was digested with increasing concentrations of EcoRI, and the digestion fragments were analyzed by Southern hybridization. Partial digestion yielded three new fragments (2.2, 1.8, and 1.6 kb) in addition to the above described three and all six fragments hybridized to the full-length rabbit intestinal H/peptide cotransporter cDNA probe. The concentration of EcoRI which generated the maximal amount of the 2.2-kb fragment was chosen for digestion in a large scale. The full-length cDNA was isolated, gene cleaned, and subcloned.

Subcloning and DNA Sequencing

The 2.2-kb full-length cDNA fragment was subcloned into pBluescript SKII. The clones were selected in which the insert was oriented in such a way that its transcription was under the control of the T7 promotor in the vector. The three fragments arising from the complete digestion were also subcloned into pBluescript SKII. These three clones as well as the full-length clone were used for nucleotide sequencing, and the full-length clone was used for functional expression.

Sequencing was done by the dideoxy chain termination method(13) . Synthetic oligonucleotide primers were used whenever necessary to complete the sequencing of both the sense and the antisense strands. Sequence analysis was performed by the software package GCG version 7.B (Genetics Computer Groups, Inc. Madison, WI). Multiple sequence alignment was done using the Genbank Program PILEUP.

Functional Expression in HeLa Cells

This was done using the vaccinia virus expression system (14) as described previously(15, 16) . Subconfluent HeLa cells were first infected with a recombinant (VTF) vaccinia virus encoding T7 RNA polymerase and then transfected with the plasmid carrying the full-length cDNA. The strategy behind this approach is that the virus-encoded T7 RNA polymerase catalyzes the transcription of the cDNA under the control of the T7 promotor allowing transient expression of the cDNA-encoded protein in the HeLa cell plasma membrane. After 8-10 h post-transfection, transport measurements were made at room temperature with [2-^14C]glycyl-[1-^14C]sarcosine (specific radioactivity 109 mCi/mmol; Cambridge Research Biochemicals, United Kingdom). The uptake medium was 25 mM Mes/Tris (pH 6.0) or 25 mM Hepes/Tris (pH 7.5), containing 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl(2), 0.8 mM MgSO(4), and 5 mM glucose. In most experiments, the time of incubation for transport measurements was 2 min. Nonspecific transport was determined in parallel experiments with the plasmid vector. This transport was negligible and represented only 3-5% of the transport measured in cells which were transfected with the vector carrying the cDNA insert. Moreover, this value was not due to carrier-mediated transport as concluded from the lack of inhibition by excess unlabeled glycylsarcosine. Thus there was no detectable endogenous glycylsarcosine transport activity in HeLa cells. Therefore, the transport values measured in cells transfected with the vector-cDNA construct were analyzed directly without correcting for the values obtained in cells which were transfected with the vector alone.

Functional Expression in X. laevis Oocytes

This was done as described previously (11) by microinjecting cRNA (25 ng) derived from the clone. The plasmid DNA was linearized with NotI and transcribed using T7 RNA polymerase. Capping was done with the cap analog m^7G(5`)PPP(5`)G (Pharmacia). Water-injected oocytes served as controls. Transport measurements were made with individual oocytes 4 days after injection. The concentration of [2-^14C]glycyl-[1-^14C]sarcosine was 30 µM, and the uptake medium was 3 mM Hepes/Mes/Tris (pH 5.5) or 3 mM Hepes/Tris (pH 7.5), containing 100 mM NaCl, 2 mM KCl, 1 mM MgCl(2), and 1 mM CaCl(2). The incubation time for transport measurements with oocytes was 1 h.

Northern Analysis

Tissue distribution of mRNA transcripts coding for the H/peptide cotransporter was determined by Northern blot. Poly(A) mRNA was isolated from human ileum and two cell lines of human intestinal origin (Caco-2 and HT-29) using the FastTrack mRNA isolation kit (Invitrogen). Poly(A) mRNA samples (2.5-4 µg) from these sources were denatured and size-fractionated on an agarose gel. The fractionated RNA was transferred onto a nylon membrane (Hybond N, Amersham Corp.) and probed with the full-length human intestinal H/peptide cotransporter (hPEPT 1) cDNA under high stringency conditions (hybridization: overnight incubation at 42 °C in 50% formamide and 10% dextran sulfate; washing: once with 3 times SSC-0.5% SDS at room temperature for 30 min, twice with 3 times SSC, 0.5% SDS at 55 °C for 30 min and once with 0.1 times SSC, 0.1% SDS at 55 °C for 30 min). Following stripping of the blot, similar hybridization was conducted with human beta-actin cDNA (Clontech) to assess RNA loading and transfer efficiency.

Northern analysis with hPEPT 1 cDNA was also done using a commercially available membrane blot containing size-fractionated poly(A) mRNA from several tissues of human origin (Clontech). This blot was also reprobed with human beta-actin cDNA for assessment of RNA loading and transfer efficiency.

Chromosomal Localization

The 2.2-kb full-length hPEPT 1 cDNA was used for chromosomal localization of the gene coding for the human intestinal H/peptide cotransporter. This was done by somatic cell hybrid analysis and by in situ hybridization to human metaphase chromosomes. A mapping panel (panel no. 1) consisting of 17 mouse-human and one Chinese hamster-human hybrids was obtained from the National Institute of General Medical Sciences' Human Genetic Mutant Cell Repository and used in somatic cell hybrid analysis. DNA samples obtained from these cells were digested with EcoRI, separated by electrophoresis on agarose gels, transferred onto a nylon membrane, and hybridized to [P]hPEPT 1 cDNA as described previously(17) . In situ hybridization of [^3H]hPEPT 1 cDNA to human chromosomes and emulsion autoradiography were carried out by the method of Harper and Saunders (18) . Chromosomes were G-banded using Wright's stain, and G-banded chromosomes were analyzed for silver grain localization. The hPEPT 1 cDNA was labeled by nick translation either in the presence of [alpha-P]dCTP (Southern blot hybridization) or in the presence of [^3H]dCTP and [^3H]dTTP (in situ hybridization) and used in these experiments.


RESULTS AND DISCUSSION

Isolation of the Full-length H/Peptide Cotransporter cDNA

Screening of approximately 3 times 10^5 plaques from a human intestinal cDNA library with a 5` end fragment (0.6 kb) of the rabbit intestinal H/peptide cotransporter cDNA as a probe yielded four positive clones. All of these clones were related as judged from EcoRI digestion pattern. One of them was characterized in detail in terms of its structure and function. Digestion of the phage DNA with EcoRI gave three fragments indicating the presence of two internal EcoRI sites in the insert. The full-length cDNA was prepared from the phage DNA by partial digestion with EcoRI. A pilot experiment was run to determine the optimal concentration of the enzyme which would give the maximal amount of the full-length cDNA insert. After digesting the phage DNA with increasing concentrations of EcoRI, the digestion products were size-fractionated and then probed with the full-length rabbit intestinal H/peptide cotransporter cDNA by Southern blot (Fig. 1). Complete digestion with the maximal amount of the enzyme used generated three hybridizing signals (1.2, 0.6, and 0.4 kb in size). With lesser concentrations of the enzyme, the amounts of these three fragments decreased while larger size fragments appeared. The size of the largest fragment was 2.2 kb. Two other partial digestion products (1.8 and 1.6 kb) were also generated. This digestion pattern indicated that the 2.2-kb full-length insert consisted of the 1.2-kb fragment flanked on one side by the 0.6-kb fragment and on the other side by the 0.4-kb fragment. The concentration of EcoRI giving the maximal amount of the 2.2-kb fragment was chosen for a large scale preparation. The full-length fragment was then isolated and subcloned into pBluescript SK II.


Figure 1: EcoRI digestion and Southern blot analysis of the phage DNA containing the hPEPT 1 cDNA insert. The phage DNA was digested with increasing concentrations of EcoRI, and the digestion products were electrophoresed and probed with P-labeled full-length rabbit intestinal H/peptide cotransporter cDNA. The arrow at the top indicates the lane containing the maximal amount of the full-length (2.2 kb in size) hPEPT 1 cDNA. The concentration of EcoRI corresponding to this lane was chosen for a large scale preparation of the full-length hPEPT 1 cDNA.



The cDNA is 2,263 bp long with an open reading frame of 2,127 bp (including termination codon) encoding a protein of 708 amino acids (Fig. 2). The open reading frame is flanked by a 56-bp long sequence on the 5` end and by a 80-bp long sequence on the 3` end. The predicted initiation codon is preceded by a Kozak consensus sequence (GCC GCC)(19) . The encoded protein is predicted to have a core molecular size of 78,810 Da and an isoelectric point of 8.6. Hydropathy analysis of the primary amino acid sequence of the predicted protein shows the presence of 12 putative transmembrane domains with a long (200 amino acids) hydrophilic segment between the transmembrane domains 9 and 10. This hydrophilic segment contains seven putative N-linked glycosylation sites. When modeled to accommodate all the transmembrane domains and to allow the long hydrophilic loop on the extracellular side, the model places both the amino terminus and the carboxyl terminus on the cytoplasmic side. There are two potential sites for protein kinase C-dependent phosphorylation (Ser-357 and Ser-704) but no site for protein kinase A-dependent phosphorylation. Comparison of the amino acid sequence between this clone and the rabbit intestinal H/peptide cotransporter reveals a high degree of homology (81% identity and 92% similarity). Most of the conserved sequences occur within the putative transmembrane domains. The human homolog is 1 amino acid residue longer than the rabbit counterpart. An important difference between these two proteins is that the rabbit intestinal H/peptide cotransporter possesses a site for protein kinase A-dependent phosphorylation (Thr-362) whereas the human homolog does not. The threonine residue present at this site in the rabbit transporter is replaced by alanine in the human homolog.


Figure 2: hPEPT 1 cDNA and predicted primary amino acid sequence.



Functional Expression

To assess the transport function of the cloned human cDNA, it was expressed in HeLa cells and in X. laevis oocytes, and its function was monitored by the transport of the dipeptide glycylsarcosine. The construct used in these expression studies contains the H/peptide cotransporter cDNA (hPEPT 1) insert cloned into pBluescript SK II in such a way that its transcription is under the control of the T7 promotor. When this plasmid was transfected into HeLa cells expressing a recombinant vaccinia virus T7 RNA polymerase, the cells were able to transport glycylsarcosine in a H-dependent manner (Fig. 3). The H dependence was evident from the 4- to 5-fold greater transport when measured at an extracellular pH of 6.0 instead of 7.5. Control cells transfected with the empty vector showed negligible transport when measured at pH 6.0. An incubation period of 2 min and an extracellular pH of 6.0 were used in subsequent experiments with hPEPT 1-transfected cells to approximate the initial transport rate.


Figure 3: Transport of glycylsarcosine in HeLa cells transfected with pBluescript SK II alone or with hPEPT 1 cDNA. Cells were transfected with either vector alone &cjs1260; or with vector carrying the hPEPT 1 cDNA (circle, bullet). Transport of [^14C]glycylsarcosine (20 µM) was measured for different time periods either at pH 6.0 (bullet, &cjs1260;) or at pH 7.5 (circle). Values represent means ± S.E. for three determinations.



Table 1describes the results of competition experiments performed to determine the substrate specificity of the hPEPT 1. In these experiments, the ability of unlabeled amino acids, dipeptides, tripeptides, and amino beta-lactam antibiotics to compete with [^14C]glycylsarcosine (20 µM) for the transport process induced by hPEPT 1 was studied. At a concentration of 10 mM, free amino acids had no effect on the transport. On the other hand, dipeptides, tripeptides, as well as the three amino beta-lactam antibiotics (cyclacillin, cefadroxil, and cephalexin) were found to be potent inhibitors of the transport. The hPEPT 1-induced transport system thus appears to be specific for small peptides and peptide-like compounds with no affinity toward free amino acids.



The induced transport process is saturable as evident from transport measurements done at varying concentrations of glycylsarcosine in the range of 0.05-5 mM (Fig. 4). Under these conditions, the transport of radiolabel from 20 µM [^14C]glycylsarcosine in hPEPT 1-transfected cells was reduced in the presence of 25 mM unlabeled glycylsarcosine to a level observed in vector-transfected cells in the absence of unlabeled glycylsarcosine, indicting that the transport induced by hPEPT 1 was almost completely carrier-mediated. The experimental data were found to fit best for a model describing the uptake as the result of a single carrier plus a diffusional component. The diffusional coefficient was 0.35 ± 0.06. The Eadie-Hofstee transformation of the data for the carrier-mediated uptake yielded a linear plot (r = -0.98) (Fig. 4). The kinetic parameters for the carrier-mediated uptake, K(t) (Michaelis-Menten constant) and V(max) (maximal velocity), were 0.29 ± 0.04 mM and 4.7 ± 0.3 nmol/2 min/10^6 cells. The diffusional component represented 2 and 28% of total uptake measured at 0.05 and 5 mM glycylsarcosine, respectively.


Figure 4: Kinetics of glycylsarcosine transport induced by the hPEPT 1 cDNA in HeLa cells. Transport of glycylsarcosine in hPEPT 1 cDNA-transfected HeLa cells was measured over a concentration range of 0.05-5 mM. The pH of the medium was 6.0, and the incubation time was 2 min. Values represent means ± S.E. for four determinations. Inset, Eadie-Hofstee transformation of the data for carrier-mediated uptake.



The transport function of hPEPT 1 was also assessed by expressing the protein in X. laevis oocytes (Fig. 5). The oocytes injected with cRNA derived from the hPEPT 1 cDNA showed 8-fold greater activity for glycylsarcosine transport than water-injected oocytes. The expressed transporter was H dependent because the dipeptide transport was significantly higher when measured at pH 5.5 than at pH 7.5. In addition, unlabeled glycylsarcosine was able to compete with [^14C]glycylsarcosine for the transport process induced by hPEPT 1.


Figure 5: Transport of glycylsarcosine in X. laevis oocytes injected either with hPEPT 1 cRNA or with water. Oocytes were microinjected with 25 ng of hPEPT 1 cRNA and transport of [^14C]glycylsarcosine (30 µM) was measured with individual oocytes on day 4 post-injection. Water-injected oocytes served as controls. Transport in water-injected oocytes was measured at pH 5.5 (A) whereas transport in cRNA-injected oocytes was measured at either pH 5.5 (B and D) or pH 7.5 (C). When present (D), concentration of unlabeled glycylsarcosine was 10 mM. Values represent means ± S.E. for 12-16 oocytes from two separate experiments.



The magnitude of stimulation of glycylsarcosine transport induced in X. laevis oocytes by hPEPT 1 is five to six times smaller than the corresponding value observed with the rabbit H/peptide cotransporter cDNA, although the amino acid sequences in the coding region of these two clones exhibit 81% identity and 92% similarity. This difference is most likely due to the fact that the rabbit clone was complete with its poly(A) tail whereas the human clone has a truncated 3`-noncoding region comprising of only 80 bp with no poly(A) sequence. The poly(A) tail is known to stabilize mRNA. Therefore, it is likely that the cRNA derived from the rabbit clone is more stable in X. laevis oocytes than the cRNA derived from the human clone.

Tissue Distribution and Heterogeneity of hPEPT 1 mRNA

Distribution of PEPT 1 mRNA in human tissues was studied using a commercially available membrane blot containing size-fractionated poly(A) mRNA from eight tissues: heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas (Fig. 6). Among these tissues, a 3.3-kb hybridizing signal was present in placenta, liver, kidney, and pancreas. The transcript was absent in other tissues. The functional characteristics of the H/peptide cotransporter have been investigated in detail only in intestine and kidney. Human placenta possesses peptide transport activity (20) but whether this activity is mediated by the H-coupled peptide transporter is not known. Therefore, it is interesting that the placenta expresses the PEPT 1 mRNA though at very low levels. The presence of 3.3-kb mRNA transcript in kidney is not unexpected, but the observation that the levels of the transcript in this tissue are manyfold lower than the levels in intestine is surprising. Brush border membrane vesicles prepared from kidney exhibit robust H/peptide cotransport activity(21, 22, 23) . It is therefore possible that a major portion of the H/peptide cotransport activity expressed in kidney is not due to PEPT 1. The existence of multiple forms of H/peptide cotransporter in mammalian tissues thus seems very likely. The levels of the 3.3-kb mRNA transcript in liver and pancreas are significantly higher than in kidney. Interestingly, it has been reported that liver does not have the ability to take up peptides from the circulation(24) . Liver contains heterogeneous population of cells, and therefore the possibility that the peptide transport activity is expressed in cells other than hepatocytes cannot be excluded. Whether pancreas expresses H/peptide cotransport activity is not known.


Figure 6: Northern blot analysis of PEPT 1 mRNA transcripts in human tissues. A commercially available hybridization-ready blot containing poly(A) mRNA from different human tissues (Multiple Tissue Northern blot; Clontech) was used to hybridize with the hPEPT 1 cDNA probe. In addition, poly(A) mRNA samples isolated from human ileum and the human colon carcinoma cell line Caco-2 were also analyzed in a similar manner. Each lane contained poly(A) mRNA and lanes 1-10 represent heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas (2 µg each), ileum (2.5 µg), and Caco-2 cells (4 µg), respectively. The same blot was stripped and reprobed with the beta-actin cDNA. The sizes of hybridizing bands were determined using RNA standards run in parallel in an adjacent lane.



Northern analysis of poly(A) mRNA isolated from human ileum revealed the presence of a major RNA species, 3.3 kb in size, which hybridized to the hPEPT 1 cDNA (Fig. 6). There are several minor hybridizing RNA species in the human intestine. We also analyzed poly(A) mRNA from two cell lines of human intestinal origin for the presence of PEPT 1 mRNA. Caco-2 cells which are known to possess H/peptide cotransporter activity (25) contains the primary 3.3-kb transcript as well as the other minor transcripts. Thus, the distribution of the different PEPT 1 mRNA transcripts in this cell line is similar to that in the human intestine. In contrast, HT-29 cells which are also commonly used as a model for intestinal transport studies do not contain any detectable hybridizing signal (data not shown). There was a report by Dantzig and Bergin (26) describing expression of peptide transport activity in HT-29 cells. We investigated the transport of glycylsarcosine in HT-29 cells in our laboratory and found no evidence for the expression of the H/peptide cotransporter in these cells. (^1)It is not known at this time whether this discrepancy is due to the possibility that different clones of the HT-29 cell line might have been used in these two studies or due to the possibility that the transport activity measured in the study by Dantzig and Bergin (26) is catalyzed by a carrier other than the H/peptide cotransporter.

Chromosomal Localization

We have mapped the chromosomal location of the gene encoding the cloned intestinal H/peptide cotransporter. Southern blot hybridization with the hPEPT 1 cDNA probe detected six human-specific fragments of 24, 17, 5.4, 3.8 and 2.6 kb, two mouse-specific fragments of 20.5 and 5.6 kb, and three Chinese hamster-specific fragments of 26, 23, and 17 kb. All human-specific fragments showed concordant segregation with chromosome 13 (Table 2). Regional localization of the hPEPT 1 gene was carried out by in situ hybridization with ^3H-labeled hPEPT 1 cDNA. Of 133 grains over 50 metaphases analyzed, 25 (18.8%) were located at chromosome 13 q33q24 (Fig. 7). No other chromosomal site was labeled above background.




Figure 7: Chromosomal localization of the hPEPT 1 gene. Position and relative abundance of silver grains on chromosome 13 as determined by in situ hybridization using ^3H-labeled hPEPT 1 cDNA as a probe are indicated.



Homology of hPEPT 1 to Other Cloned Transport Proteins

The hPEPT 1 is highly homologous to the rabbit intestinal H/peptide cotransporter(11) . Interestingly, the protein sequences of these transporters do not show strong homology with other known classes of transport proteins. However, they do show weak but definite homology with certain transport proteins from nonmammalian sources (Fig. 8). These proteins are the peptide transporter from Saccharomyces cerevisiae (Yeast Ptr 2)(27) , a protein encoded by S. cerevisiae chromosome X1 DNA (Yeast X1 DNA), the chlorate/nitrate transporter from Arabidopsis thaliana (CHL 1) (28) and the di- and tripeptide transporter from Lactococcus Lactis (L. Lact. Ptr)(29) .


Figure 8: Comparison of primary amino acid sequences among the cloned human H/peptide cotransproter (hPEPT 1), a peptide transporter from S. cerevisiae (yeast Ptr 2), the chromosome XI DNA from S. cerevisiae (yeast XI DNA), a peptide transporter from L. lactis (L. lact. Ptr), and a chlorate/nitrate transporter from A. thaliana (CHL 1).



The yeast Ptr 2 and the L. lactis Ptr catalyze transport of small peptides via a mechanism energized by an electrochemical H gradient(27, 29) . It is interesting to note that even though there is a high degree of similarity in the nature of the driving force and the transported substrates among the yeast Ptr 2, L. lactis Ptr, and hPEPT 1, the homology of the primary amino acid sequence of hPEPT 1 to the other two proteins is not very high. Transport of nitrate in A. thaliana catalyzed by CHL 1 occurs via a H-dependent mechanism(28, 30) . The significant, even though weak, homology in the amino acid sequence among these transport systems is indicative of the similarity in the nature of the substrate and/or driving force involved in transport processes mediated by these systems. There are other known transport systems which either utilize an electrochemical H gradient as the energy source or mediate the transport of peptides, but there is no significant homology between these proteins and the hPEPT 1. Examples of these transport systems include the prokaryotic H-coupled lactose permease (31) and the peptide transport systems described in several Gram-positive and Gram-negative bacteria (32, 33, 34, 35, 36) . With the exception of L. lactis Ptr, the peptide transport systems described thus far in bacteria belong to the super family of ABC transporters or traffic ATPases. This class of transport systems directly utilizes the energy derived from ATP hydrolysis to energize active transport of solutes. Another transport system which surprisingly shows no structural similarity to hPEPT 1 is the transporter associated with antigen processing(37) . This transporter is a heterodimer consisting of two proteins TAP 1 and TAP 2 and is involved in the transport of peptide antigens, normally consisting of about 9 amino acids, across the membrane of endoplasmic reticulum to be subsequently presented to the major histocompatibility complex class I molecules. This system, sometimes referred to as a peptide transporter, is not found in the plasma membrane and is not coupled to an electrochemical H gradient. It is a member of the ABC transporter family.

Recently, Dantzig et al.(38) reported on the isolation of a cDNA encoding a protein associated with intestinal peptide transport. There is no significant structural similarity between hPEPT 1 and this protein designated as hpt-1 (16% identity; 41% similarity). This protein contains a single putative transmembrane domain in contrast to hPEPT 1 which contains 12 putative transmembrane domains. Tissue distribution of hpt-1 and hPEPT 1 is also different. hpt-1 is expressed in the gastrointestinal tract and pancreas but is absent in kidney and liver whereas hPEPT 1 is expressed in all of these tissues. Similarly, hpt-1 is present in Caco-2 as well as HT-29 cells whereas hPEPT 1 is present in Caco-2 cells but not in HT-29 cells. Transfection of hpt-1 cDNA into mammalian cells leads to induction of H-dependent transport of aminocephalosporins. The induced transport is inhibitable by glycyl-D-proline. Whether there is a functional relationship between hpt-1 and hPEPT 1 remains to be determined.

The observation that hPEPT 1 contains two putative sites for protein kinase C-mediated phosphorylation may be relevant to our recent studies (25) which showed that the activity of the H/peptide cotransporter expressed in the human intestinal cell line Caco-2 is regulated by protein kinase C. Site-directed mutagenesis studies are required to determine the role of these two sites in this regulation. The findings that hPEPT 1 does not possess any site for potential phosphorylation by protein kinase A are interesting and may have physiological relevance. cAMP is an important modulator of intestinal function and its role in clinical disorders caused by pathogens such as vibrio cholerae and enterotoxigenic strains of Escherichia coli is well known. The importance, if any, of the absence of potential sites in hPEPT 1 for protein kinase A-mediated phosphorylation in physiological and pathological conditions remains to be determined.

In summary, we have isolated a cDNA (hPEPT 1) from a human intestinal cDNA library which when expressed in X. laevis oocytes and in HeLa cells induces H gradient-dependent peptide transport activity. The functional characteristics of the induced activity are similar to those of the peptide transport activity described in mammalian small intestine. The deduced primary amino acid sequence of hPEPT 1 is highly homologous to the rabbit intestinal peptide transporter but is only distantly related to the peptide transporters present in yeast and L. lactis. It has no significant homology to hpt-1, a recently reported protein apparently associated with intestinal peptide transport. The gene which codes for hPEPT 1 has been localized to human chromosome 13 q33q34.


FOOTNOTES

*
This work was supported by the National Institutes of Health Grant DK 28389. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U13173[GenBank].

§
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100. Tel.: 706-721-7661; Fax: 706-721-6608.

(^1)
R. Liang, Y.-J. Fei, P. D. Prasad, S. Ramamoorthy, H. Han, T. L. Yang-Feng, M. A. Hediger, V. Ganapathy, and F. H. Leibach, unpublished data.


ACKNOWLEDGEMENTS

We thank J. C. Liu for nucleotide sequencing and Joyce Hobson for secretarial assistance.


REFERENCES

  1. Ganapathy, V., Brandsch, M., and Leibach, F. H. (1994) in Physiology of the Gastrointestinal Tract (Johnson, L. R., ed) pp. 1773-1794, Raven Press, New York
  2. Tsuji, A. (1987) Adv. Biosci. 65, 125-131
  3. Amidon, G. L., and Lee, H. J. (1994) Annu. Rev. Pharmacol. Toxicol. 34, 321-341 [CrossRef][Medline] [Order article via Infotrieve]
  4. Ganapathy, V., and Leibach, F. H. (1985) Am. J. Physiol. 249, G153-G160
  5. Ganapathy, V., Miyamoto, Y., and Leibach, F. H. (1987) Contr. Infusion Ther. Clin. Nutr. 17, 54-68
  6. Ganapathy, V., and Leibach, F. H. (1991) Curr. Opin. Cell Biol. 3, 695-701 [CrossRef][Medline] [Order article via Infotrieve]
  7. Ganapathy, V., and Leibach, F. H. (1982) Life Sci. 30, 2137-2146 [CrossRef][Medline] [Order article via Infotrieve]
  8. Ganapathy, V., and Leibach, F. H. (1987) Am. J. Physiol. 251, F945-F953
  9. Ganapathy, V., Miyamoto, Y., and Leibach, F. H. (1987) Adv. Biosci. 65, 91-98
  10. Miyamoto, Y., Thompson, Y. G., Howard, E. F., Ganapathy, V., and Leibach, F. H. (1991) J. Biol. Chem. 266, 4742-4745 [Abstract/Free Full Text]
  11. Fei, Y. J., Kanai, Y., Nussberger, S., Ganapathy, V., Leibach,, F. H., Romero, M. F., Singh, S. K., Boron, W. F., and Hediger, M. A. (1994) Nature 368, 563-566 [CrossRef][Medline] [Order article via Infotrieve]
  12. Hediger, M. A., Turk, E., and Wright, E. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5748-5752 [Abstract]
  13. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5468 [Abstract]
  14. Blakely, R. D., Clark, J. A., Rudnick, G., and Amara, S. G. (1991) Anal. Biochem. 194, 302-308 [Medline] [Order article via Infotrieve]
  15. Ramamoorthy, S., Bauman, A. L., Moore, K. R., Han, H., Yang-Feng, T. L., Chang, A. S., Ganapathy, V., and Blakely, R. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2542-2546 [Abstract]
  16. Ramamoorthy, S., Leibach, F. H., Mahesh, V. B., Han, H., Yang-Feng. T. L., Blakely, R. D., and Ganapathy, V. (1994) Biochem. J. 300, 893-900 [Medline] [Order article via Infotrieve]
  17. Yang-Feng, T. L., Floyd-Smith, G., Nemer, M., Drouin, J., and Francke, U. (1985) Am. J. Hum. Genet. 37, 1117-1128 [Medline] [Order article via Infotrieve]
  18. Harper, M. E., and Saunders, G. F. (1981) Chromosoma 83, 431-439 [Medline] [Order article via Infotrieve]
  19. Kozak, M. (1986) Cell 44, 283-292 [Medline] [Order article via Infotrieve]
  20. Ganapathy, M. E., Mahesh, V. B., Devoe, L. D., Leibach, F. H., and Ganapathy, V. (1985) Am. J. Obstet. Gynecol. 153, 83-86 [Medline] [Order article via Infotrieve]
  21. Takuwa, N., Shimada, T., Matsumoto, H., and Hoshi, T. (1985) Biochim. Biophys. Acta 814, 186-190 [Medline] [Order article via Infotrieve]
  22. Tiruppathi, C., Kulanthaivel, P., Ganapathy, V., and Leibach, F. H. (1990) Biochem. J. 268, 27-33 [Medline] [Order article via Infotrieve]
  23. Tiruppathi, C., Ganapathy, V., and Leibach, F. H. (1990) J. Biol. Chem. 265, 14870-14874 [Abstract/Free Full Text]
  24. Lombardo, Y. B., Morse, E. L., and Adibi, S. A. (1988) J. Biol. Chem. 263, 12920-12926 [Abstract/Free Full Text]
  25. Brandsch, M., Miyamoto, Y., Ganapathy, V., and Leibach, F. H. (1994) Biochem. J. 299, 253-260 [Medline] [Order article via Infotrieve]
  26. Dantzig, A. H., and Bergin, L. (1988) Biochem. Biophys. Res. Commun. 155, 1082-1087 [Medline] [Order article via Infotrieve]
  27. Perry, J. R., Basrai, M. A., Steiner, H. Y., Naider, F., and Becker, J. M. (1994) Mol. Cell. Biol. 14, 104-115 [Abstract]
  28. Tsay, Y. F., Schroeder, J. I., Feldmann, K. A., and Crawford, N. M. (1993) Cell 72, 705-713 [Medline] [Order article via Infotrieve]
  29. Hagting, A., Kunji, E. R. S., Leenhouts, K. J., Poolman, B., and Konings, W. N. (1994) J. Biol. Chem. 269, 11391-11399 [Abstract/Free Full Text]
  30. McClure, P. R., Kochian, L. V., Spanswick, R. M., and Shaff, J. E. (1990) Plant Physiol. 93, 281-289
  31. Kaback, H. R. (1992) Int. Rev. Cytol. 137A, 97-125 [Medline] [Order article via Infotrieve]
  32. Higgins, C. F., and Gibson, M. M. (1986) Methods Enzymol. 125, 365-377 [Medline] [Order article via Infotrieve]
  33. Alloing, G., Trombe, M. C., and Claverys, J. P. (1990) Mol. Microbiol. 4, 633-644 [Medline] [Order article via Infotrieve]
  34. Kunji, E. R. S., Smid, E. J., Plapp, R., Poolman, B., and Konings, W. N. (1993) J. Bacteriol. 175, 2052-2059 [Abstract]
  35. Perego, M., Higgins, C. F., Pearce, S. R., Gallagher, M. P., and Hoch, J. A. (1991) Mol. Microbiol. 5, 173-185 [Medline] [Order article via Infotrieve]
  36. Rudner, D. J., LeDeaux, J. R., Ireton, K., and Grossman, A. D., (1991) J. Bacteriol. 173, 1388-1398 [Medline] [Order article via Infotrieve]
  37. Monaco, J. J. (1992) Immunol. Today 13, 173-179 [CrossRef][Medline] [Order article via Infotrieve]
  38. Dantzig, A. H., Hoskins, J., Tabas, L. B., Bright, S., Shepard, R. L., Jenkins, I. L., Duckworth, D. C., Sportsman, J. R., Mackensen, D., Rosteck, P. R., Jr., and Skatrud, P. L. (1994) Science 264, 430-433 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.