Urtti A, Johns SJ and Sadee W Genomic Structure of Proton-Coupled Oligopeptide Transporter hPEPT1 and pH-Sensing Regulatory Splice Variant. AAPS PharmSci 2001; 3 (1) article 6 (https://www.pharmsci.org/scientificjournals/pharmsci/journal/01_06.html).

Genomic Structure of Proton-Coupled Oligopeptide Transporter hPEPT1 and pH-Sensing Regulatory Splice Variant.

Submitted: October 23, 2000; Accepted: January 19, 2001; Published: February 6, 2001

Arto Urtti^1,2, Susan Jean Johns³ and Wolfgang Sadee¹

¹Department of Biopharmaceutical Sciences, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143-0446, USA;

²Department of Pharmaceutics, University of Kuopio, POB 1627, 70211 Kuopio, Finland

³Sequence Analysis & Consulting Service, Computer Graphics Laboratory, Department of Pharmaceutical Chemistry, University of California San Francisco, 513 Parnassus Ave, San Francisco, CA 94143-0446, USA;

Abstract

Proton-coupled oligopeptide transporter PEPT1 facilitates the transport of dipeptides and peptoid drugs (including antibiotics) across the cell membranes of endothelial and epithelial cells. Substrate transport by the proton symport is driven by pH gradients, while the profile of pH sensitivity is regulated by a closely related protein, hPEPT1-RF. We investigated the genomic structure of hPEPT1 and hPEPT1-RF . Analysis of the high-throughput genomic sequence (HTGS) database revealed that hPEPT1 and hPEPT1-RF are splice variants encoded by the same gene located in chromosome 13, consisting of 24 exons. hPEPT1 is encoded by 23 exons and hPEPT1-RF by 6 exons. Coding sequences of hPEPT1-RF share 3 exons completely and 2 exons partially with hPEPT1. The genomic organization of hPEPT1 shows high similarity with its mouse orthologue. Exon-intron boundaries occur mostly in the loops connecting transmembrane segments (TMSs), suggesting a modular gene structure reflecting the TMS-loop repeat units in hPEPT1. The putative promoter region of hPEPT1 contains TATA boxes and GC-rich regions and a potential insulin responsive element.

Introduction

Proton-coupled oligopeptide transporters (POTs) comprise the transport family 2.A.17 (for transporter classification see https://www.biology.ucsd.edu/~msaier/transport/titlepage.html ). Oligopeptide transporters are symporters driven by the flux of protons; they have a molecular architecture consisting of ~12 predicted TMSs¹ . Members of the POT family include peptide transporter 1 (PEPT1)^2,3 , peptide transporter 2 (PEPT2) (4), peptide/histidine transporter 1 (PHT-1)^5,6 , and peptide/histidine transporter 2 (PHT-2)⁶ .  Recently, a cDNA termed PET3 (NM_016582), which is largely identical to PHT-2, has been deposited into the nr database (https://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi ).

The peptide transporter 1 gene of rabbits was cloned in 1994 (2), and the human orthologue (hPEPT1 ) was cloned shortly after³ .  Human PEPT1 cDNA contains 3105 base pairs (bp), and the predicted protein consists of 708 amino acids. The transporter protein has 12 predicted TMSs and 2 putative protein kinase C phosphorylation sites.  The membrane topology of the human dipeptide transporter, hPEPT1, was determined by epitope insertions by Covitz et al⁷ . PEPT1 is expressed in the intestine (brush border), early proximal kidney tubuli, liver, placenta, and pancreas^3,8 . In the intestines, PEPT1 facilitates absorption of digested dipeptides so that most of the dietary nitrogen is absorbed as dipeptides rather than as amino acids⁹ .

Human PEPT1 has broad substrate specificity. The substrates include di- and tripeptides and peptoid drugs. Thus, PEPT1 mediates the high bioavailability of many hydrophilic beta-lactam antibiotics¹⁰ . In addition, PEPT1 is suggested to play a role in intracellular peptide transport, including lysosomal transport¹¹ .

Saito et al¹² have described a highly related transcript, termed hPEPT1-RF, which modulates the activity of human PEPT1. The cDNA for the regulatory factor encodes an open reading frame of 208 amino acids. Residues 18-195 are identical to residues 8-185 in hPEPT1, while sequences 1-17 and 196-208 are unique. Both hPEPT1 and hPEPT1-RF are expressed in Caco-2 cells. Expression studies in Xenopus oocytes and Caco-2 cells showed that the regulatory factor shifted the pH-sensitivity profile of hPEPT1-mediated peptide transport¹² .

Although somatic cell hybrid analysis and in situ hybridization studies of Liang et al³ positioned hPEPT1 to chromosome 13 q33-q34, the genomic structures of human PEPT1 and hPEPT1-RF were not known. Genomic organization of the mouse PEPT1 gene has been reported recently¹³ as having a length of 38 kb with 23 exons. 

The aim of our study was to determine the genomic structure of hPEPT1 and hPEPT1-RF . We identified a common gene with 24 exons encoding both hPEPT1 and the regulatory factor in clones representing chromosome 13. hPEPT1 and hPEPT1-RF are splice variants of the same gene.

Materials and Methods

Advanced (BLAST) analysis was carried out using the National Center for Biotechnology Information (NCBI) Web server (https://ncbi.nlm.nih.gov ).  BLOSUM62 matrix was used with default parameters.  The analysis was done with and without filtering of the low-complexity sequences and without masking of repetitive elements. Queries used the cDNA sequences of human PEPT1 (accession number: NM_005073) and hPEPT1-RF (AB001328) and the high-throughput genomic sequence (HTGS) database. 

Using the accession number of the mRNA sequence, we retrieved the (CDS) sequence from NCBI and performed a BLAST search of the HTGS database.  Results were filtered using the blastflt.py code written by Arne Mueller (BLAST2 Parser ver. 1.2, © Arne Mueller, https://www.bmm.icnet.uk/people/mueller . The obtained hits were filtered to ensure that only data from the same species and chromosome were used. The alignments served to locate the exons in the genomic sequence. When problems arose, the sequences were examined by hand to attempt to resolve or identify possible alternative splice sites. Membrane topological prediction was done using the (TOPPRED) program.

The sequences 2 kb upstream from the transcription start sites of hPEPT1 and hPEPT1-RF were investigated using programs FindPatterns and FitConsensus (Genetics Computer Group, Madison, WI)to locate possible promoters and enhancer sites.

Results

Bioinformatic analysis revealed that hPEPT1 and hPEPT1-RF are encoded by the same gene located in chromosome 13, clone RP11-56D6 (accession: AL357553).  hPEPT1 contains 23 exons (Table 1 , Figure 1 ), and hPEPT1-RF contains 6 exons (Table 2 , Figure 1 ).  Human PEPT1 and hPEPT1-RF share 3 exons completely, and 2 exons are partially shared (Figure 1 ). Therefore, hPEPT1-RF and hPEPT1 are splice variants of the same gene that has in total 24 exons. Over the course of the study, additional genomic clones became available containing all hPEPT1 exons in several contiguous fragments, and these served to verify the order and intronic sizes provided in Tables 1 and 2 .

All the exon-intron boundaries for hPEPT1 conform to the consensus splice junction sequences (gt/ag) for eukaryotic genes¹⁴ .  The 9 conserved nucleotides in the 5' donor side are (A/C)AG/gt(a/g)agt. These are conserved at 64%, 73%, 50%, 100%, 100%, 86%, 70%, 83%, and 77%, respectively, in hPEPT1 .  Similarly, we found positions of the 3'-acceptor site, (c/a)ag/(A/G), to be conserved at 73%, 100%, 100%, and 73%, respectively (Table 1 ).  Splice sites are classified in phase 0 (13 sites), phase 1 (4 sites), and phase 2 (5 sites) (Table 1 ).

The hPEPT1 gene structure shows several interesting features. The start sites of the transcripts for hPEPT1 and pH-regulatory factor are located in different exons (Figure 1 ). Moreover, exon 1 located >20 kb upstream of exon 2 contains only the first 4 nucleotides of the hPEPT1 coding region. Alternative splicing occurs in exon 3, and 118 bases in the 5' end of exon 3 are spliced out of the mRNA of hPEPT1 . Another site for differential splicing is exon 7 of hPEPT1-RF . In this case, 41 bases in the 3' end of the exon are spliced out of hPEPT1 hmRNA (Figure 1 ).

Membrane topology predictions of hPEPT1 and hPEPT1-RF proteins are shown in Figures 2 and 3 . The transmembrane topology schematics were rendered using TOPO (S.J. Johns and R.C. Speth, Transmembrane protein display software, https://www.sacs.ucsf.edu/TOPO/topo.html , unpublished data). The figures show the peptide sequences that are encoded by each exon. In accordance with earlier information, hPEPT1 is predicted to have 12 transmembrane segments (TMSs). Interestingly, comparison of membrane topology with gene structure shows possible functional modularity.  Few if any exon-intron boundaries are found within the TMSs, and in most cases each exon encodes for a single TMS-loop unit (Figure 2 ).  Topological predictions suggest that hPEPT1-RF has 5 TMSs with a cytoplasmic N-terminal and extracellular C-terminal.

The upstream region (2 kb) from the transcription start sites of hPEPT1 is shown in Figure 4 .  TATA boxes were found about 520 bp upstream from the transcription start site in hPEPT1 .  The putative regulatory region also contains GC boxes, so several GC boxes are located within 300 bp from the transcription site in hPEPT1 .  Binding sites for transcription factors did not include any amino acid responsive element. Some other transcription factor binding sites of the regulatory regions are illustrated in Figure 4 .

Discussion

The genomic structure of hPEPT1 and hPEPT1-RF presented here is based on a sequence in the HTGS database. The HTGS contains yet unordered pieces of genomic sequences. We used the August 11, 2000, version of the clone AL357553 in our analysis. It contained 11 contigs, but the true order of these pieces is still unknown, and the size of the gaps between them may change. Three introns of hPEPT1 include such gaps (indicated by > signs in Table 1 ), while hPEPT1-RF exons are all located in one contig. Within the contigs the sequences are likely to be unaffected, and intron sizes are reliable (Table 1 ). Note also that the order of the exons in the clone matches perfectly the nucleotide sequence of cDNA.  Where possible, these predictions have subsequently been verified and the intronic sizes adjusted where needed, on the basis of additional genomic clones deposited in the HTGS database.

Human PEPT1 is encoded by 23 exons, and the entire gene contains 24 exons.  Likewise, mouse PEPT1 is encoded by 23 exons¹³ . Comparison of mouse and human genes shows that the sizes of the exons and their relative locations are similar. Identity of mouse and human cDNA for PEPT1 s is 83%¹³ . A high degree of similarity in both gene clustering and coding sequence confirm that human and mouse PEPT1 genes are orthologues. In this study the comparison of membrane topological prediction and genomic structure indicates that human PEPT1 gene is modular with each TMS-loop unit encoded by a different exon (Figures 2 and 3 ). This is in accordance with earlier analysis of peptide transporters that suggested modular structure of transporter genes may have evolved by exon shuffling and rearrangements of functional modules¹⁵ .

The hPEPT1 gene also encodes the splice variant hPEPT1-RF. PEPT1-RF and PEPT1 share 5 identical TMSs, while the extramembraneous terminals differ (Figures 1 , 2 , and 3 ).  PEPT1-RF is not capable of transporting substrates across the membrane, but it is thought to sense pH changes and modulate the response of PEPT1 to these changes¹² .  Fei et al¹⁶ have shown by using chimeric PEPT1-PEPT2 proteins that the TMSs 7-9 are important for substrate recognition by hPEPT1.  PEPT1-RF does not have these TMSs and does not transport substrates.  However, the mechanisms of proton and substrate transfer and the interplay between PEPT1 and PEPT1-RF are still elusive.

The putative regulatory region of hPEPT1 (Figure 4 ) revealed some similarities with the mouse PEPT1   gene¹³ .  TATA boxes are located in unusual locations (511 bp and 517 bp upstream from the transcription start site), while GC boxes are located near the start site (at -29 bp and several others within 300 bp).  The location of TATA boxes so far upstream from the transcription start site is not optimal. Therefore, this kind of structure suggests that the GC box is a more important promoter in the regulation of hPEPT1 than is the TATA box. Note also that there may be more than one transcription start site for a gene, as shown previously¹⁷ . Unlike in the mouse genome, amino acid responsive element was not found within 1983 bp from the transcription start site in PEPT1 . Human PEPT1 expression is known to be upregulated by its substrates, dipeptides, as shown by Walker et al¹⁸ , but the mechanism of this upregulation remains unclear. Insulin regulates the activity of PEPT1 in Caco-2 cells¹⁹ . Insulin regulation was mediated by transporter translocation to the basolateral side of the cells upon release of hPEPT1 from the translated intracellular pool to the plasma membrane. Changes in hPEPT1 mRNA were not seen in that study. However, the putative insulin responsive element is located upstream from the transcription start site (Figure 4 ), suggesting that insulin might be involved in the regulation of hPEPT1 transcriptional activity.

Conclusion

The genomic organization of hPEPT1 and hPEPT1-RF indicates that they are splice variants of the same gene (Figure 1 ). Expression of hPEPT1-RF has not been studied in detail. Nevertheless, the splice variants may be expressed in different proportions depending on, for example, the stage of differentiation, hormonal regulation signals, and cell type. Human PEPT1 is expressed in several tissues (intestine, kidney, brain, liver) where the pH environment is quite different. Also, an intracellular pool of hPEPT1 may be associated with peptide trafficking in lysosomes and endosomes that have different pH depending on the maturity of the vesicle¹¹ .

Finally, the genomic organization of hPEPT1 paves the way for studies of the relationships between PEPT1 genotype and pharmacokinetics.

Acknowledgements

This study was partly supported by the Academy of Finland (AU) and a grant from the National Institutes of Health, Bethesda, MD, General Medical Sciences (GM61390).

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