cDNA structure, genomic organization, and promoter analysis of the mouse intestinal peptide transporter PEPT1

Abstract

We describe in this report the cDNA structure, functional characteristics, genomic organization, and promoter analysis of the mouse H⁺-coupled low-affinity peptide transporter PEPT1. The mouse PEPT1 cDNA cloned from a kidney cDNA library is ∼3.1 kb long and encodes a protein of 709 amino acids. When expressed heterologously in mammalian cells and in Xenopus laevis oocytes, mouse PEPT1 mediates H⁺-coupled electrogenic transport of the dipeptide glycylsarcosine. The mouse pept1 gene, cloned from a genomic DNA library in bacterial artificial chromosome, is ∼38 kb long and consists of 23 exons and 22 introns. 5′-Rapid amplification of cDNA ends with poly(A)⁺ RNA from mouse intestine has identified the transcription start site that lies 31 bp upstream of the translation start site. The promoter region upstream of the transcription start site does not contain the TATA box but possesses three GC boxes which are the binding sites for the transcription activator SP1. Functional analysis of the promoter region using the luciferase reporter assay in Caco-2 cells (a human intestinal cell line that express PEPT1 constitutively) and five different 5′-deletion fragments of the promoter has shown that essential promoter/enhancer elements are present within 1140 bp upstream of the transcription start site.

Introduction

The peptide transport system in the mammalian intestine is responsible for the absorption of small peptides (dipeptides and tripeptides) that arise from the digestion of dietary proteins [1], [2]. This transport system is expressed in the brush border membrane of the absorptive cells and has been characterized in detail at the functional level [3]. It is a H⁺-coupled transporter, mediating the electrogenic co-transport of peptides and H⁺. This system also recognizes a variety of peptidomimetic drugs as transportable substrates and thus plays an important role in the oral bioavailability of these drugs [4]. Several years ago, we cloned this peptide transporter, designated PEPT1, from a rabbit intestinal cDNA library using an expression cloning strategy and elucidated its structural and functional characteristics [5]. Rabbit PEPT1 showed no homology to any of the transporters previously cloned and thus represented the first member of a new transporter gene family. Since then, human and rat homologs of this transporter have been cloned and characterized [6], [7]. A related peptide transport system is expressed in the brush border membrane of renal absorptive cells that has relatively much higher affinity for peptides than the intestinal peptide transporter [8]. This high-affinity peptide transporter, called PEPT2, has been cloned from different animal species (rabbit, rat, and human) [9], [10], [11]. There is significant homology between PEPT1 and PEPT2 [12]. Available evidence indicates that the intestine expresses only PEPT1 whereas the kidney expresses PEPT2 as well as PEPT1 [13].

Digestion of dietary proteins in the mammalian intestine results in the generation of small peptides as well as free amino acids. Since PEPT1 is the only peptide transporter expressed in the intestine, it is solely responsible for the absorption of the protein digestion products from the lumen in the form of small peptides. Free amino acids are absorbed by a variety of amino acid transporters expressed in the brush border membrane of the intestine [2]. These amino acid transporters have overlapping substrate specificity. A physiologically relevant question regarding the absorption of protein digestion products is: What is the relative importance of peptide transport versus amino acid transport across the brush border membrane? It is generally assumed that peptide transport is more important than amino acid transport primarily based on the findings that the end products of protein digestion in the intestinal lumen exist predominantly in the form of peptides rather than free amino acids [1], [2]. However, there is no direct evidence to support this assumption. There are no reports of genetic defects of intestinal peptide transport. If there were, the status of protein nutrition in patients with such defects would reveal the importance of the peptide transport process. Genetic defects in intestinal amino acid transport across the brush border membrane are known, but these defects (e.g., Hartnup disease and cystinuria) are not generally associated with protein malnutrition under conditions of adequate protein intake [2]. This does not necessarily mean that amino acid transport is not important for the maintenance of protein nutrition. This may simply be due to the fact that there are multiple amino acid transport systems in the intestinal brush border membrane with overlapping substrate specificity. An ideal way to address the issue of relative importance of peptide transport versus amino acid transport is to generate an animal model with a defect in intestinal peptide transport. Since PEPT1 is solely responsible for intestinal peptide transport, targeted disruption of the pept1 gene in mouse would provide a PEPT1 knockout animal model suitable for these studies. With this goal in mind, we undertook the present project to clone the mouse PEPT1 cDNA and pept1 gene and to elucidate the structural organization of the gene. In this paper, we report on the cDNA structure, genomic organization, and promoter analysis of mouse PEPT1.