Expression of human intestinal dipeptide transporter in Xenopus laevis oocytes

doi:10.1016/0006-2952(93)90156-Q

Biochemical Pharmacology

Volume 45, Issue 3, 9 February 1993, Pages 776-779

https://doi.org/10.1016/0006-2952(93)90156-Q Get rights and content

Abstract

The human colon adenocarcinoma cell line Caco-2 retains the H⁺/dipeptide cotransporter. To identify the structure of the human dipeptide transporter, we have examined the expression of the transporter in Xenopus laevis oocytes injected with Caco-2 poly(A)⁺RNA, by monitoring the uptake of bestatin, a dipeptide-like anticancer agent. The bestatin uptake in the poly(A)⁺RNA-injected oocytes was inhibited by excess glycyl-l-leucine, and showed pH dependence (optimal pH of 5.5–6.0). These observations suggest that the human intestinal dipeptide transporter can be expressed functionally in Xenopus oocytes.

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Is intestinal peptide transport energized by a proton gradient?

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Cited by (17)

Cloning and characterization of a pH-sensing regulatory factor that modulates transport activity of the human H<sup>+</sup>/peptide cotransporter, PEPT1
1997, Biochemical and Biophysical Research Communications
We have isolated a cDNA encoding a pH-sensing regulatory factor protein that modulates transport activity of the human H⁺/peptide cotransporter, hPEPT1, from the human duodenum cDNA library. The cDNA (1,724 bp) for the regulatory factor (hPEPT1-RF) had an open reading frame encoding a 208-amino acid. The 18-195 amino acid residues of hPEPT1-RF were completely consistent with the 8-185 amino acid residues of hPEPT1, whereas the 1-17 and 196-208 residues were unique sequences. Using a reticulocyte lysate, thein vitrosynthesized hPEPT1-RF RNA translated a product of ∼23 kDa. Northern blot analysis and reverse transcription-coupled PCR revealed that both hPEPT1 and hPEPT1-RF mRNA transcripts are expessed in Caco-2 cells. When expressed inXenopusoocytes, hPEPT1-RF showed no transport activity of glycylsarcosine, but shifted pH profile of the dipeptide transport mediated by the coexpressed hPEPT1. The pH profile of glycylsarcosine uptake in oocytes coexpressing hPEPT1 and hPEPT1-RF was almost similar to that in the Caco-2 cells. This is the first demonstration of cDNA isolation of a regulatory factor which modulates hPEPT1 activity.
Oral absorption of β-lactams by intestinal peptide transport proteins
1997, Advanced Drug Delivery Reviews
The oral β-lactams are an extremely well-absorbed class of antibiotics. They share a common uptake mechanism with small peptides for entry into the enterocyte and exit through the basolateral membrane. Transport proteins involved in the absorption of these antibiotics have been identified. Three strategies were employed to identify proteins important for the proton dependent uptake of β-lactams. First, a 127-kDa β-lactam transporter in rabbit intestinal brush border membranes was identified by photoaffinity labeling, protein purification and reconstitution. Second, an immunological approach resulted in the identification of HPT-1, a 120-kDa protein in apical membranes of human intestinal Caco-2 cells. The hpt-1 cDNA confers the ability to transport both cephalexin and bestatin in a proton-dependent fashion. Third, functional cloning of poly(A)⁺ RNA of rabbit intestine identified a PepT1 protein that cotransports protons with small peptides, β-lactams, and angiotensin-converting enzyme inhibitors. The kidney also expresses a transporter, PepT2, with high homology to PepT1. The expression and regulation of the intestinal transporters is reviewed. The usefulness of these transport proteins to the discovery of new oral therapeutics is discussed.
The intestinal peptide carrier: A potential transport system for small peptide derived drugs
1996, Advanced Drug Delivery Reviews
Several laboratories have recently shown that many peptides and peptide-type drugs are absorbed in the small intestine via the peptide transporter for nutrient di-and tripeptides. The peptide carrier has a broad substrate specificity and can provide an efficient route for the absorption of peptide drugs that may not readily penetrate the lipophilic intestinal membrane. The peptide carrier is an active transport system that is H⁺-coupled, dependent on metabolic energy, saturable and concentration dependent, and that exhibits mutual inhibition of transport among the compounds. Studies of peptide transport are less advanced than for amino acids and there is no general agreement about the absolute number of transporters, the mechanism for basolateral efflux, and the distribution of the carrier(s) along the gastrointestinal tract. The recent successful cloning of the intestinal peptide transport system will contribute to characterize the structural requirements for potential substrates and may aid the development of effective oral drugs in this structural class.
Molecular mechanism for the relative binding affinity to the intestinal peptide carrier. Comparison of three ACE-inhibitors: enalapril, enalaprilat, and lisinopril
1995, BBA - Biomembranes
The affinity of three substrates for the intestinal peptide carrier is explained based on their three-dimensional (3D) structural data. The kinetic transport parameters of three ACE-inhibitors, enalapril, enalaprilat, and lisinopril, have been determined in an in vitro system using rat intestine. The observed kinetic transport parameters (±asymptotic standard error) of enalapril are: 0.81 (±0.23) mM, 0.58 (±0.37) μmol/h per cm 2, and 0.56 (±0.04) cm/h for the half-maximal transport concentration (K_T), the maximal transport flux (J_max) and the passive permeability constant (P_m). Enalaprilat was transported by passive diffusional with a P_m, of 0.51 (±0.04) cm/h. For lisinopril the kinetic transport parameters were 0.38 (±0.19) mM, 0.12 (±0.07) μmol/h per cm², and 0.18 (±0.02) cm/h for K_T, J_max, and P_m, respectively. The affinity of the ACE-inhibitors for the intestinal peptide carrier has been evaluated based on their ability to inhibit the transport rate of cephalexin. The inhibition constants (K_i) of enalapril, enalaprilat and lisinopril were 0.15, 0.28 and 0.39 mM, respectively. 3D structural analysis of lisinopril using molecular modelling techniques reveals that intramolecular hydrogen bond formation is responsible for decreased carrier affinity.
Functional expression of intestinal dipeptide/β-lactam antibiotic transporter in Xenopus laevis oocytes
1994, Biochemical Pharmacology
An intestinal active transport system specific to small peptides and peptide-like drugs such as β-lactam antiobiotics was functionally expressed in Xenopus laevis oocytes after microinjection of messenger RNA (mRNA) derived from rat intestinal cells. The transport activity was evaluated by measuring the uptake of a tripeptide-like cephalosporin antibiotic, ceftibuten, which has high affinity for the intestinal peptide/H⁺ co-transporter and is resittant to peptidases. Ceftibuten transport in mRNA-injected oocytes was pH dependent (a proton gradient is the driving force), stereo selective (uptake of the cis-isomer of ceftibuten was about 4-fold higher than that of the trans-isomer), saturable and temperature dependent. Furthermore, various dipeptides showed cis-inhibitory and trans-stimulatory effects on the uptake of ceftibuten by mRNA-injected oocytes, suggesting that ceftibuten and dipeptides are transported by a common carrier protein. These results are in accordance with the functional properties of native proton-coupled peptide transporter previously clarified by studies with isolated intestinal brush-border membrane vesicles and other experimental systems. A protein with a molecular mass of about 130 kDa expressed in the membrane of mRNA-injected oocytes was identified as the transport protein by specific labeling with a photoreactive β-lactam antibiotic, [³h]benxylpenicillin, followed by SDS-PAGE analysis of the radiolabeled protein. Furthermore, an experiment with mRNA size-fractionated by sucrose density gradient centrifugation indicated that the peptide transporter is encoded by mRNA of between 1.8 and 3.6 kb. These results, obtained using a heterologous gene expression technique, confirm that intestinal absorption of β-lactam antibiotics occurs through a carrier-mediated mechanism and show that biologically stable β-lactam antibiotics can be useful probes for molecular analysis of intestinal peptide transporter.
PDZK1 regulates two intestinal solute carriers (Slc15a1 and Slc22a5) in mice
2008, Drug Metabolism and Disposition

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Biochemical Pharmacology

Abstract

References (23)

Role of pH gradient and membrane potential in dipeptide transport in intestinal and renal brush-border membrane vesicles from the rabbit

J Biol Chem

H⁺ coupled uphill transport of aminocephalosporins via the dipeptide transport system in rabbit intestinal brush-border membranes

J Biol Chem

Cloning of a Na⁺- and Cl⁻-dependent betaine transporter that is regulated by hypertonicity

J Biol Chem

Functional expression of the intestinal peptide-proton co-transporter in Xenopus laevis oocytes

J Biol Chem

Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability

Gastroenterology

Transport of a large neutral amino acid (phenylalanine) in a human intestinal epithelial cell line: Caco-2

Biochim Biophys Acta

A rapid and sensitive method for the quantitation of micorogram quantities of protein utilizing the principle of protein-dye binding

Anal Biochem

The use of Xenopus oocytes for the expression of cloned genes

Expression of mammalian renal transporters in Xenopus laevis oocytes

Arch Biochem Biophys

Expression of the mouse macrophage cystine transporter in Xenopus laevis oocytes

Arch Biochem Biophys

Is intestinal peptide transport energized by a proton gradient?

Am J Physiol

Cited by (17)

Cloning and characterization of a pH-sensing regulatory factor that modulates transport activity of the human H<sup>+</sup>/peptide cotransporter, PEPT1

Oral absorption of β-lactams by intestinal peptide transport proteins

The intestinal peptide carrier: A potential transport system for small peptide derived drugs

Molecular mechanism for the relative binding affinity to the intestinal peptide carrier. Comparison of three ACE-inhibitors: enalapril, enalaprilat, and lisinopril

Functional expression of intestinal dipeptide/β-lactam antibiotic transporter in Xenopus laevis oocytes

PDZK1 regulates two intestinal solute carriers (Slc15a1 and Slc22a5) in mice

Short communicationExpression of human intestinal dipeptide transporter in Xenopus laevis oocytes

Abstract

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Gastroenterology

Biochim Biophys Acta

Anal Biochem

Arch Biochem Biophys

Arch Biochem Biophys

Is intestinal peptide transport energized by a proton gradient?

Am J Physiol

Short communication
Expression of human intestinal dipeptide transporter in Xenopus laevis oocytes