Mechanisms of 5-aminolevulinic acid uptake at the choroid plexus

5-Aminolevulinic acid (5-ALA) is a precursor of porphyrins and heme that has been implicated in the neuropsychiatric symptoms associated with porphyrias. It is also being used clinically to delineate malignant gliomas. The blood-CSF barrier may be an important interface for 5-ALA transport between blood and brain as in vivo studies have indicated 5-ALA is taken up by the choroid plexuses whereas the normal blood-brain barrier appears to be relatively impermeable. This study examines the mechanisms of 5-[(3)H]ALA uptake into isolated rat lateral ventricle choroid plexuses. Results suggest that there are two uptake mechanisms. The first was a Na(+)-independent uptake system that was pH dependent (being stimulated at low pH). Uptake was inhibited by the dipeptide Gly-Gly and by cefadroxil, an alpha-amino-containing cephalosporin. These properties are the same as the proton-dependent peptide transporters PEPT1 and PEPT2, which have recently been shown to transport 5-ALA in frog oocyte expression experiments. Choroid plexus uptake was not inhibited by captopril, a PEPT1 inhibitor, suggesting PEPT2-mediated uptake. The presence of PEPT2 and absence of PEPT1 in the choroid plexus were confirmed by western blotting. The second potential mechanism was both Na(+) and HCO(3)(-) dependent and appears to be an organic anion transporter, although it is possible that removal of Na(+) and HCO(3)(-) may indirectly affect PEPT2 by affecting intracellular pH. The presence of PEPT2 and a putative Na(+)/HCO(3)(-)-dependent organic anion transporter is important not only for an understanding of 5-ALA movement between blood and brain but also because these transporters may affect the distribution of a number of drugs between blood and CSF.     

Carnosine uptake in rat choroid plexus primary cell cultures and choroid plexus whole tissue from PEPT2 null mice

PEPT2 is functionally active and localized to the apical membrane of rat choroid plexus epithelial cells. However, little is known about the transport mechanisms of endogenous neuropeptides in choroid plexus, and the role of PEPT2 in this process. In the present study, we examined the uptake kinetics of carnosine in rat choroid plexus primary cell cultures and choroid plexus whole tissue from wild-type (PEPT2(+/+)) and null (PEPT2(-/-)) mice. Our results indicate that carnosine is preferentially taken up from the apical as opposed to basolateral membrane of cell monolayers, and that basolateral efflux in limited. Transepithelial flux of carnosine was not distinguishable from that of paracellular diffusion. The apical uptake of carnosine was characterized by a high affinity (K(m) = 34 microM), low capacity (V(max) = 73 pmol/mg protein/min) process, consistent with that of PEPT2. The non-saturable component was small (K(d) = 0.063 microL/mg protein/min) and, under linear conditions, was only 3% of the total uptake. Studies in transgenic mice clearly demonstrated that PEPT2 was responsible for over 90% of carnosine's uptake in choroid plexus whole tissue. These findings elucidate the unique role of PEPT2 in regulating neuropeptide homeostasis at the blood-cerebrospinal fluid interface.     

Targeted disruption of the peptide transporter Pept2 gene in mice defines its physiological role in the kidney

The peptide transporter PEPT2 mediates the cellular uptake of di- and tripeptides and selected drugs by proton-substrate cotransport across the plasma membrane. PEPT2 was functionally identified initially in the apical membrane of renal tubular cells but was later shown to be expressed in other tissues also. To investigate the physiological importance of PEPT2 and for a detailed analysis of the protein expression sites, we generated a Pept2 knockout mouse line in which the Pept2 gene was disrupted by insertion of a beta-galactosidase gene under the control of the PEPT2 promoter. The Pept2(-/-) mice showed no obvious phenotypic abnormalities but also no adaptive upregulation in the expression level of related genes in the kidney. The importance of PEPT2 in the reabsorption of filtered dipeptides was demonstrated in knockout animals by significantly reduced renal accumulation of a fluorophore-labeled and a radiolabeled dipeptide after in vivo administration of the tracers. This indicates that PEPT2 is the main system responsible for tubular reabsorption of peptide-bound amino acids, although this does not lead to major changes in renal excretion of protein or free amino acids.     

肽转运载体的分子特征及其分布

动物体内的肽转运载体目前发现的至少有五种,其中研究最为广泛的是：PepT1和PepT2。PepT1和PepT2都是依质子的寡肽转运载体(POT)家族的成员。PepT1是低亲和力／高容量的肽载体,PepT2高亲和力／低容量的肽载体。PepT1主要在消化道中表达,在肾脏中也有微弱的表达;PepT2主要在肾脏中表达。这些肽载体的分子结构特征主要有：(1)有12个假想的穿膜区,在9区和10区之间有一大的胞外环,且所有穿膜区内的序列都高度保守,胞外环上的序列保守的很少;(2)被编码的蛋白上有多个N-糖基化和蛋白激酶的识别位点,它们可能参与肽转运的调控;(3)PepT1上的His-57和PepT2上的His-87是最关键的组氨酸残基,它们可能是转运蛋白发挥吸收功能时最关键的结合位点;(4)不同动物肽转运蛋白的氨基酸范围在707到729之间,且不同动物相同器官肽转运载体的同源性高(大约80％),同种动物不同器官肽转运载体的同源性低(大约50％)。了解肽载体的分子特征和组织分布,可以更好地理解肽吸收的分子机制并有利于肽类药物的研发。     

Effect of Janus Kinase 3 on the Peptide Transporters PEPT1 and PEPT2

The tyrosine kinase Janus kinase 3 (JAK3) contributes to signaling regulating the proliferation and apoptosis of lymphocytes and tumor cells. Replacement of lysine by alanine in the catalytic subunit yields the inactive ^K851AJAK3 mutant that underlies severe combined immune deficiency. The gain-of-function mutation ^A572VJAK3 is found in acute megakaryoplastic leukemia and T cell lymphoma. The excessive nutrient demand of tumor cells requires upregulation of transporters in the cell membrane including peptide transporters PEPT1 and PEPT2. The carriers further accomplish intestinal peptide transport. Little is known about signaling regulating peptide transport. The present study explored whether PEPT1 and PEPT2 are upregulated by JAK3. PEPT1 or PEPT2 was expressed in Xenopus oocytes with or without additional expression of JAK3, and electrogenic peptide (glycine–glycine) transport was determined by dual-electrode voltage clamp. PEPT2-HA membrane protein abundance was analyzed by chemiluminescence. Intestinal electrogenic peptide transport was estimated from peptide-induced current in Ussing chamber experiments. In PEPT1- and PEPT2-expressing oocytes, but not in water-injected oocytes, the dipeptide gly–gly generated an inward current, which was significantly increased following coexpression of JAK3. The effect of JAK3 on PEPT1 was mimicked by ^A568VJAK3 but not by ^K851AJAK3. JAK3 increased maximal peptide-induced current in PEPT1-expressing oocytes but rather decreased apparent affinity of the carrier. Coexpression of JAK3 enhanced the PEPT2-HA protein abundance in the cell membrane. In JAK3- and PEPT1-expressing oocytes, peptide-induced current was blunted by the JAK3 inhibitor WHI-P154, 4-[(3′-bromo-4′-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline (22 μM). In intestinal segments gly–gly generated a current which was significantly smaller in JAK3-deficient mice (jak3 ^?/?) than in wild-type mice (jak3 ^+/+). In conclusion, JAK3 is a powerful regulator of peptide transporters PEPT1 and PEPT2.     

Construction, identification and application of HeLa cells stably transfected with human PEPT1 and PEPT2

The purpose of this study was to construct stably transfected HeLa cells with human peptide transporters (hPEPT1/hPEPT2) and to identify the function of the transfected cells using the substrate JBP485 (a dipeptide) and a typical substrate for PEPTs, glycylsarcosine (Gly-Sar). An efficient and rapid method was established for the preparation and transformation of competent cells of Escherichia coli. After extraction and purification, hPEPT1/hPEPT2-pcDNA3 was transfected into HeLa cells by the liposome transfection method, respectively. HeLa-hPEPT1/hPEPT2 cells were selected by measuring the protein expression and the uptake activities of JBP485 and Gly-Sar. A simple and rapid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of JBP485 and Gly-Sar in biological samples. The Michaelis-Menten constant (K(m)) values of Gly-Sar uptake by the hPEPT1 and hPEPT2-expressing transfectants were 1.03 mM and 0.0965 mM, respectively, and the K(m) values of JBP485 uptake were 1.33 mM for PEPT1 and 0.144 mM for PEPT2. The uptake of Gly-Sar was significantly inhibited by JBP485 with a K(i) value of 8.11 mM (for PEPT1) and 1.05 mM (for PEPT2). Maximal uptake of Gly-Sar were detected at pH 5.8 (for PEPT1) and pH 6.5 (for PEPT2), suggesting that both HeLa-hPEPT1 and HeLa-hPEPT2 were H(+) dependent transporters. Stably transfected HeLa-hPEPT1/HeLa-hPEPT2 cells were constructed successfully, and the functions of hPEPT1/hPEPT2 were identified using their substrates, JBP485 and Gly-Sar. The transfected cells with transporters were used to investigate drug-drug interactions (DDIs) between JBP485 and other substrates (cephalexin or lisinopril) of PEPT1 and PEPT2.     

Peptide transporter 2 (PEPT2) expression in brain protects against 5-aminolevulinic acid neurotoxicity

The proton-coupled oligopeptide transporter PEPT2 (or SLC15A2 ) is the major protein involved in the reclamation of peptide-bound amino acids and peptide-like drugs in kidney. PEPT2 is also important in effluxing peptides and peptidomimetics from CSF at the choroid plexus, thereby limiting their exposure in brain. In this study, we report a neuroprotective role for PEPT2 in modulating the toxicity of a heme precursor, 5-aminolevulinic acid (5-ALA). Our findings demonstrate that in PEPT2-deficient mice, 5-ALA administration results in reduced survivability, a worsening of neuromuscular dysfunction, and CSF concentrations of substrate that are 8–30 times higher than that in wild-type control animals. The ability of PEPT2 to limit 5-ALA exposure in CSF suggests that it may also have relevance as a secondary genetic modifier of conditions (such as acute hepatic porphyrias and lead poisoning) in which 5-ALA metabolism is altered and in which 5-ALA toxicity is important.     

Cytosolic COOH terminus of the peptide transporter PEPT2 is involved in apical membrane localization of the protein

The peptide transporter PEPT2 is a polytopic transmembrane protein that mediates the cellular uptake of di- and tripeptides and a variety of peptidomimetics. It is widely expressed in mammalian tissues, including kidney, lung, mammary gland, choroid plexus, and glia cells. In renal tubular cells, PEPT2 is exclusively found at the apical membrane. The molecular mechanisms underlying this polarized expression and targeting to the brush-border membrane are not known. We have explored the role of the 36 COOH-terminal amino acid residues in PEPT2 trafficking and apical expression. EGFP-tagged PEPT2 wild-type transporter and various truncated and mutant proteins were expressed in the polarized proximal tubule cell lines SKPT and OK, and the cellular distribution of the fusion proteins was assessed using confocal microscopy. Whereas deletion of the last seven amino acids (delC7) did not alter PEPT2 surface expression, deletion of the next residue (delC8) or up to 30 terminal amino acids resulted in impaired apical expression and distinct accumulation of mutant proteins in endosomal and lysosomal vesicles. Truncation of more amino acids (delC36) containing tyrosine-based motifs led to a rather diffuse intracellular distribution pattern. Mutations introduced at isoleucine-720 (I720A) and leucine-722 (I722A) also caused an impaired surface appearance. Internalization assays revealed a higher endocytotic rate of the PEPT2 mutants I720A, L722A, and delC36. Our data suggest that a three-amino acid stretch (INL) and tyrosine-based motifs within the COOH tail of PEPT2 are involved in PEPT2's apical membrane localization and membrane steady-state level. di- and tripeptide transport; polarized epithelial cells; lysosomes     

Influence of proton and essential histidyl residues on the transport kinetics of the H+/peptide cotransport systems in intestine (PEPT 1) and kidney (PEPT 2)

The mechanism by which H⁺ alters the kinetics of the H⁺-coupled peptide transporters PEPT 1 and PEPT 2 was investigated in two different cell lines which differentially express these transporters, namely Caco-2 cells (PEPT 1) and SKPT cells (PEPT 2). The effects of H⁺ on the affinity and the maximal velocity of Gly-Sar uptake were analyzed in these cells under identical conditions. In both cells, H⁺ influenced only the maximal velocity of uptake and not the apparent affinity. The effects of H⁺ on the IC₅₀ values (i.e., concentration necessary to cause 50% inhibition) of the cationic dipeptide Ala-Lys and the anionic dipeptide Ala-Asp for inhibition of Gly-Sar uptake were also investigated. H⁺ did not change the IC₅₀ value for Ala-Lys but did decrease the IC₅₀ value for Ala-Asp considerably. The influence of diethylpyrocarbonate (DEP) on the kinetic parameters of PEPT 1 and PEPT 2 was then studied. Histidyl residues are the most likely amino acid residues involved in H⁺ binding and translocation in H⁺-coupled transport systems and DEP is known to chemically modify histidyl residues and block their function. DEP treatment altered the maximal velocity of Gly-Sar uptake but had no effect on its K_t (Michaelis-Menten constant) or the IC₅₀ values of Ala-Lys or Ala-Asp for the inhibition of Gly-Sar uptake. It is concluded that H⁺ stimulates PEPT 1 and PEPT 2 primarily by increasing the maximal velocity of the transporters with no detectable influence on the substrate affinity.     

11C]Glycylsarcosine: synthesis and in vivo evaluation as a PET tracer of PepT2 transporter function in kidney of PepT2 null and wild-type mice

[11C]Glycylsarcosine (Gly-Sar) was synthesized as a potential radiotracer to investigate the localization and in vivo function of the peptide transporter PepT2 in mouse kidney. Its C-11 labeled diketopiperazine derivative, [11C]cyclo(Gly-Sar) [1-methylpiperazine-2,5-dione], was also evaluated as a potential tracer. [11C]Gly-Sar exhibited rapid initial uptake into kidneys with slow clearance from the medulla, consistent with uptake and retention of the radiotracer through the actions of PepT2. In contrast, the corresponding cyclized dipeptide [11C]cyclo(Gly-Sar) showed rapid clearance and accumulation only in the renal pelvis region. Involvement of PepT2 in reabsorption and delayed clearance of [11C]Gly-Sar was confirmed using the PepT2 knockout mouse, where rapid renal elimination of [11C]Gly-Sar and the absence of radioactivity in medulla were observed. This study demonstrates using in vivo imaging technique that PepT2 is primarily responsible for renal tubular active reabsorption of Gly-Sar, and provides a new tool for studying tubular peptide reabsorption and clearance.     

USP18 Sensitivity of Peptide Transporters PEPT1 and PEPT2

USP18 (Ubiquitin-like specific protease 18) is an enzyme cleaving ubiquitin from target proteins. USP18 plays a pivotal role in antiviral and antibacterial immune responses. On the other hand, ubiquitination participates in the regulation of several ion channels and transporters. USP18 sensitivity of transporters has, however, never been reported. The present study thus explored, whether USP18 modifies the activity of the peptide transporters PEPT1 and PEPT2, and whether the peptide transporters are sensitive to the ubiquitin ligase Nedd4-2. To this end, cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding USP18. Electrogenic peptide (glycine-glycine) transport was determined by dual electrode voltage clamp. As a result, in Xenopus laevis oocytes injected with cRNA encoding PEPT1 or PEPT2, but not in oocytes injected with water or with USP18 alone, application of the dipeptide gly-gly (2 mM) was followed by the appearance of an inward current (I_gly-gly). Coexpression of USP18 significantly increased I_gly-gly in both PEPT1 and PEPT2 expressing oocytes. Kinetic analysis revealed that coexpression of USP18 increased maximal I_gly-gly. Conversely, overexpression of the ubiquitin ligase Nedd4-2 decreased I_gly-gly. Coexpression of USP30 similarly increased I_gly-gly in PEPT1 expressing oocytes. In conclusion, USP18 sensitive cellular functions include activity of the peptide transporters PEPT1 and PEPT2.     

Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di‐ and tri‐peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT_So, a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand‐bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide‐binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT_So represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.     

Uptake, transport and regulation of JBP485 by PEPT1 in vitro and in vivo

Cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) is a dipeptide with anti-hepatitis activity that has been chemically synthesized. Previous experiments in rats showed that JBP485 was well absorbed by the intestine after oral administration. The human peptide transporter (PEPT1) is expressed in the intestine and recognizes compounds such as dipeptides and tripeptides. The purposes of this study were to determine if JBP485 acted as a substrate for intestinal PEPT1, and to investigate the characteristics of JBP485 uptake and transepithelial transport by PEPT1. The uptake of JBP485 was pH dependent in human intestinal epithelial cells Caco-2. And JBP485 uptake was also significantly inhibited by glycylsarcosine (Gly-Sar, a typical substrate for PEPT1 transporters), JBP923 (a derivative of JBP485), and cephalexin (CEX, a β-lactam antibiotic and a known substrate of PEPT1) in Caco-2 cells. The rate of apical-to-basolateral transepithelial transport of JBP485 was 1.84 times higher than that for basolateral-to-apical transport. JBP485 transport was obviously inhibited by Gly-Sar, JBP923 and CEX in Caco-2 cells. The uptake of JBP485 was increased by verapamil but not by cyclosporin A (CsA) and inhibited by the presence of Zn²⁺ or the toxic metabolite of ethanol, acetaldehyde (AcH) in Caco-2 cells. The in vivo uptake of JBP485 was increased by verapamil and decreased by ethanol in vivo, which was consisted with the in vitro study. PEPT1 mRNA levels were enhanced after exposure of the cells to JBP485 for 24 h, compared to control. In conclusion, JBP485 was actively transported by the intestinal oligopeptide transporter PEPT1. This mechanism is likely to contribute to the rapid absorption of JBP485 by the gastrointestinal tract after oral administration.     

Synthesis and characterization of high affinity inhibitors of the H+/peptide transporter PEPT2.

In this study, we describe the rational synthesis and functional analysis of novel high affinity inhibitors for the mammalian peptide transporter PEPT2. Moreover, we demonstrate which structural properties convert a transported compound into a non-translocated inhibitor. Starting from Lys[Z(NO(2))]-Pro (where Z is benzyloxycarbonyl), which we recently identified as the first competitive high affinity inhibitor of the intestinal peptide transporter PEPT1, a series of different lysine-containing dipeptide derivatives was synthesized and studied for interaction with PEPT2 based on transport competition assays in Pichia pastoris yeast cells expressing PEPT2 heterologously and in renal SKPT cells expressing PEPT2. In addition, the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT2 was used to determine whether the compounds are transported electrogenically or block the uptake of dipeptides. Synthesis and functional analysis of Lys-Lys derivatives containing benzyloxycarbonyl or 4-nitrobenzyloxycarbonyl side chain protections provided a set of inhibitors that reversibly inhibited the uptake of dipeptides by PEPT2 with K(i) values as low as 10 +/- 1 nm. This is the highest affinity of a ligand of PEPT2 ever reported. Moreover, based on the structure-function relationship, we conclude that the spatial location of the side chain amino protecting group in a dipeptide containing a diaminocarbonic acid and its intramolecular distance from the Calpha atom are key factors for the transformation of a substrate into an inhibitor of PEPT2.     

Modified amino acids and peptides as substrates for the intestinal peptide transporter PepT1.

The binding affinities of a number of amino-acid and peptide derivatives by the mammalian intestinal peptide transporter PepT1 were investigated, using the Xenopus laevis expression system. A series of blocked amino acids, namely N-acetyl-Phe (Ac-Phe), phe-amide (Phe-NH2), N-acetyl-Phe-amide (Ac-Phe-NH2) and the parent compound Phe, was compared for efficacy in inhibiting the uptake of the peptide [3H]-D-Phe-L-Gln. In an equivalent set of experiments, the blocked peptides Ac-Phe-Tyr, Phe-Tyr-NH2 and Ac-Phe-Tyr-NH2 were compared with the parent compound Phe-Tyr. Comparing amino acids and derivatives, only Ac-Phe was an effective inhibitor of peptide uptake (Ki = 1.81+/- 0.37 mM). Ac-Phe-NH2 had a very weak interaction with PepT1 (Ki = 16.8+/-5.64 mM); neither Phe nor Phe-NH2 interacted with PepT1 with measurable affinity. With the dipeptide and derivatives, unsurprisingly the highest affinity interaction was with Phe-Tyr (Ki = 0.10+/-0.04 mM). The blocked C-terminal peptide Phe-Tyr-NH2 also interacted with PepT1 with a relatively high affinity (Ki = 0.94+/-0.38 mM). Both Ac-Phe-Tyr and Ac-Phe-Tyr-NH2 interacted weakly with PepT1 (Ki = 8.41+/-0.11 and 9.97+/-4.01 mM, respectively). The results suggest that the N-terminus is the primary binding site for both dipeptides and tripeptides. Additional experiments with four stereoisomers of Ala-Ala-Ala support this conclusion, and lead us to propose that a histidine residue is involved in binding the C-terminus of dipeptides. In addition, a substrate binding model for PepT1 is proposed.     

Preferential recognition of zwitterionic dipeptides as transportable substrates by the high-affinity peptide transporter PEPT2.

We investigated the interaction of rat PEPT2, a high-affinity peptide transporter, with neutral, anionic, and cationic dipeptides using electrophysiological approaches as well as tracer uptake methods. D-Phe-L-Gln (neutral), D-Phe-L-Glu (anionic), and D-Phe-L-Lys (cationic) were used as representative, non-hydrolyzable, dipeptides. All three dipeptides induced H+-dependent inward currents in Xenopus laevis oocytes heterologously expressing rat PEPT2. The H+:peptide stoichiometry was 1:1 in each case. A simultaneous measurement of radiolabeled dipeptide influx and charge transfer in the same oocyte indicated a transfer of one net positive charge into the oocyte per transfer of one peptide molecule irrespective of the charged nature of the peptide. We conclude that the zwitterionic peptides are preferentially recognized by PEPT2 as transportable substrates and that the proton/peptide stoichiometry is 1 for the transport process.     

Identification of Drosophila Yin and PEPT2 as Evolutionarily Conserved Phagosome-associated Muramyl Dipeptide Transporters

NOD2 (nucleotide-binding oligomerization domain containing 2) is an important cytosolic pattern recognition receptor that activates NF-κB and other immune effector pathways such as autophagy and antigen presentation. Despite its intracellular localization, NOD2 participates in sensing of extracellular microbes such as Staphylococcus aureus. NOD2 ligands similar to the minimal synthetic ligand muramyl dipeptide (MDP) are generated by internalization and processing of bacteria in hydrolytic phagolysosomes. However, how these derived ligands exit this organelle and access the cytosol to activate NOD2 is poorly understood. Here, we address how phagosome-derived NOD2 ligands access the cytosol in human phagocytes. Drawing on data from Drosophila phagosomes, we identify an evolutionarily conserved role of SLC15A transporters, Drosophila Yin and PEPT2, as MDP transporters in fly and human phagocytes, respectively. We show that PEPT2 is highly expressed by human myeloid cells. Ectopic expression of both Yin and PEPT2 increases the sensitivity of NOD2-dependent NF-κB activation. Additionally, we show that PEPT2 associates with phagosome membranes. Together, these data identify Drosophila Yin and PEPT2 as evolutionarily conserved phagosome-associated transporters that are likely to be of particular importance in delivery of bacteria-derived ligands generated in phagosomes to cytosolic sensors recruited to the vicinity of these organelles.