The renal type H+/peptide symporter PEPT2: structure-affinity relationships

Summary. The H⁺/peptide cotransporter PEPT2 is expressed in a variety of organs including kidney, lung, brain, mammary gland, and eye. PEPT2 substrates are di- and tripeptides as well as peptidomimetics, such as β-lactam antibiotics. Due to the presence of PEPT2 at the bronchial epithelium, the aerosolic administration of peptide-like drugs might play a major role in future treatment of various pulmonary and systemic diseases. Moreover, PEPT2 has a significant influence on the in vivo disposition and half-life time of peptide-like drugs within the body, particularly in kidney and brain. PEPT2 is known to have similar but not identical structural requirements for substrate recognition and transport compared to PEPT1, its intestinal counterpart. In this review we compiled available affinity constants of 352 compounds, measured at different mammalian tissues and expression systems and compare the data whenever possible with those of PEPT1.     

The contribution of proline residues to protein stability is associated with isomerization equilibrium in both unfolded and folded states

It has long been understood that the proline residue has lower configurational entropy than any other amino acid residue due to pyrrolidine ring hindrance. The peptide bond between proline and its preceding amino acid (Xaa-Pro) typically exists as a mixture of cis- and trans-isomers in the unfolded protein. Cis–trans isomerization of Xaa-Pro peptide bonds are infrequent, but still occur in folded proteins. Therefore, the effects of the cis–trans isomerization equilibrium in both unfolded and folded states should be taken into account when estimating the stability contribution of a specific proline residue. In order to study the stability contribution of the four proline residues to the hyperthermophilic protein Ssh10b, in this work, we expressed and purified a series of Pro→Ala mutants of Ssh10b, and performed correlative unfolding experiments in detail. We proposed a new unfolding model including proline isomerization. The model predicts that the contribution of a proline residue to protein stability is associated with the thermodynamic equilibrium between cis- and trans-isomers both in the unfolded and folded states, agreeing well with the experimental results.     

Electrophysiological characteristics of the proton-coupled peptide transporter PEPT2 cloned from rat brain

We have cloned a peptide transporter from rat brain and found itto be identical to rat kidney PEPT2. In the present study wecharacterize the transport function of the rat brain PEPT2, withspecial emphasis on electrophysiological properties and interaction withN-acetyl-L-aspartyl-L-glutamate(NAAG). When heterologously expressed in HeLa cells and in SK-N-SHcells, PEPT2 transports several dipeptides but not free amino acids inthe presence of a proton gradient. NAAG competes with other peptidesfor the PEPT2-mediated transport process. When PEPT2 is expressed inXenopus laevis oocytes, substrate-induced inward currents are detectable with dipeptides ofdiffering charge in the presence of a proton gradient. Proton activation kinetics are similar for differently charged peptides. NAAGis a transportable substrate for PEPT2, as evidenced by NAAG-induced currents. The Hill coefficient for protons for the activation of thetransport of differently charged peptides, including NAAG, is 1. Although the peptide-to-proton stoichiometry for negatively chargedpeptides is 1, the transport nonetheless is associated with transfer ofpositive charge into the oocyte, as indicated by peptide-induced inwardcurrents.

     

Functional characterisation of LKT1, a K+ uptake channel from tomato root hairs, and comparison with the closely related potato inwardly rectifying K+ channel SKT1 after expression in Xenopus oocytes

A cDNA encoding a novel inwardly rectifying potassium (K⁺ _in) channel, LKT1, was cloned from a root-hair-specific cDNA library of tomato (Lycopersicon esculentum Mill.). The LKT1 mRNA was shown to be most strongly expressed in root hairs by Northern blot analysis. The LKT1 channel is a member of the AKT family of K⁺ _in channels previously identified in Arabidopsis thaliana (L.) Heynh. and potato (Solanum tuberosum L.). Moreover, LKT1 is closely related (97% identical amino acids) to potato SKT1. An electrophysiological comparison of the two channels should therefore assist the identification of possible molecular bases for functional differences. For this comparison, both channels were functionally expressed and electrophysiologically characterised within the same expression system, i.e. Xenopus laevis oocytes. Voltage-clamp measurements identified LKT1 as a K⁺-selective inward rectifier which activates with slow kinetics upon hyperpolarising voltage pulses to potentials more negative than −50 mV. The activation potential of LKT1 is shifted towards positive potentials with respect to SKT1 which might be due to single amino acid exchanges in the rim of the channel's pore region or in the S4 domain. Like SKT1, LKT1 reversibly activated upon shifting the external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K⁺ _in channels. The pharmacological inhibitor Cs⁺, applied externally, inhibited K⁺ _in currents mediated by LKT1 and SKT1 half-maximally with a concentration (IC₅₀) of 21 μM and 17 μM, respectively. In conclusion, LKT1 may serve as a low-affinity influx pathway for K⁺ into root hair cells. Comparison of homologous K⁺ _in rectifiers from different plant species expressed in the same heterologous system allows conclusions to be drawn in respect to structure-function relationships. Received: 3 August 1999 / Accepted: 2 November 1999     

Amino acid transport in the renal proximal tubule

Summary. In the kidney the proximal tubule is responsible for the uptake of amino acids. This occurs via a variety of functionally and structurally different amino acid transporters located in the luminal and basolateral membrane. Some of these transporters show an ion-dependence (e.g. Na⁺, Cl⁻ and K⁺) or use an H⁺-gradient to drive transport. Only a few amino acid transporters have been cloned or functionally characterized in detail so far and their structure is known, while little is known about a majority of amino acid transporters. Only few attempts have been untertaken looking at the regulation of amino acid transport. We summarized more recent information on amino acid transport in the renal proximal tubule emphasizing functional and regulatory aspects. Received August 8, 1999; Accepted April 20, 2000     

Transport of Charged Dipeptides by the Intestinal H+/Peptide Symporter PepT1 Expressed in Xenopus laevis Oocytes

The cloned intestinal peptide transporter is capable of electrogenic H⁺-coupled cotransport of neutral di- and tripeptides and selected peptide mimetics. Since the mechanism by which PepT1 transports substrates that carry a net negative or positive charge at neutral pH is poorly understood, we determined in Xenopus oocytes expressing PepT1 the characteristics of transport of differently charged glycylpeptides. Transport function of PepT1 was assessed by flux studies employing a radiolabeled dipeptide and by the two-electrode voltage-clamp-technique. Our studies show, that the transporter is capable of translocating all substrates by an electrogenic process that follows Michaelis Menten kinetics. Whereas the apparent K_0.5 value of a zwitterionic substrate is only moderately affected by alterations in pH or membrane potential, K_0.5 values of charged substrates are strongly dependent on both, pH and membrane potential. Whereas the affinity of the anionic dipeptide increased dramatically by lowering the pH, a cationic substrate shows only a weak affinity for PepT1 at all pH values (5.5–8.0). The driving force for uptake is provided mainly by the inside negative transmembrane electrical potential. In addition, affinity for proton interaction with PepT1 was found to depend on membrane potential and proton binding subsequently affects the substrate affinity. Furthermore, our studies suggest, that uptake of the zwitterionic form of a charged substrate contributes to overall transport and that consequently the stoichiometry of the flux-coupling ratios for peptide: H⁺/H₃O⁺ cotransport may vary depending on pH. Received: 19 August 1996/Revised: 10 October 1996     

Polyamines are absorbed through a y+ amino acid carrier in rat intestinal epithelial cells

Summary. Due to the similarity in transport characteristics of polyamines and the y⁺ basic amino acid system, we hypothesized that both substrates could be moving through a common carrier site. Competitive and cross inhibition experiments in intestinal epithelial cells revealed the possibility of a common transport site. N-ethylmalemide (NEM) inhibited both lysine and putrescine transport, confirming that both were carried by a y⁺ transporter. Overexpressing the y⁺ transporter CAT-1 in a polyamine transport-deficient cell line, CHO-MG, did not reconstitute polyamine-transport. Thus, polyamines are not traveling through CAT-1. To determine if lysine is carried by a polyamine transport site, an antizyme-overexpressing cell line was used. Antizyme overexpression decreased polyamine uptake by 50%; in contrast, lysine transport was unaffected. Therefore, lysine is not traveling through a polyamine transport site. It appears that polyamines and lysine are likely traveling through a common unknown y⁺ transport site.     

Two Novel Toxins from the Venom of the Scorpion Pandinus imperator Show that the N-terminal Amino Acid Sequence is Important for their Affinities towards Shaker B K+ Channels

Two novel peptides were purified from the venom of the scorpion Pandinus imperator, and were named Pi2 and Pi3. Their complete primary structures were determined and their blocking effects on Shaker B K⁺ channels were studied. Both peptides contain 35 amino acids residues, compacted by three disulfide bridges, and reversibly block the Shaker B K⁺ channels. They have only one amino acid changed in their sequence, at position 7 (a proline for a glutamic acid). Whereas peptide Pi2, containing the Pro7, binds the Shaker B K⁺ channels with a K _d of 8.2 nm, peptide Pi3 containing the Glu7 residue has a much lower affinity of 140 nm. Both peptides are capable of displacing the binding of ¹²⁵I-noxiustoxin to brain synaptosome membranes. Since these two novel peptides are about 50% identical to noxiustoxin, the present results support previous data published by our group showing that the amino-terminal region of noxiustoxin, and also the amino-terminal sequence of the newly purified homologues: Pi2, and Pi3, are important for the recognition of potassium channels. Received: 13 November 1995/Revised: 11 March 1996     

Expression and function of PEPT2 during transdifferentiation of alveolar epithelial cells

Aims

The purpose of this study was to clarify the expression and function of peptide transporter 2 (PEPT2) in primary cultured alveolar type II epithelial cells and in transdifferentiated type I-like cells.

Main methods

Real-time PCR analysis, uptake study of [³H]Gly-Sar, and immunostaining were performed in alveolar epithelial cells.

Key findings

The expression of PEPT2 mRNA in type II cells isolated from rat lungs was highest at day 0, and decreased rapidly during culture of the cells. In accordance with this change, PEPT2 activity estimated as cefadroxil-sensitive [³H]Gly-Sar uptake also decreased along with transdifferentiation. The expression of PEPT2 protein in type II cells was confirmed by immunostaining and Western blot analysis. The uptake of [³H]Gly-Sar in type II cells was time- and pH-dependent. In contrast, minimal time-dependence and no pH-dependence of [³H]Gly-Sar uptake were observed in type I-like cells. The maximal [³H]Gly-Sar uptake was observed at pH 6.0, and the uptake decreased at higher pHs in type II cells. The uptake of [³H]Gly-Sar in type II cells was inhibited by cefadroxil in a concentration-dependent manner, the IC₅₀ value being 4.3 μM. On the other hand, no significant inhibition by cefadroxil was observed in type I-like cells. In addition, [³H]Gly-Sar uptake in type II cells was saturable, the Km value being 72.0 μM.

Significance

PEPT2 is functionally expressed in alveolar type II epithelial cells, but the expression decreases along with transdifferentiation, and PEPT2 would be almost completely lost in type I cells.     

Prediction of Xaa-Pro peptide bond conformation from sequence and chemical shifts

We present a program, named Promega, to predict the Xaa-Pro peptide bond conformation on the basis of backbone chemical shifts and the amino acid sequence. Using a chemical shift database of proteins of known structure together with the PDB-extracted amino acid preference of cis Xaa-Pro peptide bonds, a cis/trans probability score is calculated from the backbone and ¹³C^β chemical shifts of the proline and its neighboring residues. For an arbitrary number of input chemical shifts, which may include Pro-¹³C^γ, Promega calculates the statistical probability that a Xaa-Pro peptide bond is cis. Besides its potential as a validation tool, Promega is particularly useful for studies of larger proteins where Pro-¹³C^γ assignments can be challenging, and for on-going efforts to determine protein structures exclusively on the basis of backbone and ¹³C^β chemical shifts.     

Importance of proline and other amino acids during honeybee flight

Summary. The levels of proline and other amino acids in the haemolymph and other body parts of honeybee foragers were investigated by HPLC analysis. The concentrations of proline in the blood of glucose-fed or -injected bees finishing their exhaustive tethered flights on a roundabout were significantly reduced compared to bees that were fed and rested for one hour. This indicates some utilization of proline during flight metabolism. The levels of essential amino acids and of the sum of all amino acids except proline remained roughly constant, indicating that the decrease of proline did not result from a changed haemolymph volume. ¹⁴C-labelled proline was injected into bees either shortly before starting their flight or before a resting period of equal duration in an incubator at the same temperature. Bees that rested had incorporated more proline into thorax body protein, and less of the labelled substance was unrecovered ("missing") and considered to be respired or less probably defecated. If the entire amount of missing ¹⁴C-proline is regarded as exhaled, the oxidative breakdown of proline reached higher levels after flight than in rested bees. This is another hint that proline is utilized during flight. Usually the exhaled amount did not exceed 10 μg proline in half an hour of flight. Although our data indicate involvement of proline in flight metabolism, the amount metabolized is low compared to the utilization of carbohydrates. Received December 5, 1998, Accepted February 1, 1999     

Membrane Topology Motifs in the SGLT Cotransporter Family

Homologues of the Na⁺/glucose cotransporter, the SGLT family, include sequences of mammalian, eubacterial, yeast, insect and nematode origin. The cotransported substrates are sugars, inositol, proline, pantothenate, iodide, urea and undetermined solutes. It is reasonable to expect that the SGLT family members share a similar or identical topology of membrane spanning elements, by virtue of their common ancestry and similar coupling of solute transport to downhill sodium flux. Here we examine their membrane topologies as deduced from diverse analyses of their primary sequences, and from their sequence correlations with the experimentally determined topology of the human Na⁺/glucose cotransporter SGLT1. Our analyses indicate that all family members share a common core of 13 transmembrane helices, but that some, like SGLT1 itself, have one additional span appended to the C-terminus, and still others, two. One bacterial member incorporates an additional span at the N-terminus. Sequence comparisons indicative of common ancestry of the SGLT and the [Na⁺+ Cl⁻] transporter families are introduced, and evaluated in light of their topologies. New evidence concerning the previously asserted common ancestry of SGLT1 and an N-acetylglucosamine permease of the bacterial phosphotransferase system is considered. Finally, we analyze observations which lead us to conjecture that the experimental strategy most commonly employed to reveal the topology of bacterial transporters (i.e., the fusion of reporter enzymes such as phoA alkaline phosphatase, beta-lactamase or beta-galactosidase, to progressively C-truncated fragments of the transporter) has often instead so perturbed local topology as to have entirely missed pairs of adjacent membrane spans. Received: 18 May 1996     

A method for simple identification of signature peptides derived from polyUb-K48 and K63 by MALDI-TOF MS and chemically assisted MS/MS fragmentation

Summary. A simple method is described to identify signature peptides derived from polyubiquitin (polyUb) chains. The method is based on MALDI-TOF MS/MS analysis after chemically assisted fragmentation, and works on peptides isolated from polyacrylamide gels. PolyUb chains branched at K48 and K63 were chosen as models for Ub-protein conjugates. They were resolved by SDS-PAGE, and their tryptic peptides (in-gel-trypsinolysis) derivatized with 3-sulfopropinic acid NHSester to obtain chemically assisted fragmentation during the MS/MS analysis. PolyUb-K63 produced a single peptide identified as ⁵⁵TLSDYNIQK⁶³ (GG)ESTLHLVLR⁷². PolyUb-K48 produced two branched signature peptides identified as ⁴³LIFAGK⁴⁸(GG)QLEDGR⁵⁴ and ⁴³LIFAGK⁴⁸(LRGG)QLEDGR⁵⁴. The recovery of signature peptide with LRGG as branched chain underscores the need to take limited proteolysis into account in the search for detection of ubiquitinated peptides in proteomics studies. In conclusion, a simple method has been described allowing the identification of signature peptides, which are diagnostic markers of the majority of polyUb-conjugated proteins. In principle, the method should be applicable also for other more rare signature peptides.     

Free radical scavenging potential of L-proline: evidence from in vitro assays

Summary. An assessment of the potential of proline to scavenge free radicals was made in a couple of in vitro assay systems, namely graft co-polymerization and autooxidation of pyrogallol. Both these assays are essentially dependent upon free radical mechanisms. Graft co-polymerization involved a ceric (Ce⁴⁺) ion- or γ-radiation-induced grafting of methyl acrylate (MA) onto a cellulose backbone. The degree of grafting, measured gravimetrically, was taken as a measure of free radical generation. The γ-radiation-dependent grafting was far greater than that due to Ce⁴⁺ ions. Inclusion of proline in the assay, irrespective of the initiator used, led to suppression of grafting in a concentration-dependent manner indicating the ability of proline to scavenge free radicals. The γ-radiation-dependent grafting was also suppressed by hydroquinone and glutathione but not by ascorbate, glycine and spermine. In contrast to graft co-polymerization, proline did not inhibit the autooxidation of pyrogallol, a reaction involving superoxide radical generation. A subset of data constitutes an evidence for the ability of proline to scavenge free radicals in vitro. It is implied by extension that free proline, known to accumulate in plant tissues during abiotic stresses, would contribute to scavenging of surplus free radicals produced under a variety of abiotic stresses. Authors’ address: Shanti S. Sharma, Department of Biosciences, Himachal Pradesh University, Shimla 171 005, India     

Substrate specificity of the amino acid transporter PAT1

Summary. The proton coupled amino acid transporter PAT1 expressed in intestine, brain, and other organs accepts L- and D-proline, glycine, and L-alanine but also pharmaceutically active amino acid derivatives such as 3-amino-1-propanesulfonic acid, L-azetidine-2-carboxylic acid, and cis-4-hydroxy-D-proline as substrates. We systematically analyzed the structural requirements for PAT1 substrates by testing 87 amino acids, proline homologs, indoles, and derivatives. Affinity data and effects on membrane potential were determined using Caco-2 cells. For aliphatic amino acids, a blocked carboxyl group, the distance between amino and carboxyl group, and the position of the hydroxyl group are affinity limiting factors. Methylation of the amino group enhances substrate affinity. Hetero atoms in the proline template are well tolerated. Aromatic α-amino acids display low affinity. PAT1 interacts strongly with heterocyclic aromatic acids containing an indole scaffold. The structural requirements of PAT1 substrates elucidated in this study will be useful for the development of prodrugs.     

Relationship between NO synthesis, arginine transport, and intracellular arginine levels in vascular smooth muscle cells

Summary. The present study was designed to evaluate the relevance of arginine transport in nitric oxide (NO) synthesis in vascular smooth muscle cells. For this purpose, NO synthesis and arginine transport (system B^0,+ and y⁺) were evaluated in cells treated with IL-1β or angiotensin II (Ang II). In addition, the effects of 5 mM lysine and glutamine, competitive inhibitors of systems y⁺ and B^0,+ respectively, were examined. L-arginine transport was estimated with ³H-labelled arginine and NO was determined with the Griess reagent. These studies were done in control conditions, arginine-starved cells, and in cells incubated in media containing 10 mM arginine. Our data indicate that induction of NO biosynthesis by IL-1β depends on external arginine when cells are arginine-depleted for 24 hours. The concentration of arginine producing half maximal activation of NO synthesis in arginine-depleted cells ([arginine]_i < 10 μM) was 41.1 ± 18 μM. By contrast, in normal culture conditions, NO synthesis occurred independently of arginine transport. Neither 5 mM lysine or glutamine which abolished arginine transport through systems y⁺ and B^0,+, respectively, reduced nitrite release in cells incubated in normal media. This suggests that the relevance of arginine uptake to NO synthesis depends on the status of intracellular arginine pools. Intracellular arginine concentrations were not affected by the stimulation of NO production using IL-1β or its inhibition using Ang II, but were markedly reduced by arginine starvation for 48 h. Aspartate levels were also reduced by arginine-depletion, but were not affected in cells incubated with 10 mM arginine. By contrast, glutamate levels were reduced in arginine-starved cells and were increased in cells incubated in arginine-supplemented medium. Ornithine levels were markedly increased by arginine supplementation. Altogether, these findings indicate that NO synthesis is normally independent of membrane transport. However in arginine-depleted cells, membrane transport is essential for NO synthesis. It is concluded that arginine transport is required for the long-term maintenance of intracellular arginine pools. Received February 7, 1999; Accepted June 21, 1999     

Concise preparation of <Emphasis Type="Italic">N</Emphasis><Superscript>α</Superscript>-Fmoc-<Emphasis Type="Italic">N</Emphasis><Superscript>ɛ</Superscript>-(Boc,methyl)-lysine and its application in the synthesis of site-specifically lysine monomethylated peptide

Summary. A concise preparation of N ^α-Fmoc-N ^ɛ-(Boc, methyl)-lysine and its application in the synthesis of site-specifically lysine monomethylated peptide is described. N ^α-Fmoc-N ^ɛ-(Boc, methyl)-lysine is obtained, via consecutive reductive benzylation and reductive methylation in a one-pot reaction, followed by debenzylation through catalytic hydrogenolysis and Boc protection in another one-pot reaction. A peptide containing monomethylated lysine is successfully synthesized by incorporating N ^α-Fmoc-N ^ɛ-(Boc, methyl)-lysine as a building block via solid-phase peptide synthesis.     

Evidence for Negative Charge in the Conduction Pathway of the Cardiac Ryanodine Receptor Channel Provided by the Interaction of K+ Channel N-type Inactivation Peptides

We have investigated the interaction of two peptides (ShB — net charge +3 and ShB:E12KD13K — net charge +7) derived from the NH₂-terminal domain of the Shaker K⁺ channel with purified, ryanodine-modified, cardiac Ca²⁺-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a single-site binding scheme with 50% saturation occurring at 5.92 ± 1.06 μm for ShB and 0.59 ± 0.14 nm for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 ± 0.4 sec⁻¹μm ⁻¹ for ShB and 2000 ± 200 sec⁻¹μm ⁻¹ for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied potential with the bulk of the voltage-dependence residing in K_off. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As is the case with inactivation and block of K⁺ channels, this is mediated by a large increase in K_on. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites which will contribute to the ion handling properties of this channel. Received: 15 December 1997/Revised: 13 March 1998     

Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures

Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv. using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (¹⁵NH₄)₂SO₄ and K¹⁵NO₃. By using two-dimensional ¹⁵N-¹H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences, an enhanced resolution of the incorporation of ¹⁵N label into a range of compounds could be detected. Thus, in addition to the amino acids normally observed in one-dimensional ¹⁵N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline. Furthermore, it was found that the peak normally assigned to the non-protein amino-acid γ-aminobutyric acid in the one-dimensional ¹⁵N NMR spectrum was resolved into a several components. A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into γ-aminobutyric acid, N-acetylputrescine. The occurrence of ¹⁵N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored. For the first time, labelling of the peptide glutathione and of the nucleotide uridine could be seen. Received: 29 March 1999 / Accepted: 15 July 1999