Frog oocytes to unveil the structure and supramolecular organization of human transport proteins

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

The FGFRL1 receptor is shed from cell membranes, binds fibroblast growth factors (FGFs), and antagonizes FGF signaling in Xenopus embryos

FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs.

Monepantel allosterically activates DEG-3/DES-2 channels of the gastrointestinal nematode Haemonchus contortus

Monepantel is the first drug of a new family of anthelmintics, the amino acetonitrile derivatives (AAD), presently used to treat ruminants infected with gastrointestinal nematodes such as Haemonchus contortus. Monepantel shows an excellent tolerability in mammals and is active against multidrug-resistant parasites, indicating that its molecular target is absent or inaccessible in the host and is different from those of the classic anthelmintics. Genetic approaches with mutant nematodes have suggested acetylcholine receptors of the DEG-3 subfamily as the targets of AADs, an enigmatic clade of ligand-gated ion channels that is specific to nematodes and does not occur in mammals. Here we demonstrate direct interaction of monepantel, its major active metabolite monepantel sulfone, and other AADs with potential targets of the DEG-3 subfamily of acetylcholine receptors. H. contortus DEG-3/DES-2 receptors were functionally expressed in Xenopus laevis oocytes and were found to be preferentially activated by choline, to permeate monovalent cations, and to a smaller extent, calcium ions. Although monepantel and monepantel sulfone did not activate the channels by themselves, they substantially enhanced the late currents after activation of the channels with choline, indicating that these AADs are type II positive allosteric modulators of H. contortus DEG-3/DES-2 channels. It is noteworthy that the R-enantiomer of monepantel, which is inactive as an anthelmintic, inhibited the late currents after stimulation of H. contortus DEG-3/DES-2 receptors with choline. In summary, we present the first direct evidence for interaction of AADs with DEG-3-type acetylcholine receptors and discuss these findings in the context of anthelmintic action of AADs.

Reduced pretreatment ovarian reserve in premenopausal female patients with Hodgkin lymphoma or non-Hodgkin-lymphoma--evaluation by using antimüllerian hormone and retrieved oocytes

To study whether the ovarian reserve in female lymphoma patients is already reduced before the start of chemotherapy.

Ovarian stimulation to cryopreserve fertilized oocytes in cancer patients can be started in the luteal phase

OBJECTIVE: To analyze if oocytes can be obtained in all patients before cancer treatment within 2 weeks by initiating ovarian stimulation during the follicular or luteal phase. DESIGN: Prospective controlled multicenter trial. SETTING: Four university-based centers. PATIENT(S): Forty cancer patients before chemotherapy. INTERVENTION(S): Twenty-eight patients were stimulated with gonadotropins in the follicular phase (group I). In 12 patients (group II), ovarian stimulation was initiated in the luteal phase, and these received GnRH antagonists and recombinant FSH. In 14 patients, 143 oocytes were further processed for fertilization by intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Number of oocytes aspirated after ovarian stimulation, cumulative FSH/hMG dosage, viability and maturity of oocytes, and fertilization rate by ICSI. RESULT(S): Patients in group I (age 27.6 +/- 4.9 yrs) were stimulated on average for 10.6 days, and patients in group II (age 31.2 +/- 5.7 yrs) for 11.4 days. Total amount of FSH was on average 2,255 IU (I) and 2,720 IU (II) per patient. Average and median numbers of aspirated oocytes were, respectively, 13.1 and 11.5 (I) versus 10.0 and 8.5 (II); 83.7% (I) and 80.4% (II) of the oocytes were mature and viable and could be treated by ICSI. Fertilization rate was 61.0% (I) versus 75.6% (II). CONCLUSION(S): This pilot study suggests that oocytes can be obtained before cancer treatment efficiently irrespective of the phase of the menstrual cycle.

Expression, purification and low-resolution structure of human vitamin C transporter SVCT1 (SLC23A1)

Expression and purification of human membrane proteins for structural studies represent a great challenge. This is because micro- to milligram amounts of pure isolated protein are required. To this aim, we successfully expressed the human vitamin C transporter-1 (hSVCT1; SLC23A1) in Xenopus laevis oocytes and isolated highly pure protein in microgram amounts. Recombinant hSVCT1 was functional when expressed in oocytes and glycosylated. Structural analysis of purified hSVCT1 by transmission electron microscopy and single particle analysis unveiled its shape, dimensions and low-resolution structure as well as the existence of a major monomeric and minor dimeric population. Chemical crosslinking of isolated oocyte membranes containing expressed hSVCT1 indicated similar oligomeric states of hSVCT1 in lipid bilayers. This work reports the first purification and structural analysis of a human SVCT protein and opens the way for future functional and structural studies using purified hSVCT1.

AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis

For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids. A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant. AtPTR1 mediated growth of S. cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin. The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions. AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond. However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1. At pH 5.5 AtPTR1 had an apparent lower affinity (K-0.5 = 416 mum) for Ala-Asp compared with Ala-Ala (K-0.5 = 54 mum) and Ala-Lys (K-0.5 = 112 mum). Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane. In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides.

Trypanosoma brucei eflornithine transporter AAT6 is a low-affinity low-selective transporter for neutral amino acids

Amino acid transporters are crucial for parasite survival since the cellular metabolism of parasitic protozoa depends on the uptake of exogenous amino acids. Amino acid transporters are also of high pharmacological relevance because they may mediate uptake of toxic amino acid analogues. In the present study we show that the eflornithine transporter AAT6 from Trypanosoma brucei (TbAAT6) mediates growth on neutral amino acids when expressed in Saccharomyces cerevisiae mutants. The transport was electrogenic and further analysed in Xenopus laevis oocytes. Neutral amino acids, proline analogues, eflornithine and acivicin induced inward currents. For proline, glycine and tryptophan the apparent affinities and maximal transport rates increased with more negative membrane potentials. Proline-induced currents were dependent on pH, but not on sodium. Although proline represents the primary energy source of T. brucei in the tsetse fly, down-regulation of TbAAT6-expression by RNAi showed that in culture TbAAT6 is not essential for growth of procyclic form trypanosomes in the presence of glucose or proline as energy source. TbAAT6-RNAi lines of both bloodstream and procyclic form trypanosomes showed reduced susceptibility to eflornithine, whereas the sensitivity to acivicin remained unchanged, indicating that acivicin enters the cell by more than one transporter

Placental Uric Acid Transport System: Glucose Transporter 9 (SLC2A9)

Placental Uric Acid Transport System: Glucose Transporter 9 (SLC2A9). INTRODUCTION: Pre-eclampsia, a pregnancy-specific disease, contributes substantially to perinatal morbidity and mortality of both the mother and her child. Pre-eclampsia is often associated with high maternal urate serum levels, which in turn has been shown to play a role in the pathogenesis of this disease. The aim of this study was to investigate the glucose transporter GLUT9-mediated placental uric acid transport system. METHODS: In this study western blot, immunofluorescence techniques as well as a transepithelial transport (Transwell) model were used to assess GLUT9 protein expression and, respectively, uric acid transport activity. Electrophysiological techniques and transmission electron microscopy (TEM) were used to characterize the properties and the structure of GLUT9. RESULTS: Uric acid is transported across a BeWo choriocarcinoma cell monolayer with 530 pmol/min. We could successfully overexpress and for the first time purify the GLUT9b isoform using the Xenopus laevis oocytes expression system. Chloride seems to modulate the urate transport system. TEM revealed that GLUT9b isoform is present as monomer and dimmer in the Xenopus laevis overexpression model. A class average of all the particles allowed us to develop a first model of human GLUT9b structure, which was derived from the published crystal structure of the bacterial homologue of GLUT1-4. CONCLUSIONS: In vitro the “materno-fetal” transport of uric acid is slow indicating that in vivo the fetus might be protected from short-term fluctuations of maternal urate serum levels. The low-resolution structure obtained from TEM validates the proposed homology model regarding the structure of human GLUT9b. In ongoing studies this model is used to perform virtual screening to identify novel modulators of the urate transport system enabling the development of novel therapies in pregnancy complications.

Rapid method to express and purify human membrane protein using the Xenopus oocyte system for functional and low-resolution structural analysis.

Progress toward elucidating the 3D structures of eukaryotic membrane proteins has been hampered by the lack of appropriate expression systems. Recent work using the Xenopus oocyte as a novel expression system for structural analysis demonstrates the capability of providing not only the significant amount of protein yields required for structural work but also the expression of eukaryotic membrane proteins in a more native and functional conformation. There is a long history using the oocyte expression system as an efficient tool for membrane transporter and channel expression in direct functional analysis, but improvements in robotic injection systems and protein yield optimization allow the rapid scalability of expressed proteins to be purified and characterized in physiologically relevant structural states. Traditional overexpression systems (yeast, bacteria, and insect cells) by comparison require chaotropic conditions over several steps for extraction, solubilization, and purification. By contrast, overexpressing within the oocyte system for subsequent negative-staining transmission electron microscopy studies provides a single system that can functionally assess and purify eukaryotic membrane proteins in fewer steps maintaining the physiological properties of the membrane protein.

Isolation of an active gene and of two pseudogenes for mouse U7 small nuclear RNA

Three U7 RNA-related sequences were isolated from mouse genomic DNA libraries. Only one of the sequences completely matches the published mouse U7 RNA sequence, whereas the other two apparently represent pseudogenes. The matching sequence represents a functional gene, as it is expressed after microinjection into Xenopus laevis oocytes. Sequence variations of the conserved cis-acting 5' and 3' elements of U RNA genes may partly explain the low abundance of U7 RNA.

Primary structure and evolutionary relationship between the adult alpha-globin genes and their 5'-flanking regions of Xenopus laevis and Xenopus tropicalis

To investigate the evolution of globin genes in the genus Xenopus, we have determined the primary structure of the related adult alpha I- and alpha II-globin genes of X. laevis and of the adult alpha-globin gene of X. tropicalis, including their 5'-flanking regions. All three genes are comprised of three exons and two introns at homologous positions. The exons are highly conserved and code for 141 amino acids. By contrast, the corresponding introns vary in length and show considerable divergence. Comparison of 900 bp of the 5'-flanking region revealed that the X. tropicalis gene contains a conserved proximal 310-bp promoter sequence, comprised of the canonical TATA and CCAAT motifs at homologous positions, and five conserved elements in the same order and at similar positions as previously shown for the corresponding genes of X. laevis. We therefore conclude that these conserved upstream elements may represent regulatory sequences for cell-specific regulation of the adult Xenopus globin genes.

Conserved sequences and cell-specific DNase I hypersensitive sites upstream from the co-ordinately expressed alpha I- and alpha II-globin genes of Xenopus laevis

The globin gene family of Xenopus laevis comprises pairs of closely related genes that are arranged in two clusters, each pair of genes being co-ordinately and stage-specifically expressed. To get information on putative regulatory elements, we compared the DNA sequences and the chromatin conformation 5' to the co-ordinately expressed adult alpha-globin genes. Sequence analysis revealed a relatively conserved region from the cap site up to position -289, and further upstream seven distinct boxes of homology, separated by more diverged sequences or deletions/insertions. The homology boxes comprise 22 to 194 base-pairs showing 78 to 95% homology. Analysis of chromatin conformation showed that DNase I preferentially cuts the upstream region of both genes at similar positions, 5' to the T-A-T-A and the C-C-A-A-T boxes, only in chromatin of adult erythroblasts and erythrocytes, where adult globin genes are expressed, but not in chromatin of adult liver cells or larval erythrocytes, where these genes are silent. This suggests that cell- and stage-specific activation of these genes coincides with specific changes in chromatin conformation within the proximal upstream region. No difference was found in the nucleotide sequence within the DNase I hypersensitive region proximal to the adult alpha 1-globin gene in DNA from embryonic cells, in which this gene is inactive, and adult erythrocytes, expressing this gene.

The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous GltPh coordinates l-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na(+) over Li(+). S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for l-aspartate over d-aspartate and l-glutamate, and lost their selectivity for Na(+) over Li(+). Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.