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PubMed1994

Heterologous expression of delta F508 CFTR results in decreased sialylation of membrane glycoconjugates.

The cystic fibrosis transmembrane conductance regulator (CFTR) is commonly mutated in cystic fibrosis to the delta F508 CFTR. CFTR has been shown to function as a adenosine 3',5'-cyclic monophosphate-dependent Cl- channel at the cell surface, and there is evidence to suggest that CFTR may also have a role in transmembrane Cl- conductance in intracellular membrane compartments. Studies using cells from cystic fibrosis patients or heterologous expression systems have demonstrated that defective Cl- conductance at the cell surface and defective acidification of the Golgi compartment are associated with the presence of mutant forms of CFTR. It is possible that mutation of CFTR could also result in altered Golgi function, consistent with reports of changes in the glycosylation of cell surface and secreted glycoproteins in cystic fibrosis. Glycosylation of cell surface glycoproteins, particularly levels of sialylation, may also be related to the increased binding of Pseudomonas to cystic fibrosis cells. The current study was undertaken to compare the sialylation of cell surface glycoconjugates in heterologous cells overexpressing normal CFTR or delta F508 CFTR. The presence of sialylated residues on cells was assessed by the surface binding of the specific lectins, wheat germ agglutinin and elderberry bark lectin. A fluorescent cholera toxin B subunit probe was used to measure surface binding to sialylated gangliosides in transfected cells. Our studies show that cells lacking CFTR and cells expressing normal CFTR have unaltered levels of sialylation. In contrast, cells expressing the delta F508 CFTR have significantly decreased amounts of sialylated glycoproteins and gangliosides on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2000

Transport of valganciclovir, a ganciclovir prodrug, via peptide transporters PEPT1 and PEPT2.

In clinical trials, valganciclovir, the valyl ester of ganciclovir, has been shown to enhance the bioavailability of ganciclovir when taken orally by patients with cytomegalovirus infection. We investigated the role of the intestinal peptide transporter PEPT1 in this process by comparing the interaction of ganciclovir and valganciclovir with the transporter in different experimental systems. We also studied the interaction of these two compounds with the renal peptide transporter PEPT2. In cell culture model systems using Caco-2 cells for PEPT1 and SKPT cells for PEPT2, valganciclovir inhibited glycylsarcosine transport mediated by PEPT1 and PEPT2 with K(i) values (inhibition constant) of 1.68+/-0.30 and 0.043+/- 0.005 mM, respectively. The inhibition by valganciclovir was competitive in both cases. Ganciclovir did not interact with either transporter. Similar studies done with cloned PEPT1 and PEPT2 in heterologous expression systems yielded comparable results. The transport of valganciclovir via PEPT1 was investigated directly in PEPT1-expressing Xenopus laevis oocytes with an electrophysiological approach. Valganciclovir, but not ganciclovir, induced inward currents in PEPT1-expressing oocytes. These results demonstrate that the increased bioavailability of valganciclovir is related to its recognition as a substrate by the intestinal peptide transporter PEPT1. This prodrug is also recognized by the renal peptide transporter PEPT2 with high affinity.

Animals↗

PubMed2004

Molecular mechanisms underlying calcium current modulation by nociceptin.

Nociceptin is a non-opioid peptide that modulates pain response. One of mechanism underlying its analgesic action is the inhibition of voltage-dependent calcium current (ICa), similar to that of opioids. We investigated the molecular mechanism by which nociceptin inhibits ICa using sensory neurons and a heterologous expression system. ICa inhibition by nociceptin was voltage-dependent, exhibited the slowing of activation kinetics and prepulse facilitation, and was blocked by N-ethylmaleimide, indicating the involvement of Gi/Go protein. ICa inhibition by nociceptin was primarily mediated through binding to its own receptor, ORL-1, but not through affecting other mu-opioid receptors. Thus, our results strongly demonstrate that heterologous cross-talk between ORL1 and muOR is not involved in the ICa inhibition by nociceptin.

Calcium Channel Blockers↗

PubMed1994

Expression of neuronal nicotinic acetylcholine receptor subunit genes in the rat autonomic nervous system.

In the autonomic nervous system efferent signals are relayed in sympathetic and parasympathetic ganglia. Fast synaptic transmission between pre- and postsynaptic neurons is achieved by neuronal nicotinic acetylcholine receptors (nAChRs). There is still little known about the subunit composition of these receptors. Establishing the subunit composition of native neuronal nAChRs is important for the understanding of their functional properties both in vivo and after expression in heterologous expression systems. We have combined in situ hybridization and autoradiography to detect the presence of mRNAs encoding subunits of neuronal nAChRs in sympathetic and parasympathetic ganglia. Inspection of the autoradiographs showed that the hybridization signal of five riboprobes (alpha 3, alpha 4-1, alpha 7, beta 2 and beta 4) was significantly higher than the unspecific signal obtained with sense riboprobes. The distribution of alpha 7 was tissue-dependent: alpha 7 riboprobe binding was detected in the neurons of the superior cervical ganglion, adrenal medulla and ciliary ganglion. In contrast, the alpha 7 hybridization signal was found only in a small fraction (1-3%) of the neurons of the sphenopalatine and otic ganglia. Our results are consistent with the idea that alpha 3 mRNA expression levels are somewhat higher than those of alpha 7, alpha 4-1, beta 2 and beta 4.

Animals↗

PubMed2002

Improved system for protein engineering of the hydroxylase component of soluble methane monooxygenase.

Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O(2)- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (alphabetagamma)(2) complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M. trichosporium OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hydroxylase component of sMMO. This new system has been used to investigate the functions of Cys 151 and Thr 213 of the alpha subunit, which are the only nonligating protonated side chains in the hydrophobic active site. Both residues were found to be critical for the stability and/or activity of sMMO, but neither was essential for oxygenation reactions. The T213S mutant was purified to >98% homogeneity. It had the same iron content as the wild type and had 72% wild-type activity toward toluene but only 17% wild-type activity toward propene; thus, its substrate profile was significantly altered. With these results, we have demonstrated proof of the principle for protein engineering of this uniquely versatile enzyme.

Amino Acid Substitution↗

PubMed2005

Role of the second intradiscal loop of peripherin/rds in homo and hetero associations.

Peripherin/rds (P/rds) is a disk rim protein that assembles into homo and hetero complexes with its nonglycosylated homologue, Rom-1, to maintain the integrity of the photoreceptor outer segment. Mutations in the rds gene have been identified in a variety of human retinal degenerative diseases. More than 70% of these mutations are located in the second intradiscal (D2) loop, highlighting the functional importance of this region. This study examines the involvement of different regions of the D2 loop in protein associations using a GST pull-down assay and a heterologous coexpression system. The pull-down assay suggests an association of the N-terminal portion (Phe(120)-Phe(187)) of the D2 loop with Rom-1 as well as with other P/rds molecules. Through peptide competition experiments, the region between Cys(165) and Asn(182) of the D2 loop has been identified as the domain for these associations. In a COS-1 cell heterologous expression system, coexpression of the D2 loop along with the intact P/rds and Rom-1 hindered the association of the two full-length proteins. In contrast to the homo association of P/rds molecules, it seems that the hetero association of P/rds with Rom-1 has a more stringent structural requirement. This work defines the crucial domain of the D2 loop, which mediates homo and hetero associations, specifically the regions that lay between Cys(165) and Asn(182). Elucidation of the molecular mechanisms behind the protein-protein associations of P/rds and its partners may reveal the pathogenic defects arising from the most common mutations in this gene.

Amino Acid Sequence↗

PubMed2003

Expression of Helicobacter pylori vacuolating toxin in Escherichia coli.

VacA is a secreted toxin that plays a role in Helicobacter pylori colonization of the stomach and that contributes to the pathogenesis of peptic ulcer disease. Studies of VacA structure and function have been hindered by the lack of an efficient system for expression and genetic manipulation of this toxin. In this study, we developed methodology for expression of a functionally active VacA toxin in Escherichia coli. We then used a high-throughput screen to analyze a library of mutant toxins with pentapeptide insertions and identified six mutants that lacked the capacity to induce vacuolation of HeLa cells. The capacity to analyze VacA in this heterologous-expression system should greatly facilitate efforts to elucidate the structure and function of this toxin.

Bacterial Proteins↗

PubMed1991

A Mycoplasma hyorhinis protein with sequence similarities to nucleotide-binding enzymes.

We have determined the nucleotide (nt) and deduced amino acid (aa) sequence of a unique 115-kDa Mycoplasma hyorhinis protein (P115) with an N-terminal region containing a highly conserved consensus sequence characteristics of nt-binding domains of several ATPase and GTPase enzymes. However, P115 lacked additional conserved features characteristic of some classes of nt-binding proteins. Based on the hydropathy profile of the deduced aa sequence, the absence of a leader peptide, its exclusive partitioning into the hydrophilic phase during Triton X-114 phase fractionation of M. hyorhinis, and immunofluorescence analysis indicating no surface-exposed domains, it was concluded that P115 is a cytoplasmic protein lacking intrinsic membrane interaction. M. hyorhinis P115 appears to be a species-specific protein, since it was not detected in any other mycoplasmal or bacterial species examined with specific antibody or genomic probes. Since genetic systems for direct mutational analysis are currently unavailable in this organism, sequence analysis provides critical information in establishing the possible function of this protein. Moreover, the nt sequence encoding P115 reported here supports a previously proposed model, based on synthesis of P115-related proteins in Escherichia coli, suggesting that multiple polypeptide products can be generated from mycoplasma genes by promiscuous translation initiation in this heterologous expression system.

Adenosine Triphosphatases↗

PubMed1997

Functional expression of the murine Golgi CMP-sialic acid transporter in saccharomyces cerevisiae.

We have functionally expressed the murine Golgi putative CMP-sialic acid transporter in Saccharomyces cerevisiae. Using a galactose-inducible expression system, S. cerevisiae vesicles were able to transport CMP-sialic acid. Transport was dependent on galactose induction and was temperature-dependent and saturable with an apparent Km of 2.9 microM. Transport was inhibited by CMP, and upon vesicle disruption with Triton X-100 parameters were very similar to the previously described CMP-sialic acid transport characteristics observed with mammalian Golgi vesicles. CMP-sialic acid transport induction was specific as no transport of UDP-galactose was observed even though the latter putative transporter has a high degree of amino acid sequence identity with the CMP-sialic acid transporter. Together, the above results demonstrate that the previously described cDNA encoding the putative CMP-sialic acid transporter encodes the transporter protein per se and suggests that this heterologous expression system may be used for further structural and functional studies of other Golgi membrane transporter proteins.

Animals↗

PubMed1998

Expression of a plastidic ATP/ADP transporter gene in Escherichia coli leads to a functional adenine nucleotide transport system in the bacterial cytoplasmic membrane.

Recently, a second type of eucaryotic adenine nucleotide transporter located in the inner envelope membrane of higher plants has been identified at the molecular level (Neuhaus, H. E., Thom, E., Möhlmann, T., Steup, M., and Kampfenkel, K. (1997) Plant J. 11, 73-82). Here we have analyzed the biochemical properties of this ATP/ADP transporter from Arabidopsis thaliana (AATP1, At). This analysis was carried out by expressing a cDNA encoding this carrier as a histidine-tagged chimeric protein heterologously in Escherichia coli. Isopropyl-1-thio-beta-D-galactopyranoside (IPTG)-induced E. coli cells were able to import radioactively labeled [alpha-32P]ATP. Uninduced E. coli cells did not import [alpha-32P]ATP. Further control experiments revealed that IPTG induction did not promote import of other phosphorylated or unphosphorylated metabolites into the bacterial cell indicating the specificity of [alpha-32P]ATP transport. [alpha-32P]ATP uptake into induced E. coli cells was linear with time for several minutes allowing for determination of kinetic constants. The apparent Km for ATP was 17 microM which is close to values reported on the authentic protein in isolated plastids. ADP was a strong competitive inhibitor of -alpha-32P-ATP uptake (Ki ADP 3.6 microM). Other metabolites like AMP, ADP glucose, UTP, UDP, NAD, and NADP did not influence [alpha-32P]ATP uptake. IPTG-induced E. coli cells preloaded with [alpha-32P]ATP exported radioactively labeled adenylates after exogenous addition of unlabeled ATP or ADP indicating a counter exchange mechanism of transport. The biochemical properties of the heterologously expressed AATP1 gene product demonstrated that the protein is functionally integrated in the cytoplasmic membrane of E. coli. This is the first report of the functional expression of a plant membrane protein in E. coli leading to new transport properties across the cytoplasmic membrane. The functional integration of a plant membrane protein in the cytoplasmic membrane of E. coli offers new possibilities for future studies of the structural and mechanistic properties of this transporter. Since IPTG induction allowed synthesis of a 67-kDa protein in E. coli, which was subsequently specifically enriched by metal-chelate chromatography, this procaryotic heterologous expression system might provide a suitable system for overproduction of membrane proteins of eucaryotic origin in the near future.

Adenine Nucleotides↗

PubMed2004

Renal water reabsorption: a physiologic retrospective in a molecular era.

The cloning and sequencing of the aquaporin water channels has been an enormous advance in the biomedical sciences, as recognized by the award of the Nobel Prize to Peter Agre last year. Among many other examples, expression of aquaporin proteins in Xenopus oocytes and other heterologous expression systems has confirmed two important models of renal function: the increase in the water permeability of the collecting duct by antidiuretic hormone (ADH), and the mechanism of near isosmotic volume reabsorption by the proximal tubule. These mechanisms were the subjects of intensive investigation by numerous investigators, including Thomas E. Andreoli, who is being honored by this symposium, and who developed many of the key concepts in these areas. His early work with artificial lipid bilayer membranes and the pore-forming antibiotic amphotericin provided the rigorous foundation in experimental and conceptual modeling techniques that he later applied to physiologic and pathophysiologic mechanisms in the kidney, which are summarized in this retrospective. Dr. Andreoli and his colleagues proposed a water channel mechanism for the action of ADH, which has been confirmed by the cloning and heterologous expression of aquaporin-2. They also proposed that volume reabsorption by the proximal tubule depended on a very high hydraulic conductivity and the development of luminal hypotonicity produced by active solute reabsorption. This model has also been confirmed in mice in which aquaporin-1 expression is knocked out, resulting in a low proximal tubule water permeability that exaggerates the development of luminal hypotonicity.

Absorption↗

PubMed2006

Temperature: an important experimental variable in studying PKC modulation of ligand-gated ion channels.

Amphibian oocyte and mammalian heterologous expression systems are often used to investigate the function of recombinant ion channels using electrophysiological techniques. Although both systems have yielded important information, the results obtained in these systems are sometimes conflicting. Oocytes and mammalian cells differ in their physiological temperature requirements. While room temperature is within the physiological temperature range for oocytes, this temperature is far below that required by mammalian cells. Since electrophysiological studies are often performed in both oocytes and mammalian cells at room temperature, we sought to determine if recording temperature could be a factor in some disparate results obtained in these cell types. For these studies, we examined phorbol ester modulation of GABA(A) and glycine receptors. Consistent with the literature, at room temperature, PMA (phorbol 12-myristate 13-acetate) produced a large reproducible decrease in the peak amplitude of GABA and glycine-gated currents in Xenopus oocytes. In contrast, PMA was ineffective in modulating these heterologously expressed receptors at room temperature in human embryonic kidney (HEK) 293 cells. However, when electrophysiological experiments were performed at 35 degrees C in HEK 293 cells, PMA decreased the function of these receptors. Our results indicate that the temperature at which electrophysiological studies are conducted is an important experimental variable. To determine the extent to which electrophysiological recordings are performed at physiological temperatures in HEK 293 cells, a PubMed search was conducted using the search terms "patch clamp" and "HEK" for the years 2003-2004. This search revealed that only 15% of the patch clamp studies were reported to have been conducted in the temperature range of 32-37 degrees C. The results of our study indicate that temperature is an important experimental variable that requires rational consideration in the design of electrophysiological experiments.

Analysis of Variance↗

PubMed1991

Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system.

17-alpha-Hydroxypregnenolone↗

PubMed1999

Pronounced differences between the native K+ channels and KAT1 and KST1 alpha-subunit homomers of guard cells.

Stomatal opening is the result of K(+)-salt accumulation in guard cells. Potassium uptake in these motor cells is mediated by voltage-dependent, K(+)-selective ion channels. Here we compare the invitro properties of two guard-cell K(+)-channel alpha-subunits from Arabidopsis thaliana (L.) Heynh. (KAT1) and Solanum tuberosum L. (KST1) after heterologous expression with the respective K(+)-transport characteristics in their mother cell. The KAT1 and KST1 subunits when expressed in Xenopus oocytes shared the basic features of the K(+)-uptake channels in the corresponding guard cells, including voltage dependence and single-channel conductance. Besides these similarities, the electrophysiological comparison of K+ channels in the homologous and the heterologous expression systems revealed pronounced differences with respect to modulation and block by extracellular cations. In the presence of 1 mM Cs+, 50% of the guard-cell K(+)-uptake channels (GCKClin) in A. thaliana and S. tuberosum, were inhibited upon hyperpolarization to -90 mV. For a similar effect on KAT1 and KST1 in oocytes, voltages as negative as -155 mV were required. In contrast, compared to the K+ channels in vivo the functional alpha-subunit homomers almost lacked a voltage-dependent block by extracellular Ca2+. Similar to the block by Cs+ and Ca2+, the acid activation of the alpha-homomers was less pronounced in oocytes. Upon acidification the voltage-dependence shifted by 82 and 90 mV for GCKCLin in A. thaliana and S. tuberosum, respectively, but only by 25 mV for KAT1 and KST1. From the differences in K(+)-channel modulation in vivo and after heterologous expression we conclude that the properties of functional guard-cell K(+)-uptake channels result either from the heterometric assembly of different alpha-subunits or evolve from cell-type specific posttranslational modification.

Animals↗

PubMed2005

Expression systems for cloned xenobiotic transporters.

One challenge of modern biology is to be able to match genes and their encoded proteins with events at the molecular, cellular, tissue, and organism levels, and thus, provide a multi-level understanding of gene function and dysfunction. How well this can be done for xenobiotic transporters depends on a knowledge of the genes expressed in the tissue, the cellular locations of the gene products (do they function for uptake or efflux?), and our ability to match substrates with transporters using information obtained from cloned transporters functioning in heterologous expression systems. Clearly, making a rational choice of expression system to use for the characterization and study of cloned xenobiotic transporters is a critical part of study design. This choice requires well-defined goals, as well as an understanding of the strengths and weaknesses of candidate expression systems.

Animals↗

PubMed1999

Cystic fibrosis transmembrane conductance regulator regulates luminal Cl-/HCO3- exchange in mouse submandibular and pancreatic ducts.

We have demonstrated previously the regulation of Cl-/HCO3- exchange activity by the cystic fibrosis transmembrane conductance regulator (CFTR) in model systems of cells stably or transiently transfected with CFTR (Lee, M. G., Wigley, W. C., Zeng, W., Noel, L. E., Marino, C. R., Thomas, P. J., and Muallem, S. (1999) J. Biol. Chem. 274, 3414-3421). In the present work we examine the significance of this regulation in cells naturally expressing CFTR. These include the human colonic T84 cell line and the mouse submandibular gland and pancreatic ducts, tissues that express high levels of CFTR in the luminal membrane. As in heterologous expression systems, stimulation of T84 cells with forskolin increased the Cl-/HCO3- exchange activity independently of CFTR Cl- channel activity. Freshly isolated submandibular gland ducts from wild type mice showed variable Cl-/HCO3- exchange activity. Measurement of [Cl-]i revealed that this was largely the result of variable steady-state [Cl-]i. Membrane depolarization with 5 mM Ba2+ or 100 mM K+ increased and stabilized [Cl-]i. Under depolarized conditions wild type and DeltaF/DeltaF mice had comparable basal Cl-/HCO3- exchange activity. Notably, stimulation with forskolin increased Cl-/HCO3- exchange activity in submandibular gland ducts from wild type but not DeltaF/DeltaF mice. Microperfusion of the main pancreatic duct showed Cl-/HCO3- exchange activity in both the basolateral and luminal membranes. Stimulation of ducts from wild type animals with forskolin had no effect on basolateral but markedly stimulated luminal Cl-/HCO3- exchange activity. By contrast, forskolin had no effect on either basolateral or luminal Cl-/HCO3- exchange activity of ducts from DeltaF/DeltaF animals. We conclude that CFTR regulates luminal Cl-/HCO3- exchange activity in CFTR-expressing cells, and we discuss the possible physiological significance of these findings regarding cystic fibrosis.

Animals↗

PubMed1993

Molecular biology of the voltage-gated potassium channels of the cardiovascular system.

K+ channels represent the most diverse class of voltage-gated ion channels in terms of function and structure. Voltage-gated K+ channels in the heart establish the resting membrane K+ permeability, modulate the frequency and duration of action potentials, and are targets of several antiarrhythmic drugs. Consequently, an understanding of K+ channel structure-function relationships and pharmacology is of great practical interest. However, the presence of multiple overlapping currents in native cardiac myocytes complicates the study of basic K+ channel function and drug-channel interactions in these cells. The application of molecular cloning technology to cardiovascular K+ channels has identified the primary structure of these proteins, and heterologous expression systems have allowed a detailed analysis of channel function and pharmacology without contaminating currents. To date six different K+ channels have been cloned from rat and human heart, and all have been functionally characterized in either Xenopus oocytes or mammalian tissue culture systems. This initial research is an important step toward understanding the molecular basis of the action potential in the heart. An important challenge for the future is to determine the cell-specific expression and relative contribution of these cloned channels to cardiac excitability.

Amino Acid Sequence↗

PubMed2003

Robo4 is a vascular-specific receptor that inhibits endothelial migration.

Guidance and patterning of axons are orchestrated by cell-surface receptors and ligands that provide directional cues. Interactions between the Robo receptor and Slit ligand families of proteins initiate signaling cascades that repel axonal outgrowth. Although the vascular and nervous systems grow as parallel networks, the mechanisms by which the vascular endothelial cells are guided to their appropriate positions remain obscure. We have identified a putative Robo homologue, Robo4, based on its differential expression in mutant mice with defects in vascular sprouting. In contrast to known neuronal Robo family members, the arrangement of the extracellular domains of Robo4 diverges significantly from that of all other Robo family members. Moreover, Robo4 is specifically expressed in the vascular endothelium during murine embryonic development. We show that Robo4 binds Slit and inhibits cellular migration in a heterologous expression system, analogous to the role of known Robo receptors in the nervous system. Immunoprecipitation studies indicate that Robo4 binds to Mena, a known effector of Robo-Slit signaling. Finally, we show that Robo4 is the only Robo family member expressed in primary endothelial cells and that application of Slit inhibits their migration. These data demonstrate that Robo4 is a bona fide member of the Robo family and may provide a repulsive cue to migrating endothelial cells during vascular development.

Activin Receptors, Type I↗