No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Biochemistry and molecular genetics of Leishmania glucose transporters
Parasitology
Abstract
Glucose is utilized as a significant source of metabolic energy by Leishmania parasites. This sugar is accumulated by the parasite via a specific carrier-mediated transport system located in the parasite membrane. Parasites may also contain another transporter that shuttles glucose between the cytoplasm and the glycosome, a membrane-bound organelle where the early steps of glycolysis occur. The transport systems of both the insect stage promastigotes and the intracellular amastigotes have been characterized and shown to have kinetic properties that are consistent with the different physiological environments of the insect gut and the macrophage phagolysosome. Several genes have been cloned from Leishmania species which encode proteins with substantial sequence similarity to glucose transporters from mammals and lower eukaryotes. Two of these genes are expressed preferentially in the promastigote stage of the life cycle, where glucose is more readily available and more rapidly transported and metabolized than in the intracellular amastigotes. One of these two developmentally-regulated genes has been functionally expressed in Xenopus oocytes and shown to encode a glucose transporter. A third gene encodes a protein that is also a member of the glucose transporter family on the basis of sequence similarity and proposed secondary structure. However, the significant differences between this protein and the other two suggest that it is likely to transport a different substrate. Functional expression will be required to define the specific biochemical role of each gene within the parasite.
... Fructose, mannose, N-acetylglucosamine, and galactose are all transported by Leishmania promastigotes' hexose transporters, which have a high affinity for glucose (km 25 M) (Langford et al., 1994). Amastigotes have a specific pH optimum and a 10-fold lower rate of glucose uptake than promastigotes (pH 5 versus 7) (Burchmore & Hart, 1995;Hart & Coombs, 1982;Mukkada et al., 1985;Seyfang & Landfear, 1999). ...
In tropical and subtropical regions of the world, leishmaniasis is endemic and causes a range of clinical symptoms in people, from severe tegumentary forms (such as cutaneous, mucocutaneous, and diffuse leishmaniasis) to lethal visceral forms. The protozoan parasite of the genus Leishmania causes leishmaniasis, which is still a significant public health issue, according to the World Health Organization 2022. The public's worry about the neglected tropical disease is growing as new foci of the illness arise, which are exacerbated by alterations in behavior, changes in the environment, and an enlarged range of sand fly vectors. Leishmania research has advanced significantly during the past three decades in a few different avenues. Despite several studies on Leishmania, many issues, such as illness control, parasite resistance, parasite clearance, etc., remain unresolved. The key virulence variables that play a role in the pathogenicity-host-pathogen relationship of the parasite are comprehensively discussed in this paper. The important Leishmania virulence factors, such as Kinetoplastid Membrane Protein-11 (KMP-11), Leishmanolysin (GP63), Proteophosphoglycan (PPG), Lipophosphoglycan (LPG), Glycosylinositol Phospholipids (GIPL), and others, have an impact on the pathophysiology of the disease and enable the parasite to spread the infection. Leishmania infection may arise from virulence factors; they are treatable with medications or vaccinations more promptly and might greatly shorten the duration of treatment. Additionally, our research sought to present a modeled structure of a few putative virulence factors that might aid in the development of new chemotherapeutic approaches for the treatment of leishmaniasis. The predicted virulence protein's structure is utilized to design novel drugs, therapeutic targets, and immunizations for considerable advantage from a higher understanding of the host immune response.
... Here, we investigated the impact of the deletion of KH1 on glucose uptake in L. infantum ( Supplementary Fig. S3). It was found that the deletion of KH1 in L. infantum did not interfere with glucose transport and this lack of phenotype is potentially a result of the presence of more than one Leishmania transporter capable of importing the hexoses glucose, mannose, fructose and galactose [31][32][33][34] . In L. infantum there is a locus encoding three glucose transporters isoforms were genes LinJ. ...
There is no safe and efficacious vaccine against human leishmaniasis available and live attenuated vaccines have been used as a prophylactic alternative against the disease. In order to obtain an attenuated Leishmania parasite for vaccine purposes, we generated L. infantum KHARON1 (KH1) null mutants (ΔLikh1). This gene was previously associated with growth defects in L. mexicana. ΔLikh1 was obtained and confirmed by PCR, qPCR and Southern blot. We also generate a KH1 complemented line with the introduction of episomal copies of KH1. Although ΔLikh1 promastigote forms exhibited a growth pattern similar to the wild-type line, they differ in morphology without affecting parasite viability. L. infantum KH1-deficient amastigotes were unable to sustain experimental infection in macrophages, forming multinucleate cells which was confirmed by in vivo attenuation phenotype. The cell cycle analysis of ΔLikh1 amastigotes showed arrested cells at G2/M phase. ΔLikh1-immunized mice presented reduced parasite burden upon challenging with virulent L. infantum, when compared to naïve mice. An effect associated with increased Li SLA-specific IgG serum levels and IL-17 production. Thus, ΔLikh1 parasites present an infective-attenuated phenotype due to a cytokinesis defect, whereas it induces immunity against visceral leishmaniasis in mouse model, being a candidate for antileishmanial vaccine purposes.
... Durante el desarrollo del parásito en el tracto digestivo del vector se han descrito varias formas morfológicas (3). Estos cambios morfológicos, los cuales se correlacionan con el desarrollo del parásito, se ha encontrado que están asociados con cambios genéticos y bioquímicos (4,5), los cuales, en algunos casos, modulan funciones biológicas. ...
La metaciclogénesis es un proceso que experimentan naturalmente los promastigotes de Leishmania en el tracto digestivo del insecto vector y cuya finalidad es transformar los promastigotes en formas altamente infectivas y capaces de sobrevivir en el hospedero vertebrado, donde es sometido a los ataques por parte del sistema inmune. Se ha demostrado que este fenómeno ocurre también en promastigotes en crecimiento en cultivos axénicos in vitro. El proceso de metaciclogénesis se asoció inicialmente con cambios morfológicos, observándose que los promastigotes cambiaban su forma y tamaño con incremento en la longitud del flagelo. Luego, se logró asociar con este fenómeno la expresión de ciertas moléculas implicadas en virulencia, como el lipofosfoglicano (LPG) y una proteasa de superficie, la gp63. Se demostró que estas moléculas experimentaban cambios tanto cuantitativos como cualitativos a medida que los promastigotes se diferencian de promastigotes procíclicos no infectivos a metacíclicos o infectivos. Hoy en día, mediante técnicas de hibridización substractiva con cADN o de amplificación diferencial, se han logrado identificar genes cuyo patrón de expresión está íntimamente ligado al proceso de metaciclogénesis. Se han identificado moléculas como el producto del gen B y una proteína asociada con la metaciclogénesis (Mat-1), las cuales se expresan exclusivamente en promastigotes metacíclicos, mientras que otras como las proteínas Meta-1, SHERP y HASP se sobrexpresan en los promastigotes metacíclicos. Sin embargo, la función y asociación de estas proteínas con este patrón particular de expresión y virulencia se está empezando a evaluar.
... This development helps to advance Leishmania as a model organism for the study of gene expression during adaptive differentiation. Several genes that exhibit developmental regulation have now been cloned, for example, the genes for histone H2B and the glucose transporter of Leishmania enriettii, both of which showed higher levels of accumulation in the promastigote stage of the parasitic life cycle (Langford et al. 1994). Similarly, other less well characterized complementary DNAs have been identified that are associated predominantly with the arnastigote stage (Joshi et al. 1993; Charest and Matlashewski 1994). ...
Leishmania is a member of the family Trypanosomatidae, which causes debilitating diseases in humans. Over the past several years, researchers have applied molecular genetic techniques to study extensively the biology of this parasitic protozoan. Many aspects of Leishmania biology are found to be unique when compared with the higher eukaryotes. This minireview highlights some recent developments in the molecular genetic analysis of this fascinating organism.Key words: Leishmania, protozoan, molecular genetics, disease.
... In recent years, more attention has been directed to the understanding of sugar transport in various systems, including Escherichia coli (12), Saccharomyces cerevisiae (23), Schizosaccharomyces pombe (18,22), plants (14), Drosophila cell lines (30), human erythrocytes (8), and some parasites, such as Leishmania spp. (17). Except for E. coli and S. cerevisiae, we know very little about the mechanism of sugar transport in other systems. ...
A wild-type strain, Sp972 h-, of Schizosaccharomyces pombe was mutagenized with ethylmethanesulfonate (EMS), and 2-deoxyglucose (2-DOG)-resistant mutants were isolated. Out of 300 independent 2-DOG-resistant mutants, 2 failed to grow on glucose and fructose (mutants 3/8 and 3/23); however, their hexokinase activity was normal. They have been characterized as defective in their sugar transport properties, and the mutations have been designated as std1-8 and std1-23 (sugar transport defective). The mutations are allelic and segregate as part of a single gene when the mutants carrying them are crossed to a wild-type strain. We confirmed the transport deficiency of these mutants by [14C]glucose uptake. They also fail to grow on other monosaccharides, such as fructose, mannose, and xylulose, as well as disaccharides, such as sucrose and maltose, unlike the wild-type strain. Lack of growth of the glucose transport-deficient mutants on maltose revealed the extracellular breakdown of maltose in S. pombe, unlike in Saccharomyces cerevisiae. Both of the mutants are unable to grow on low concentrations of glucose (10 to 20 mM), while one of them, 3/23, grows on high concentrations (50 to 100 mM) as if altered in its affinity for glucose. This mutant (3/23) shows a lag period of 12 to 18 h when grown on high concentrations of glucose. The lag disappears when the culture is transferred from the log phase of its growth on high concentrations. These mutants complement phenotypically similar sugar transport mutants (YGS4 and YGS5) reported earlier by Milbradt and Hoefer (Microbiology 140:2617-2623, 1994), and the clone complementing YGS4 and YGS5 was identified as the only glucose transporter in fission yeast having 12 transmembrane domains. These mutants also demonstrate two other defects: lack of induction and repression of shunt pathway enzymes and defective mating.
... One is the W H generated across the parasite plasma membrane by the H -AT- Pase which allegedly provides the driving force for a number of H -substrate symport systems [35,91^96]. For example, in Leishmania, the active inward movement of D-glucose and amino acids has been shown to be a¡ected by the W H [30,35,92,95,96]. However, the role of such a pump in nutrient uptake has been recently challenged ([97,98] and references therein). ...
... This is the first clear demonstration of a H ϩ -glucose symport mechanism for glucose accumulation operative in Leishmania parasites. It is important to note that developmentally regulated glucose transporter genes having significant homology with such genes of higher eucaryotes were earlier cloned from Leishmania, although their relationship to energy coupling remained uncertain (14,41). ...
Experiments from other laboratories conducted with Leishmania donovani promastigote cells had earlier indicated that the plasma membrane Mg 2+-ATPase of the parasite is an extrusion pump for H+. Taking advantage of the pellicular microtubular structure of the plasma membrane of the organism, we report procedures for obtaining sealed ghost and sealed everted vesicle of defined polarity. Rapid influx of H+ into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mM) and Mg2+ (1 mM). Excellent correspondence between rate of H+ entry and the enzyme activity clearly demonstrated the Mg 2+-ATPase to be a true H+ pump. H+ entry into everted vesicle was strongly inhibited by SCH28080 (IC50 = ∼40 μM) and by omeprazole (IC50 = ∼50 μM), both of which are characteristic inhibitors of mammalian gastric H+,K +-ATPase. H+ influx was completely insensitive to ouabain (250 μM), the typical inhibitor of Na+,K+-ATPase. Mg2+-ATPase activity could be partially stimulated with K + (20 mM) that was inhibitable (>85%) with SCH28080 (50 μM). ATP-dependent rapid efflux of 86Rb+ from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 μM) and by 5,5′-dithiobis-(2-nitrobenzoic acid) (1 mM), an inhibitor of the enzyme. Assuming Rb+ to be a true surrogate for K+, an ATP-dependent, electroneutral stoichiometric exchange of H+ and K+ (1:1) was established. Rapid and 10-fold active accumulation of [U-14C]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior alkaline) was imposed. Rapid efflux of [U- 14C]D-glucose from preloaded everted vesicles could also be initiated by activating the enzyme, with ATP. Taken together, the plasma membrane Mg2+-ATPase has been identified as an electroneutral H +/K+ antiporter with some properties reminiscent of the gastric H+,K+-ATPase. This enzyme is possibly involved in active accumulation of glucose via a H+-glucose symport system and in K+ accumulation.
... Durante el desarrollo del parásito en el tracto digestivo del vector se han descrito varias formas morfológicas (3). Estos cambios morfológicos, los cuales se correlacionan con el desarrollo del parásito, se ha encontrado que están asociados con cambios genéticos y bioquímicos (4,5), los cuales, en algunos casos, modulan funciones biológicas. ...
Metacyclogenesis is a process whereby Leishmania transforms from poorly infective procyclic promastigotes into highly infective metacyclic promastigotes. In nature, metacyclogenesis occurs in the insect vector. This transformation is accompanied by an increased ability to infect and survive in the vertebrate host, where the parasite is attacked by the host's immune system. Metacyclogenesis has also been shown to occur in axenic cultures of promastigotes. Morphological changes in size and shape, and length of flagellum were first associated with differentiation in the insect gut and in different phases of growth in culture. Later, the expression of molecules such as LPG and the surface protease gp63 were associated with this process. These two molecules were observed to undergo qualitative and quantitative modifications as the promastigotes differentiated from procyclic to metacyclic forms. Using cDNA subtractive hybridization-based methods or differential amplification, previously unknown genes tightly linked to metacyclogenesis have been identified. Gene products exclusively expressed in metacyclic promastigotes included a gene B product and Mat-1--a gene associated with metacyclogenesis. Other proteins, Meta-1, SHERP and HASP, were up-regulated during the metacyclic stage. The function and stage-regulated expression of these molecules and their relationship with infectivity are now under investigation.
... We have also used the cloned sequence to search the ongoing EST's database from T. rangeli (Snoeijer et al. 2004 ), but unfortunately no sequence corresponding to the cloned DNA was found. D I S C U S S I O N Kinetic analysis of D-glucose transport by T. rangeli epimastigotes and trypomastigotes indicates that a high-affinity transporter is present in both life-cycle stages studied, a situation similar to that found in other insect-form kinetoplastids including Crithidia luciliae and some Leishmania species (Knodler et al. 1992 ; Langford et al. 1994 ; Burchmore and Hart, 1995 ; Tetaud et al. 1997), and with no evidence of a C y t o c h a l a s i n B P h l o r e t i n P h l o r i d z i n % D-glucose uptakeTable 1. Inhibition constants (K i ) for analogues and inhibitors (The values of K i for T. rangeli forms were determined by the inhibition of D-glucose incorporation as described in the Materials and Methods section. Other values are from references: (a) (Tetaud et al. 1994) ; (b) (Tetaud et al. 1997) and (c) (Vendrene et al. 2000).) ...
Like in other trypanosomatids D-glucose is a crucial source of energy to Trypanosoma rangeli, a non-pathogenic parasite that in Central and South America infects triatomine vectors and different mammalian species, including humans. In several trypanosome species, D-glucose transporters were already described and cloned. In this study, we characterized the D-glucose transport activity present in 2 life-stage forms of T. rangeli (epimastigotes and trypomastigotes) using D-[U-14C]glucose as substrate. Our results indicate that T. rangeli transports D-glucose with high affinity in both epimastigote (Km 30 microM) and trypomastigotes (Km 80 microM) life-forms. Both transport activities were inhibited by Cytochalasin B and Phloretin, indicating that probably D-glucose uptake in T. rangeli is mediated by facilitated diffusion of the sugar. Significant differences were observed between epimastigotes and trypomastigotes in relation to their affinity for D-glucose analogues, and the predicted amino acid sequence of a putative D-glucose transporter from T. rangeli (TrHT1) showed a larger identity with the T. cruzi D-glucose transporter encoded by the TcrHT1 gene than with other transporters already characterized in trypanosomatids.
Alveolar echinococcosis (AE) is a zoonotic parasitosis caused by larvae of the fox tapeworm, Echinococcus multilocularis. E. multilocularis is distributed widely in the Northern hemisphere, causing serious health problems in various animals and humans. E. multilocularis, like other cestodes, lacks a digestive tract and absorbs essential nutrients, including glucose, across the syncytial tegument on its external surface. Therefore, it is hypothesized that E. multilocularis uses glucose transporters on its surface similar to a closely-related species, Taenia solium. Based on this hypothesis, we cloned and characterized glucose transporter homologues from E. multilocularis. As a result, we obtained full-length sequences of 2 putative glucose transporter genes (EmGLUT1 and EmGLUT2) from E. multilocularis. In silico analysis predicted that these were classified in the solute carrier family 2 group. Functional expression analysis using Xenopus oocytes demonstrated clear uptake of 2-deoxy-D-glucose (2-DG) by EmGLUT1, but not by EmGLUT2 in this experimental system. EmGLUT1 was shown to have relatively high glucose transport activity. Further analyses using the Xenopus oocyte system revealed that 2-DG uptake of EmGLUT1 did not depend on the presence or concentration of Na+ nor H+, respectively. Immunoblot analyses using cultured metacestode, ex vivo protoscolex, and adult worm samples demonstrated that both EmGLUTs were stably expressed during each developmental stage of the parasite. Based on the above-mentioned findings, we conclude that EmGLUT1 is a simple facilitated glucose transporter and possibly plays an important role in glucose uptake by E. multilocularis throughout its life cycle.
Promastigotes and amastigotes of Leishmania mexicana mexicana transported 2-deoxy-D-glucose (2-DOG) by a saturable process with a Km of 24 +/- 3 microM and Vmax of 2.21 nmol min-1 (mg protein)-1 for the promastigote and a Km of 29 +/- 8 microM and Vmax of 0.13 nmol min-1 (mg protein)-1 for the amastigote stage. Amastigotes incorporated 2-DOG maximally at pH 5.0, while for promastigotes the optimum was at pH 7.0. Mid-log phase promastigotes were found to accumulate 2-DOG via a stereospecific carrier-mediated process which was competitively inhibited by D-glucose and D-mannose but not L-glucose. Transport was dependent upon temperature, with a Q10 in promastigotes of 1.83 and an optimum rate at 35 degrees C (+/- 4 degrees C) with an activation energy of 50.12 kJ mol-1. Stationary phase promastigotes accumulated 2-DOG at approximately twice the rate of mid-log phase promastigotes. Cytochalasin B, forskolin and phloretin were all found to inhibit human erythrocyte 2-DOG uptake but only cytochalasin B was found significantly to inhibit promastigote 2-DOG uptake. Interestingly, leishmanial 2-DOG uptake was inhibited by a series of membrane potential antagonists including the ionophore monensin, the H+ATPase inhibitor N, N'-dicyclohexylcarbodiimide (DCCD) and uncoupling agent carbonylcyanide-4-(triflouromethoxy) phenylhydrazone (FCCP), as well as, the tricyclic drugs chlomipramine and imipramine, but was insensitive to the Na+/K+ATPase inhibitor ouabain and the antitrypanosomal drugs Pentostam and Suramin. We therefore conclude that there are significant structural and mechanistic differences between the D-glucose uptake systems of Leishmania and the mammalian host to merit the inclusion of glucose transporters as putative targets for rational drug design.
Intracellular killing of Leishmania parasites within activated murine macrophages is thought to result from the toxic activities of nitrogen oxidation products (referred to as NO) released by the activated cells. In order to determine possible mechanisms of NO toxicity for these microorganisms, promastigotes of Leishmania major and Leishmania enriettii were exposed to NO generated chemically from acidified nitrite, S-nitrosocysteine, diethylamine NONOate, or nitroprusside. Treatment with these agents led to loss of viability (as determined from decreased motility and inhibition of [3H]TdR uptake upon reincubation in NO-free medium) with kinetics characteristic for each compound L. major was less sensitive to these effects than L. enriettii, and amastigotes displayed the same sensitivity as promastigotes of the same species. The early effects of NO toxicity could be detected within minutes of exposure to the NO donors; they included decreased respiration rate and inhibition of glucose, proline, and adenine incorporation. Inhibition of the activities of glyceraldehyde 3-phosphate dehydrogenase and of aconitase were also evidenced. In order to determine whether these phenomena reflected the mechanisms of toxicity of bona fide NO generated by macrophages, promastigotes were exposed to IFN-gamma + LPS-activated macrophages across permeable membranes. This resulted in marked inhibition of proline and adenine uptake in the parasites, which was restored, however, to control levels when macrophages were activated in the presence of the nitric oxide synthase inhibitor NGMMA. These results indicate that several cellular targets may be subject to NO toxicity in Leishmania parasites, including enzymes of glycolysis and respiratory metabolism as well as trans-membrane transport systems.
Arsenic exposure is associated with hypertension, diabetes, and cancer. Some mammals methylate arsenic. Saccharomyces cerevisiae hexose permeases catalyze As(OH)(3) uptake. Here, we report that mammalian glucose transporter GLUT1 catalyzes As(OH)(3) and CH(3)As(OH)(2) uptake in yeast or in Xenopus laevis oocytes. Expression of GLUT1 in a yeast lacking other glucose transporters allows for growth on glucose. Yeast expressing yeast HXT1 or rat GLUT1 transport As(OH)(3) and CH(3)As(OH)(2). The K(m) of GLUT1 is to 1.2mM for CH(3)As(OH)(2), compared to a K(m) of 3mM for glucose. Inhibition between glucose and CH(3)As(OH)(2) is noncompetitive, suggesting differences between the translocation pathways of hexoses and arsenicals. Both human and rat GLUT1 catalyze uptake of both As(OH)(3) and CH(3)As(OH)(2) in oocytes. Thus GLUT1 may be a major pathway uptake of both inorganic and methylated arsenicals in erythrocytes or the epithelial cells of the blood-brain barrier, contributing to arsenic-related cardiovascular problems and neurotoxicity.
Tubulins are abundant structural proteins in the Leishmania parasite, and tubulin genes are examples of highly expressed genes, which are present in multiple copies arranged in tandem repeats. Functional analysis of such multicopy genes using genetic manipulation has not yet been possible. Here we describe a method for creating deletions of alpha-tubulin gene clusters by targeted gene replacement and report the isolation of null/+ mutants deleted for either of the two allelic tubulin clusters. We also report null/null mutants in which both clusters have been deleted from their chromosomal loci and in which alpha-tubulin genes are present as episomal elements. Characterization of tubulin mRNA expression in these mutants indicated a posttranscriptional up-regulation of alpha-tubulin mRNA stability in null/+ mutants which contained only one-third the normal number of alpha-tubulin genes. A parallel increase in beta-tubulin mRNA levels indicated coordinate regulation of tubulin mRNA in Leishmania.
Cultures of the insect stage of the protozoan parasites Leishmania donovani and Trypanosoma brucei were grown in chemostats with glucose as the growth rate-limiting substrate. L. donovani has a maximum specific growth rate (mu max) of 1.96 day-1 and a Ks for glucose of 0.1 mM; the mu max of T. brucei is 1.06 day-1 and the Ks is 0.06 mM. At each steady state (specific growth rate, mu, equals D, the dilution rate), the following parameters were measured: external glucose concentration (Glcout), cell density, dry weight, protein, internal glucose concentration (Glcin), cellular ATP level, and hexokinase activity. L. donovani shows a relationship between mu and yield that allows an estimation of the maintenance requirement (ms) and the yield per mole of ATP (YATP). Both the ms and the YATP are on the higher margin of the range found for prokaryotes grown on glucose in a complex medium. L. donovani maintains the Glcin at a constant level of about 50 mM as long as it is not energy depleted. T. brucei has a decreasing yield with increasing mu, suggesting that it oxidizes its substrate to a lesser extent at higher growth rates. Glucose is not concentrated internally but is taken up by facilitated diffusion, while phosphorylation by hexokinase is probably the rate-limiting step for glucose metabolism. The Ks is constant as long as glucose is the rate-limiting substrate. The results of this study demonstrate that L. donovani and T. brucei have widely different metabolic strategies for dealing with varying external conditions, which reflect the conditions they are likely to encounter in their respective insect hosts.
GLUT-4 is the major facilitative glucose transporter isoform in tissues that exhibit insulin-stimulated glucose transport. Insulin regulates glucose transport by the rapid translocation of GLUT-4 from an intracellular compartment to the plasma membrane. A critical feature of this process is the efficient exclusion of GLUT-4 from the plasma membrane in the absence of insulin. To identify the amino acid domains of GLUT-4 which confer intracellular sequestration, we analyzed the subcellular distribution of chimeric glucose transporters comprised of GLUT-4 and a homologous isoform, GLUT-1, which is found predominantly at the cell surface. These chimeric transporters were transiently expressed in CHO cells using a double subgenomic recombinant Sindbis virus vector. We have found that wild-type GLUT-4 is targeted to an intracellular compartment in CHO cells which is morphologically similar to that observed in adipocytes and muscle cells. Sindbis virus-produced GLUT-1 was predominantly expressed at the cell surface. Substitution of the GLUT-4 amino-terminal region with that of GLUT-1 abolished the efficient intracellular sequestration of GLUT-4. Conversely, substitution of the NH2 terminus of GLUT-1 with that of GLUT-4 resulted in marked intracellular sequestration of GLUT-1. These data indicate that the NH2-terminus of GLUT-4 is both necessary and sufficient for intracellular sequestration.
We have used double gene targeting to create homozygous gene replacements in the protozoan parasite Leishmania major, an asexual diploid. This method uses two independent selectable markers in successive rounds of gene targeting to replace both alleles of an endogenous gene. We developed an improved hygromycin B-resistance cassette encoding hygromycin phosphotransferase (HYG) for use as a selectable marker for Leishmania. HYG-containing vectors functioned equivalently to those containing the neomycin phosphotransferase (NEO) cassette previously used for extrachromosomal transformation or gene targeting. Drug resistances conferred by the NEO and HYG markers were independent, allowing simultaneous selection for both markers. A HYG targeting vector was utilized to replace the single dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene remaining in a line heterozygous for a NEO replacement at the dhfr-ts locus (+/neo), with a targeting efficiency comparable to that seen with wild-type recipients. The resultant dhfr-ts- line (hyg/neo) was auxotrophic for thymidine. The double targeted replacement method will enable functional genetic testing in a variety of asexual diploids, including cultured mammalian cells and fungi such as Candida albicans. Additionally, it may be possible to use Leishmania bearing conditionally auxotrophic gene replacements as safe, improved live vaccines for leishmaniasis.
We have characterized a cDNA encoding a cysteine-rich, acidic integral membrane protein (CRAM) of the parasitic protozoa Trypanosoma
brucei and Trypanosoma equiperdum. Unlike other membrane proteins of T. brucei, which are distributed throughout the cell
surface, CRAM is concentrated in the flagellar pocket, an invagination of the cell surface of the trypanosome where endocytosis
has been documented. Accordingly, CRAM also locates to vesicles located underneath the pocket, providing evidence of its internalization.
CRAM has a predicted molecular mass of 130 kilodaltons and has a signal peptide, a transmembrane domain, and a 41-amino-acid
cytoplasmic extension. A characteristic feature of CRAM is a large extracellular domain with a roughly 66-fold acidic, cysteine-rich
12-amino-acid repeat. CRAM is conserved among different protozoan species, including Trypanosoma cruzi, and CRAM has structural
similarities with eucaryotic cell surface receptors. The most striking homology of CRAM is to the human low-density-lipoprotein
receptor. We propose that CRAM functions as a cell surface receptor of different trypanosome species.
The insulin-regulated glucose transporter GLUT4 was immunolocalized in rat cardiac muscle under conditions of basal and stimulated glucose uptake, achieved by fasting and a combined exercise/insulin stimulus, respectively. In basal myocytes there was very little (less than 1%) GLUT4 in the different domains of the plasma membrane (sarcolemma, intercalated disk, and transverse tubular system). GLUT4 was localized in small tubulo-vesicular elements that occur predominantly near the sarcolemma and the transverse tubular system and in the trans-Golgi region. Upon stimulation approximately 42% of GLUT4 was found in the plasma membrane. Each domain of the plasma membrane contributed equally to this effect. GLUT4-positive, clathrin-coated pits were also present at each cell surface domain. The remainder of the labeling was in tubulo-vesicular elements at the same sites as in basal cells and in the intercalated disk areas. The localization of GLUT4 in cardiac myocytes is essentially the same as in brown adipocytes, skeletal muscle, and white adipocytes. We conclude that increased glucose transport in muscle and fat is accounted for by translocation of GLUT4 from the intracellular tubulo-vesicular elements to the plasma membrane. The labeling of coated pits indicates that in stimulated myocytes, as in adipocytes, GLUT4 recycles constantly between the endosomal compartment and the plasma membrane and that stimulation of the exocytotic rate constant is likely the major mechanism for GLUT4 translocation.
Glucose transport in the bloodstream form of the protozoan parasite Trypanosoma brucei was characterized by enzymatically measuring the D-glucose uptake. Uptake kinetics showed a concentration-dependent saturable process, typical for a carrier-mediated transport system, with an apparent Km = 0.49 +/- 0.14 mM and Vmax = 252 +/- 43 nmol.min-1.mg cell protein-1 (equal to 2.25 x 10(8) trypanosomes). The specificity of glucose transport was investigated by inhibitor studies. Glucose uptake was shown to be sodium independent; neither the Na+/K(+)-ATPase inhibitor ouabain (1 mM) nor the ionophor monensin (1 microM) inhibited uptake. Transport was also unaffected by the H(+)-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD; 20 microM) and the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; 1 microM). However, highly significant inhibition was obtained with both phloretin (82% at 0.13 mM; Ki = 64 microM) and cytochalasin B (77% at 0.3 mM; Ki = 0.44 mM), and partial inhibition with phlorizin (14% at 0.5 mM; Ki = 3.0 mM). In each case, inhibition was noncompetitive, partially reversible (45%) for phloretin and completely reversible for cytochalasin B and phlorizin. Measurement of the temperature-dependent glucose uptake between 25 degrees C and 37 degrees C resulted in a temperature quotient of Q10 = 1.97 +/- 0.02 and an activation energy of Ea = 52.12 +/- 1.00 kJ/mol for glucose uptake. We conclude that glucose uptake in T. brucei bloodstream forms occurs via a facilitated diffusion system, clearly distinguished from the human erythrocyte-type glucose transporter with about a 10-fold higher affinity for glucose and about a 1000-fold decreased sensitivity to the inhibitor cytochalasin B.
We have cloned a developmentally regulated gene from the parasitic protozoan Leishmania enrietti. The mRNA from this gene accumulates to a much higher level in the promastigote stage of the parasite life cycle that lives in the gut of the insect vector than in the amastigote stage of the parasite that lives inside the macrophages of the mammalian host. The predicted protein encoded by this gene is homologous to the human erythrocyte glucose transporter and to several sugar-transport proteins from Escherichia coli. These structural similarities strongly suggest that the cloned gene encodes a membrane transport protein that is developmentally induced when the parasite enters its insect vector. Regulated membrane transporters may be required for the parasite to adapt to the environment of the insect gut.
Peptides corresponding to amino acid residues 1-12 of the amino terminal and 480-492 of the carboxyl terminal of the deduced sequence of the glucose transporter were synthesized and used to produce site-specific polyclonal antipeptide sera. In a solid-phase radioimmunoassay, antiserum to the carboxyl terminal recognizes peptide 480-492 and purified human erythrocyte glucose transporter, but not peptide 1-12. Antiserum to the amino terminal recognizes peptide 1-12 but neither peptide 480-492 nor the erythrocyte transporter. The antiserum to the carboxyl terminal specifically immunoblots the Mr 55,000 glucose transporter in erythrocyte membranes and the purified erythrocyte transporter. It also recognizes a Mr 40,000-60,000 polypeptide in membranes of cells derived from different mammalian species and tissues including insulin-sensitive rat adipocytes as well as a Mr 20,000 tryptic fragment of the transporter which contains the site for photolabeling by cytochalasin B. Antiserum to the carboxyl terminal of the transporter binds specifically to leaky erythrocyte membranes but not to intact erythrocytes. This binding is saturable and competitively inhibited by peptide 480-492. Using immunofluorescence microscopy, this antiserum detects glucose transporter protein in permeabilized erythrocytes, but not in intact erythrocytes. These studies provide immunochemical evidence in support of the predicted cytoplasmic orientation of the carboxyl terminus of the glucose transporter, allow us to suggest a spatial relationship of the cytochalasin B binding site to the carboxyl terminal of the glucose transporter and suggest that antisera directed to the carboxyl terminal domain of the protein may be useful for the immunocytochemical localization of the glucose transporter.
The slender bloodstream form of Trypanosoma brucei shows receptor-mediated endocytosis of low density lipoprotein (LDL) particles of its hosts. We have purified the LDL receptor of this species nearly to homogeneity (about 1000-fold purification) and obtained monospecific polyclonal antibodies against it. As analyzed by NaDodSO4/polyacrylamide gel electrophoresis, the purified receptor consists of a single subunit, with an apparent molecular mass of 86 kDa. Its isoelectric point is 5.9. On the average, each cell exposes 52,000 copies of low-affinity receptors (Kd of 250 nM) and 1800 copies of high-affinity receptors (Kd of 5.7 nM). According to indirect en bloc immunolabeling of fixed parasites, the receptor appears to be localized to the flagellar pocket membrane and the flagellar membrane and to be completely absent from the rest of the pericellular membrane. LDL is required for optimal growth of the trypanosome in vitro: cell growth can be inhibited either by removal of LDL from the culture medium or by antibodies against the purified LDL receptors. In both cases, growth is restored by the addition of excess LDL.
The uptake of a sugar across the boundary membrane is a primary event in the nutrition of most cells, but the hydrophobic nature of the transport proteins involved makes them difficult to characterize. Their amino-acid sequences can, however, be determined by cloning and sequencing the corresponding gene (or complementary DNA). We have determined the sequences of the arabinose-H+ and xylose-H+ membrane transport proteins of Escherichia coli. They are homologous with each other and, unexpectedly, with the glucose transporters of human hepatoma and rat brain cells. All four proteins share similarities with the E. coli citrate transporter. Comparisons of their sequences and hydropathic profiles yield insights into their structure, functionally important residues and possible evolutionary relationships. There is little apparent homology with the lactose-H+ (LacY) or melibiose-Na+ (MelB) transport proteins of E. coli.
Antibodies were raised in rabbits against synthetic peptides corresponding to the N-terminal (residues 1-15) and the C-terminal (residues 477-492) regions of the human erythrocyte glucose transporter. The antisera recognized the intact transporter in enzyme-linked immunosorbent assays (ELISA) and Western blots. In addition, the anti-C-terminal peptide antibodies were demonstrated, by competitive ELISA and by immunoadsorption experiments, to bind to the native transporter. Competitive ELISA, using intact erythrocytes, unsealed erythrocyte membranes, or membrane vesicles of known sidedness as competing antigen, showed that these antibodies bound only to the cytoplasmic surface of the membrane, indicating that the C terminus of the protein is exposed to the cytoplasm. On Western blots, the anti-N-terminal peptide antiserum labeled the glycosylated tryptic fragment of the transporter, of apparent Mr = 23,000-42,000, showing that this originates from the N-terminal half of the protein. The anti-C-terminal peptide antiserum labeled higher Mr precursors of the Mr = 18,000 tryptic fragment, although not the fragment itself, indicating that the latter, with its associated cytochalasin B binding site, is derived from the C-terminal half of the protein. Antiserum against the intact transporter recognized the C-terminal peptide on ELISA, and the Mr = 18,000 fragment but not the glycosylated tryptic fragment on Western blots.
An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin (IAPS-forskolin), has been synthesized, purified, and characterized. The I50 for inhibition of 3-O-methylglucose transport in red blood cells by IAPS-forskolin was found to be 0.05 microM. The carrier free radioiodinated label is a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes (ghosts) and purified glucose transporter preparations with 1-2 nM [125I]IAPS-forskolin and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed specific derivatization of a broad band with an apparent molecular mass of 40-70 kDa. Photoincorporation into erythrocyte membranes using 2 nM [125I]IAPS-forskolin was protected with D-glucose (I50 400 mM), cytochalasin B (I50 0.5 microM), and forskolin (I50 10 microM). No protection was observed with L-glucose (600 mM). Endo-beta-galactosidase digestion of [125I] IAPS-forskolin-labeled ghosts and purified transporter resulted in a dramatic sharpening of the specifically radiolabeled transporter to 40 kDa. Trypsinization of [125I]IAPS-forskolin-labeled ghosts and purified transporter reduced the specifically radiolabeled transporter to a sharp peak at 18 kDa. [125I]IAPS-forskolin will be a useful tool to study the structural aspects of the glucose transporter.
To test the effects of post-bloodmeal nutrition of sand flies on the transmission of Leishmania major, groups of infected P. papatasi females maintained on diets of sucrose, trehalose, albumin or a mixture of sucrose and albumin, were subjected to forced feeding with capillaries. Transmission was evaluated by counting the parasites egested; numbers ranged from 0 to over 1,000 promastigotes. Infections of the anterior midgut were seen in the majority of flies from all the experimental groups but the percentage of transmitting females was significantly higher in the group maintained on a mixture of sucrose and albumin. There were no attached parasites in the pharynx and cibarium of the flies and the presence of free promastigotes in these parts was not itself indicative of infectivity. However, transmission was positively correlated with apparent inability to engorge. The parasites egested were typical infective form promastigotes and identical to those observed in the esophagus and the anterior thoracic midgut. A mechanism by which infective stage promastigotes from the esophagus and the stomodeal valve may be transmitted by bite is proposed.
The plasma membrane glucose transporter of Leishmania donovani, an obligate intracellular protozoan parasite of humans, was specifically labeled, identified, and biochemically characterized. Cytochalasin B, a known inhibitor of D-glucose transport in mammalian cells, but not cytochalasin E inhibited the transport of 2-deoxy-D-glucose in the extracellular promastigote form of this organism. Hydroxysuccinimidyl-4-azido-benzoate was used to photochemically cross-link [3H] cytochalasin B to the glucose transporter in isolated surface membranes and plasma membrane vesicles of L. donovani promastigotes. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the covalently labeled glucose transporter migrated as a 20-30-kDa protein band. This band was eluted from SDS-PAGE gels and subsequently analyzed by isoelectric focusing. The latter revealed two major peaks focusing at pH 6.8 and 6.6 [3H]Cytochalasin B-labeled membrane activity was detergent-solubilized, bound to concanavalin A-agarose beads, and specifically eluted with alpha-methyl mannoside. Analysis of the eluted material by SDS-PAGE revealed a D-glucose-inhibitable cytochalasin B peak with an apparent Mr approximately 20,000. The cumulative results indicate that the glucose transporter of L. donovani promastigotes is a glycoprotein which contains mannose as its major carbohydrate constituent.
Evidence is presented for the occurrence of glycosomes (organelles resembling peroxisomes) in four major species of Leishmania (viz. L. major, L.m. mexicana, L. b. braziliensis and L. donovani), based on latency as well as differential and isopycnic centrifugation studies. The enzymes involved in glycolysis; (hexokinase, phosphoglucose isomerase, phosphofructokinase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase); glycerol metabolism (sn-glycerol-3-phosphate dehydrogenase and glycerol kinase); carbon dioxide fixation (phosphoenolpyruvate carboxykinase and possibly malate dehydrogenase); together with an enzyme involved in the beta-oxidation of fatty acids (3-beta-hydroxybutyryl coenzyme A dehydrogenase); a key enzyme in the synthesis of ether lipids (dihydroxyacetone phosphate acyltransferase) as well as the ADP utilising enzyme adenylate kinase, were all found associated, at least in part, with a subcellular organelle which had a buoyant density in sucrose gradients of 1.21 to 1.24 g cm-3. Little variance in enzyme composition was found between the different species of Leishmania or in comparison with other members of the Trypanosomatidae, supporting the unifying principle that glycosomes are a unique characteristic of this family. The occurrence of important catabolic, anabolic and anaplerotic pathways in the glycosomes of Leishmania renders them prime targets for chemotherapy.
A tandemly arranged multigene family encoding putative hexose transporters in Trypanosoma brucei has been characterized. It
is composed of two 80% homologous groups of genes called THT1 (six copies) and THT2 (five copies). When Xenopus oocytes are
microinjected with in vitro-transcribed RNA from a THT1 gene, they express a glucose transporter with properties similar to
those of the trypanosome bloodstream-form protein(s). This THT1-encoded transport system for glucose differs from the human
erythrocyte-type glucose transporter by its moderate sensitivity to cytochalasin B and its capacity to transport D-fructose.
These properties suggest that the trypanosomal transporter may be a good target for antitrypanosomal drugs. mRNA analysis
revealed that expression of these genes was life cycle stage dependent. Bloodstream forms express 40-fold more THT1 than THT2.
In contrast, procyclic trypanosomes express no detectable THT1 but demonstrate glucose-dependent expression of THT2.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.
GLUT-4 is the major facilitative glucose transporter isoform in tissues that exhibit insulin-stimulated glucose transport. Insulin regulates glucose transport by the rapid translocation of GLUT-4 from an intracellular compartment to the plasma membrane. A critical feature of this process is the efficient exclusion of GLUT-4 from the plasma membrane in the absence of insulin. To identify the amino acid domains of GLUT-4 which confer intracellular sequestration, we analyzed the subcellular distribution of chimeric glucose transporters comprised of GLUT-4 and a homologous isoform, GLUT-1, which is found predominantly at the cell surface. These chimeric transporters were transiently expressed in CHO cells using a double subgenomic recombinant Sindbis virus vector. We have found that wild-type GLUT-4 is targeted to an intracellular compartment in CHO cells which is morphologically similar to that observed in adipocytes and muscle cells. Sindbis virus-produced GLUT-1 was predominantly expressed at the cell surface. Substitution of the GLUT-4 amino-terminal region with that of GLUT-1 abolished the efficient intracellular sequestration of GLUT-4. Conversely, substitution of the NH2 terminus of GLUT-1 with that of GLUT-4 resulted in marked intracellular sequestration of GLUT-1. These data indicate that the NH2-terminus of GLUT-4 is both necessary and sufficient for intracellular sequestration.
The utilisation of substrates by Leishmania mexicana amastigotes and promastigotes differed significantly. The rates of uptake and catabolism of nonesterified fatty acids were up to 10-fold higher with amastigotes. Almost all the available exogenous fatty acids were consumed during amastigote transformation and by stationary phase of promastigote growth. The results suggest that fatty acids are important energy substrates for amastigotes, whereas promastigote utilisation may reflect the requirement for these substrates in anabolism. Glucose was utilised by amastigotes and promastigotes but the rate of catabolism was up to 10-fold higher in promastigotes. Uptake of glucose occurred throughout amastigote transformation and growth in vitro of promastigotes. High-subpassage promastigotes exhibited markedly lower glucose but higher amino acid utilisation than low-subpassage promastigotes. Asparagine, glutamine, glutamate, leucine, lysine, methionine, and threonine were consumed in large quantities by amastigotes and promastigotes, whereas alanine and glycine were excreted. Proline was catabolised to CO2 by amastigotes and promastigotes but only at a low rate, and it was excreted in large amounts throughout promastigote growth. The major end products of energy metabolism were found to be CO2 and succinate with both forms of the parasite and there was a secretion of up to 12 and 16% of the total protein synthesised by transforming amastigotes and growing promastigotes, respectively. Catabolism in amastigotes and promastigotes was found to be sensitive to cyanide and amytal, whereas 2-mercaptoacetate and 4-pentenoate primarily affected β-oxidation in the amastigote.
Leishmania mexicana mexicana amastigotes have been shown to contain greater activities than promastigotes of the enzymes that catalyse the β-oxidation of fatty acids, but lower activities of several glycolytic enzymes, with the activity of pyruvate kinase being especially low. The results suggest the β-oxidation of fatty acids is relatively more important to Leishmania amastigotes than promastigotes, whereas the reverse is true for glycolysis. Succinic dehydrogenase and peptidase activities were much higher in promastigotes than amastigotes. The activities of glucose-6-phosphatase, fructose-1,6-bisphosphatase, acid phosphatase and glucose-6-phosphate dehydrogenase varied less, although in each case the activity was significantly lower in the mammalian stage. A method for lysing and fractionating L. m. mexicana promastigotes has been developed. Using this procedure it has been established that many of the glycolytic and functionally related enzymes are located in cell organelles, that hexokinase is intimately connected with the particulate part of the parasite, and that the microsomal fraction of L. m. mexicana is very different in composition from the microsomes of mammalian liver cells.
The glucose transport system in Leishmania tropica promastigotes was characterized by the use of labeled 2-deoxy-D-glucose (2-DOG), a nonmetabolizable glucose analog. The uptake system has a Q10 of 2 and a heat of activation of 10.2 kcal/mole. The glucose transport system is subject to competitive inhibition by 2-DOG, glucosamine, N-acetyl glucosamine, mannose, galactose, and fructose which suggests that substitutions in the hexose chain at carbons 2 and 4 do not affect carrier specificity. In contrast, changes at carbon 1 (alpha-methyl-D-glucoside, 1,5-anhydroglucitol) and carbon 3 (3-0-methyl glucose) lead to loss of carrier affinity since these sugars do not compete for the glucose carrier. Sugars that compete with the glucose carrier have one common feature--they all exist in the pyranose form in solution. The carrier for D-glucose does not interact with L-glucose or any of the pentose sugars tested. Uptake of 2-DOG is inhibited by glycerol. This inhibition, however, is noncompetitive; it is evident; therefore, that glucose and glycerol do not compete for the same carrier. Glycerol does not repress the glucose carrier since cells grown in presence of glycerol transport the sugar normally.
The consumption of glucose by trypanosomatid protozoa such as Trypanosoma brucei, Trypanosoma cruzi, Leishmania spp., and Crithidia spp. is characterized by the excretion of reduced products such as succinate, pyruvate, ethanol, L-alanine, or lactate (depending on the species) not only in anaerobiosis, but also under aerobic conditions. The "aerobic fermentation" of glucose is accompanied by a complete lack, or even a reversal, of the Pasteur effect. This peculiar catabolism is mediated by a so-far unique compartmentation of the glycolytic enzymes, most of which are placed in an organelle called the glycosome; by an almost complete lack of inhibitory controls at the level of hexokinase and phosphofructokinase; and by a central role of CO2 fixation through the reaction catalyzed by phosphoenolpyruvate carboxykinase. The production of fermentative products seems to be due to a relative inefficiency of the respiratory chain, which lacks NADH dehydrogenase and the first phosphorylation site and preferentially uses succinate as substrate.
The polymerase chain reaction was used to clone two genes from Leishmania donovani, each of which encodes a member of a superfamily of membrane transporters which include the mammalian facilitative glucose transporters. One of these transporters, designated D2, is similar in sequence and overall structural features to a previously cloned Leishmania transporter Pro-1. Both D2 and Pro-1 are developmentally regulated genes which are expressed primarily in the insect stage of the parasite life cycle. In contrast, the second novel transporter, D1, is structurally quite different from either D2 or Pro-1, and its expression is not regulated during the parasite life cycle. All three genes are located on different chromosomes in L. donovani.
Promastigotes from late log phase and 3-day stationary phase cultures of Leishmania donovani were collected, washed in buffer, and the cell pellet was treated with boiling KOH. A putative carbohydrate storage material was then precipitated and washed in ethanol/LiBr. This material did not liberate glucose when treated with amyloglucosidase, indicating that it was not glycogen. Acid hydrolysis released a hexose which was identified as mannose by several criteria. Considerably more of this mannan-like carbohydrate is present in cells from 3-day stationary phase than from late log phase cultures, consistent with the ability of 3-day stationary phase cells to survive in non-nutrient buffer and maintain oxygen consumption for longer than log phase cells. The amount of this mannan-like compound decreased by over 50% during a 3-h incubation in buffer of cells from 3-day stationary phase cultures. The presence of glucose during the incubation prevented the utilization of this carbohydrate, consistent with the possibility that it serves as an energy reserve.
A cDNA cloned from Trypanosoma brucei brucei codes for a putative membrane protein which is homologous to the erythrocyte glucose transporter and several other sugar transporters from Escherichia coli, yeast, algae and Leishmania. This cDNA hybridizes to a 2.3-kb mRNA that accumulates to a much higher degree in the bloodstream mammalian form than in the procyclic insect form of the parasite. The correlation between the expression of this gene and the hexose metabolism of Leishmania enriettii and T. brucei suggest that these 2 related genes probably encode hexose transporters. The gene encoding this mRNA is a member of a multigene family. The putative hexose transporter gene is highly conserved among Kinetoplastidae, indicating an important role for this protein in the parasite life cycle.
A membrane transport protein of the glucose transporter superfamily from Leishmania enriettii is encoded by a family of tandemly
repeated genes. The first gene in this tandem repeat codes for a structural isoform that contains a unique amino-terminal
hydrophilic domain, probably located in the cytoplasm; the remainder of the protein is identical to the polypeptide encoded
by the internal genes in the tandem repeat. The unique isoform is represented by a distinct stable RNA.
The epitope tagging approach offers advantages of economy, universality, and precision over the use of antibodies raised directly against a protein of interest. The latter strategy promises a potentially greater diversity of reagents and obviates the need to modify the protein, but it may not yield sufficiently high-affinity, abundant, or specific antibodies. The major uncertainty in an epitope-tagging strategy, namely, the ability of the altered protein to function in vivo, is readily resolved in yeast by testing complementation of a null allele by the modified gene. Modification of the protein is easily accomplished by addition of the epitope coding sequence to the gene via oligonucleotide-mediated site-directed mutagenesis. The uniqueness of the epitope in the genome and the use of the monoclonal antibody assure a high-affinity, specific, and abundant antibody. Unrelated but identically modified proteins can be immunoprecipitated and affinity purified under the same conditions. Only extraction conditions and possibly a simple initial fractionation step need vary. Moreover, otherwise identical but differentially tagged proteins can be separated. Even proteins completely defective in an essential in vivo function can be purified and studied. Finally, polypeptides coprecipitating with the protein of interest are normally difficult to distinguish from those merely cross-reactive with the antibody used. As an alternative to defining a complex of proteins using a battery of antibodies, complexes are defined as a set of immunoprecipitable polypeptides present only in extracts containing the modified protein.
In African trypanosomes the requirements for glucose and its metabolism vary in different stages of the life cycle. Here we present evidence that cultured procyclic trypanosomes of Trypanosoma brucei rhodesiense uptake glucose against a concentration gradient in a time and dose-dependent manner. Moreover, glucose transport is completely inhibited by the sulphydryl inhibitor N-ethylmaleimide, suggesting the presence of a protein moiety as the carrier molecule. Comparison of glucose uptake in bloodstream and procyclic trypanosomes point to the possibility that different transporters may function in the 2 developmental stages. Glucose uptake by bloodstream trypanosomes requires Na+ ions and is inhibited by phlorizin, an inhibitor of Na(+)-dependent glucose transporters in mammalian cells. Conversely, procyclic trypanosomes transport glucose in a Na(+)-dependent manner, and transport is not affected by phlorizin. Finally, the putative procyclic glucose transporter has a higher affinity for glucose (apparent Km 23 microM) than the bloodstream carrier (apparent Km 237 microM).
The uptake of glucose into most eukaryotic cells is accomplished by a carrier-mediated transport system, facilitative diffusion, which transports glucose down its chemical gradient in a stereospecific manner. Recent studies have shown that facilitative transport of glucose across the plasma membrane is mediated by a family of structurally related proteins. This review summarizes the structural and functional features of the family of facilitative glucose transporters.
The characteristics of glucose transport by procyclic forms of Trypanosoma brucei were examined in a rapid transport assay using the glucose analogue 2-deoxyglucose. In contrast to bloodforms where the Km for 2-deoxyglucose transport is about 1 mM, procyclic forms have a Km of about 38 microM. Procyclic forms show temperature-dependent, saturable import, and import of 2-deoxyglucose is competitive with glucose and mannose. Unlike the bloodforms which employ facilitated diffusion, the procyclic forms actively transport glucose. Use of inhibitors and ionophores suggests that a protonmotive force is required for glucose transport in procyclic forms. Unlike the human erythrocyte glucose transporter, the glucose transporter of the T. brucei procyclic form is relatively insensitive to inhibition by cytocholasin B.
We have previously cloned a gene for a developmentally regulated transport protein from the trypanosomatid protozoan Leishmania enriettil. We demonstrate here that this transporter is encoded by a single family of tandemly clustered genes containing approximately
8 copies of the 3.6 kilobase repeat unit. Transcriptional mapping defines a contiguous 3.3 kilobase region of the repeat unit
that encodes the mRNA. The 5′ end of the mature mRNA contains the spliced leader or mlni-exon previously identified in kinetoplastid
protozoa, while the 3′ ends of the mRNA are heterogeneous in sequence and in location of the polyadenylation site. We have
identified genomic restriction fragments that flank the tandem repeat on the 5′ and 3′ sides and which may be linked to sequences
required for expression of the gene family. Other species of Leishmanla also contain sequences that hybridize to the cloned L. enriettil gene at high stringency.
Molecular cloning of cDNA encoding the human erythrocyte facilitated-diffusion glucose transporter (GT) has elucidated its structure and has permitted a careful study of its tissue distribution and of its involvement in processes such as insulin-stimulated glucose uptake by adipose cells or transformation-induced increase in glucose metabolism. An important outcome of these studies was the discovery that additional isoforms of this transporter were expressed in a tissue-specific manner; these comprise a family of structurally and functionally related molecules. Their tissue distribution, differences in kinetic properties, and differential regulation by ambient glucose and insulin levels suggest that they play specific roles in the control of glucose homeostasis. Herein, we will discuss the structure of three members of the GT family: erythroid/brain GT, liver GT, and adipose cell/muscle GT. In the light of their tissue-specific expression, kinetic parameters, and susceptibility to insulin action, we discuss their possible specific functions.
Organic substrates (sugars, amino acids, carboxylic acids and neutrotransmitters) are actively transported into eukaryotic cells by Na+ co-transport. Some of the transport proteins have been identified--for example, intestinal brush border Na+/glucose and Na+/proline transporters and the brain Na+/CI-/GABA transporter--and progress has been made in locating their active sites and probing their conformational states. The archetypical Na+-driven transporter is the intestinal brush border Na+/glucose co-transporter (see ref. 8), and a defect in the co-transporter is the origin of the congenital glucose-galactose malabsorption syndrome. Here we describe cloning of this co-transporter by a method new to membrane proteins. We have sequenced the cloned DNA and have found no homology between the Na+/glucose co-transporter and either the mammalian facilitated glucose carrier or the bacterial sugar transport proteins. This suggests that the mammalian Na+-driven transporter has no evolutionary relationship to the other sugar transporters.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme,
isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed
at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments
up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule
present only once in a sample of 10(5) cells.
Transport of 6-deoxy-D-glucose was studied in Trypanosoma brucei in order to characterise the kinetics of hexose transport in this organism using a nonphosphorylated sugar. Kinetic parameters for efflux and entry, measured using zero-trans and equilibrium exchange protocols, indicate that the transporter is probably kinetically symmetrical. Comparison of the kinetic constants of D-glucose metabolism with those for 6-deoxy-D-glucose transport shows that transport across the plasma membrane is likely to be the rate-limiting step of glucose utilisation. The transport rate is nevertheless very fast and 6-deoxy-D-glucose, at concentrations below Km, enters the cells with a half filling time of less than 2 s at 20 degrees C. Thus the high metabolic capacity of these organisms is matched by a high transport rate. The structural requirements for the trypanosome hexose transporter were explored by measuring inhibition constants (Ki) for a range of D-glucose analogues including fluoro and deoxy sugars as well as epimeric hexoses. The relative affinities shown by these analogues indicated H-bonds from the carrier to the C-3, C-4 and C-5 hydroxyl oxygens and from the C-1 and C-3 hydroxyl hydrogens to the binding site. Hydrophobic interactions are likely at the C-2 and C-6 regions of the glucose molecule. Spatial constraints appear to occur around C-4 indicating that the transport site at this position is not freely open to the external solution as is the case with the mammalian hexose transporter. However, the trypanosome transporter appears to accept D-fructose but the common mammalian (erythrocyte type) hexose transporter does not.
Midlogarithmic phase Leishmania donovani promastigotes accumulate 2-deoxy-D-glucose (2-dGlc) and L-proline, maintaining concentration gradient factors across the surface membrane of 78.7 and 60, respectively. Cyanide (1 mM) and iodoacetate (0.5 mM) inhibited the transport of both substrates. L-proline uptake was also inhibited by 2-dGlc (10 mM). Transport of neither substrate was affected by Na+, phlorizin, or ouabain, indicating the sodium-independent transport of both systems. However, N',N'-dicyclohexylcarbodiimide (DCCD; 20 microM) significantly inhibited the transport of both 2-dGlc and L-proline (70% and 90%, respectively). The ionophores valinomycin (1 microM) and nigericin (5 microM) each partially inhibited the uptake of both substrates. In parallel experiments, nigericin and valinomycin were added concomitantly to promastigotes, each at a concentration that individually inhibited the transport of 2-dGlc and L-proline by less than 30%. Under such conditions, the transport of 2-dGlc and L-proline was inhibited by 69% and 78%, respectively. However, these ionophores had no significant effect on the promastigotes cellular ATP level. Carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 1 microM) inhibited 2-dGlc (79%) and L-proline (85%) transport, whereas ATP levels of such cells were diminished by only 20%. Symport of D-glucose/H+ and L-proline/H+ was measured directly in cells pretreated with KCN and DCCD. Upon addition of D-glucose to such cells, a rapid movement of protons into the organisms occurred and was reversed upon addition of FCCP. Conversely, no proton movement was observed when L-glucose was added to such cells. L-proline, as D-glucose, caused a rapid influx of protons into the promastigotes, indicating that both substrates were cotransported with protons. We conclude that transport of D-glucose and L-proline in L. donovani promastigotes is protonmotive force-driven and is coupled to both delta pH and delta psi.
Recent advances in characterizing the molecular structure and kinetic properties of the human erythrocyte glucose transporter are now starting to give an insight into the dynamics of the transporter.
A transport system for ribose in Leishmania donovani promastigotes was identified and characterized by measuring the uptake of radioisotope-labeled ribose. The pentoses arabinose, 2-deoxyribose and xylose inhibited ribose uptake, whereas hexoses (glucose, alpha-methylglucoside, thioglucose, galactose, lactose, maltose, mannose), adenosine, and proline did not inhibit uptake, indicating that the transporter exhibited substrate specificity. Intracellular ribose exchanged with 2-deoxyribose. Uptake of ribose showed saturation kinetics with an apparent Km = 2 mM and Vmax = 11 nmol (mg protein)-1 min-1. Both N-ethylmaleimide and p-hydroxymercuribenzoate inhibited ribose uptake which was prevented by dithiothreitol. The uncoupling agents 2,4-dinitrophenol and carbonylcyanide p-(trifluoromethoxy)phenylhydrazone and a variety of inhibitors of energy-driven transport had no significant effect on ribose uptake. Following transport, the intracellular ribose pool contained two-thirds of the sugar in the phosphorylated form and one-third in the neutral form. These cumulative results indicate that a specific carrier mediates ribose uptake via a facilitated diffusion system in L. donovani promastigotes.
The SNF3 gene is required for high-affinity glucose transport in the yeast Saccharomyces cerevisiae and has also been implicated in control of gene expression by glucose repression. We report here the nucleotide sequence of the cloned SNF3 gene. The predicted amino acid sequence shows that SNF3 encodes a 97-kilodalton protein that is homologous to mammalian glucose transporters and has 12 putative membrane-spanning regions. We also show that a functional SNF3-lacZ gene-fusion product cofractionates with membrane proteins and is localized to the cell surface, as judged by indirect immunofluorescence microscopy. Expression of the fusion protein is regulated by glucose repression.
The amino acid sequence of the glucose transport protein from human HepG2 hepatoma cells was deduced from analysis of a complementary DNA clone. Structural analysis of the purified human erythrocyte glucose transporter by fast atom bombardment mapping and gas phase Edman degradation confirmed the identity of the clone and demonstrated that the HepG2 and erythrocyte transporters are highly homologous and may be identical. The protein lacks a cleavable amino-terminal signal sequence. Analysis of the primary structure suggests the presence of 12 membrane-spanning domains. Several of these may form amphipathic alpha helices and contain abundant hydroxyl and amide side chains that could participate in glucose binding or line a transmembrane pore through which the sugar moves. The amino terminus, carboxyl terminus, and a highly hydrophilic domain in the center of the protein are all predicted to lie on the cytoplasmic face. Messenger RNA species homologous to HepG2 glucose transporter messenger RNA were detected in K562 leukemic cells, HT29 colon adenocarcinoma cells, and human kidney tissue.
A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.
Mid-log phase Leishmania donovani promastigotes accumulated 2-deoxy-D-glucose (2-DOG) via a carrier mediated transport system, maintaining an apparent Km of 24.4 microM and a Vmax of 3.12 nmol mg-1 protein min-1. D-Glucose but not L-glucose competitively inhibited the 2-DOG transport with an apparent Ki of 18.7 microM. Transport of 2-DOG was inhibited by 2,4-dinitrophenol and NaN3. The parasites maintained a 2-DOG gradient of at least 79 fold across the surface membrane, demonstrating the active nature of the transport system.
An algorithm has been developed which identifies alpha-helices involved in the interactions of membrane proteins with lipid bilayers and which distinguishes them from helices in soluble proteins. The membrane-associated helices are then classified with the aid of the hydrophobic moment plot, on which the hydrophobic moment of each helix is plotted as a function of its hydrophobicity. The magnitude of hydrophobic moment measures the amphiphilicity of the helix (and hence its tendency to seek a surface between hydrophobic and hydrophilic phases), and the hydrophobicity measures its affinity for the membrane interior. Segments of membrane proteins in alpha-helices tend to fall in one of three regions of a hydrophobic moment plot: (1) monomeric transmembrane anchors (class I HLA transmembrane sequences) lie in the region of highest hydrophobicity and smallest hydrophobic moment; (2) helices presumed to be paired (such as the transmembrane M segments of surface immunoglobulins) and helices which are bundled together in membranes (such as bacteriorhodopsin) fall in the adjacent region with higher hydrophobic moment and smaller hydrophobicity; and (3) helices from surface-seeking proteins (such as melittin) fall in the region with still higher hydrophobic moment. alpha-Helices from globular proteins mainly fall in a region of lower mean hydrophobicity and hydrophobic moment. Application of these methods to the sequence of diphtheria toxin suggests four transmembrane helices and a surface-seeking helix in fragment B, the moiety known to have transmembrane function.
A simple technique, developed for the isolation of clones derived from single, promastigote cells of Leishmania donovani and Leishmania tropica, involved the use of semisolid agar. Both species of Leishmania promastigotes formed discrete colonies at high efficiency either in semidefined medium containing 10% fetal calf serum or in completely-defined medium lacking serum. Visible colonies appeared between 8 and 14 days in growth medium containing 10% fetal calf serum. Replacement of the fetal calf serum with bovine serum albumin and Tween-80 increased the time of colony formation by 50% but did not affect the cloning efficiency. Viability of colonies transferred from semisolid agar to liquid suspension culture was 100%.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.