RESUMEN
Glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) is an integral membrane protein in the protozoan parasite Trypanosoma brucei. Enzyme activity appears to be suppressed in T. brucei, although the polypeptide is readily detectable. The basis for the apparent quiescence of GPI-PLC is not known. Protein oligomerization was investigated as a possible mechanism for post-translational regulation of GPI-PLC activity. An equilibrium between monomers, dimers, and tetramers of purified GPI-PLC was detected by molecular sieving and shown to be perturbed with specific detergents. Homotetramers dominated in Nonidet P-40, and dimers and monomers of GPI-PLC were the major species in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The detergents were exploited as tools to study the effect of oligomerization on enzyme activity. Tetrameric GPI-PLC was 3. 6-20-fold more active than the monomeric enzyme. Tetramer existence was confirmed by chemical cross-linking. In vivo cross-linking revealed the oligomeric state of GPI-PLC during latency and after enzyme activation. During quiescence, monomers were the predominant species in T. brucei. Assembly of tetrameric GPI-PLC occurred when parasites were subjected to conditions known to activate the enzyme. In Leishmania where heterologous expression of GPI-PLC causes a GPI deficiency, the enzyme existed as a tetramer. Hence, oligomerization of GPI-PLC is associated with high enzyme activity both in vivo and in vitro.
Asunto(s)
Trypanosoma brucei brucei/enzimología , Fosfolipasas de Tipo C/metabolismo , Animales , Biopolímeros , Ácidos Cólicos , Reactivos de Enlaces Cruzados/química , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Glicosilfosfatidilinositol Diacilglicerol-Liasa , Fosfatidilinositol Diacilglicerol-Liasa , Conformación Proteica , Procesamiento Proteico-Postraduccional , Fosfolipasas de Tipo C/químicaRESUMEN
Stable transformation depends on the efficient delivery of DNA into cells and the robust expression of genes that encode proteins which provide resistance to selective (cytotoxic) compounds. We have examined the possibility that altering the 5'untranslated region (UTR) of a selectable marker may increase transformation efficiency. A 15-nucleotide synthetic UTR (the so-called universal translational enhancer [UTE]) was placed upstream of a kanamycin/neomycin phosphotransferase (kanaR) gene to create a novel expression cassette, UTE-kanaR. In comparison to a wild-type version of kanaR, UTE-kanaR produced up to 30-fold more transformants in E. coli. The superior performance of UTE-kanaR was independent of the promoter strength, indicating that the gene may find general use in routine transformation experiments.
Asunto(s)
Kanamicina Quinasa/genética , Transformación Genética/genética , Regiones no Traducidas 5'/genética , Cartilla de ADN , Escherichia coli , Resistencia a la Kanamicina/genética , Plásmidos , Regiones Promotoras GenéticasRESUMEN
Glycosylphosphatidylinositol-specific phospholipase C (GPtdIns-PLC) is found in the protozoan parasite Trypanosoma brucei. A region of protein sequence similarity exists between the protozoan enzyme and eubacterial phosphatidylinositol-phospholipases C. The functional relevance of Cys80 and Gln81 of GPtdIns-PLC, both in this region, was tested with a panel of mutations at each position. Gln81Glu, Gln81Ala, Gln81Gly, Gln81Lys and Gln81Leu mutants were inactive. Cleavage of GPtdIns was detectable in Gln81Asn, although the specific activity decreased 500-fold, and kcat was reduced 50-fold. Thus an amide side-chain at residue 81 is essential for catalysis by GPtdIns-PLC. Sulfhydryl reagents inactivate GPtdIns-PLC, suggesting that a Cys could be close to the enzyme active site. Surprisingly, p-chloromercuriphenyl sulfonate (p-CMPS) is significantly more potent than N-ethylmaleimide, the less bulky compound. This knowledge prompted us to test whether replacement of Cys80 with an amino acid possessing a bulky side-chain would inactivate GPtdIns-PLC: Cys80Ala, Cys80Thr, Cys80Phe, Cys184Ala, and Cys269-270-273Ser were constructed for that purpose. Cys80Phe lacked enzyme activity, while Cys80Ala, Cys80Thr and Cys269-270-273Ser retained 33 to 100% of wild-type activity. Interestingly, the Cys80Ala and Cys80Thr mutants became resistant to p-CMPS, as predicted if the sulfhydryl reagent reacted with Cys80 in the wild-type enzyme to form a cysteinyl mercurylphenylsulfonate moiety, a bulky adduct that inactivated GPtdIns-PLC, similar to the Cys80Phe mutation. We conclude that a bulky side-chain (or adduct) at position 80 of GPtdIns-PLC abolishes enzyme activity. Together, these observations place Cys80 and Gln81 at, or close to, the active site of GPtdIns-PLC from T. brucei.
Asunto(s)
Trypanosoma brucei brucei/enzimología , Fosfolipasas de Tipo C/química , Fosfolipasas de Tipo C/metabolismo , 4-Cloromercuribencenosulfonato/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Dominio Catalítico/genética , Cisteína/química , Cartilla de ADN/genética , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Glutamina/química , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfatidilinositol Diacilglicerol-Liasa , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Trypanosoma brucei brucei/genética , Fosfolipasas de Tipo C/genéticaRESUMEN
Glycosylated phosphatidylinositols (GPIs) are abundant cell surface molecules of the Leishmania. Amastigote-specific GPIs AmGPI-Y and AmGPI-Z, both ethanolamine (EtN)-containing glycolipids, were identified in Leishmania amazonensis. A paucity of GPI-anchored proteins in amastigotes of L. amazonensis made the kinetoplastid suitable for evaluating the importance of free (i.e. unconjugated to protein or polysaccharide) GPIs. A strain deficient in both AmGPI-Y and AmGPI-Z was produced by stable transfection of wild-type Leishmania with a GPI-phospholipase C gene. Phosphatidylinositol deficiency was not detected in the transfectants. GPI-deficient promastigotes infected murine macrophages in vitro and differentiated into amastigotes whose growth was arrested within the host cells. Cytostasis of amastigotes was also observed during axenic culture of GPI-deficient parasites. In a hamster model of leishmaniasis, GPI-deficient promastigotes produced smaller lesions with 20-fold fewer amastigotes than infections with control parasites. Together, these observations indicate that EtN-GPIs may be essential for amastigote viability, replication, and/or virulence. Implicit in these observations is the notion that drugs targeted against the GPI biosynthetic pathway might be of value in the management of human leishmaniasis.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Leishmania mexicana/crecimiento & desarrollo , Leishmaniasis Cutánea/parasitología , Macrófagos/parasitología , Animales , Cricetinae , Glucolípidos/análisis , Glucolípidos/aislamiento & purificación , Glicosilfosfatidilinositoles/química , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Leishmania mexicana/patogenicidad , Mesocricetus , Ratones , Polisacáridos/análisis , Transfección , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismo , VirulenciaRESUMEN
Reversible esterification of myristic acid to cysteine residue(s) (S-myristoylation) was documented recently in the protozoan Trypanosoma brucei. Unlike N-myristoylation, S-myristoylation appears to be rare (or non-existent) in animal cells and has not been documented in any other trypanosome. Reasoning that a lack of knowledge of appropriate substrates may have contributed to this state of affairs, we devised an assay to test for protein S-myristoylation in the ancient eukaryote Leishmania. A cDNA encoding a glycosylphosphatidylinositol-phospholipase C (GPI-PLC) from T. brucei was transfected into Leishmania and the expressed protein analyzed for covalent lipid modifications. Leishmania modified the reporter with myristate in a thio-ester linkage. From these observations, we infer that (i) GPI-PLC may be used as a reporter of this lipid modification in eukaryotes, and (ii) protein S-myristoylation might have ancient origins.
Asunto(s)
Leishmania major/metabolismo , Ácido Mirístico/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Membrana Celular/metabolismo , Cisteína/metabolismo , Evolución Molecular , Ácidos Grasos/análisis , Ácidos Grasos/metabolismo , Genes Reporteros , Glicosilfosfatidilinositol Diacilglicerol-Liasa , Hidroxilamina/farmacología , Leishmania major/enzimología , Leishmania major/genética , Metabolismo de los Lípidos , Lípidos/química , Peso Molecular , Ácido Mirístico/análisis , Fosfatidilinositol Diacilglicerol-Liasa , Pruebas de Precipitina , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfección , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genética , Fosfolipasas de Tipo C/química , Fosfolipasas de Tipo C/genéticaRESUMEN
Covalent modification with lipid can target cytosolic proteins to biological membranes. With intrinsic membrane proteins, the role of acylation can be elusive. Herein, we describe covalent lipid modification of an integral membrane glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from the kinetoplastid Trypanosoma brucei. Myristic acid was detected on cysteine residue(s) (i.e. thiomyristoylation). Thiomyristoylation occurred both co- and post-translationally. Acylated GPI-PLC was active against variant surface glycoprotein (VSG). The half-life of fatty acid on GPI-PLC was 45 min, signifying the dynamic nature of the modification. Deacylation in vitro decreased activity of GPI-PLC 18-30-fold. Thioacylation, from kinetic analysis, activated GPI-PLC by accelerating the conversion of a GPI-PLC.VSG complex to product. Reversible thioacylation is a novel mechanism for regulating the activity of a phospholipase C.
Asunto(s)
Ácido Mirístico/metabolismo , Trypanosoma brucei brucei/enzimología , Fosfolipasas de Tipo C/metabolismo , Acilación , Animales , Esterificación , Ácidos Grasos/metabolismo , Glicosilfosfatidilinositol Diacilglicerol-Liasa , Cinética , Ácido Palmítico/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa , Procesamiento Proteico-Postraduccional , Ratas , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
Efficient ribosomal protein synthesis is dependent on cis-acting elements in the 5' untranslated region (UTR) of mRNAs. Between prokaryotes and eukaryotes, the sequence and location of these elements differ to the extent of not being functionally interchangeable. We explored the possibility of constructing bifunctional UTRs that could direct translation in both prokaryotes and eukaryotes. A variant of a UTR from ner of phage Mu (ner-ACC) enhanced protein synthesis in a rabbit reticulocyte lysate, and it was compared to a lacZ-CTA, containing the lambda cro RBS and the Escherichia coli lacZ spacer. Several mutants in the -3 to -1 regions of both lacZ-CTA and ner-ACC were tested in rabbit reticulocyte lysate and E. coli to select UTRs that were optimized simultaneously for both biological kingdoms. The lacZ-ATC proved 217-fold more effective than ner-ACC in this cross-species ability to enhance translation. The lacZ-ACC and ner-ATC were 83- and 78-fold, respectively, better than ner-ACC. We conclude that short UTRs (12-15 nt in length) can be fine-tuned in the -9 to -1 regions to enhance protein synthesis concurrently in prokaryotes and eukaryotes. In related studies, we show that nt at the -3 to -1 region of mRNAs exert an enormous impact on synthesis of proteins in E. coli.
Asunto(s)
Regiones no Traducidas 5' , Biosíntesis de Proteínas , Animales , Bacteriófago mu , Sitios de Unión , Sistema Libre de Células , Escherichia coli , Células Eucariotas , Células Procariotas , Conejos , Reticulocitos , Ribosomas/metabolismoRESUMEN
N-Terminal signal peptides direct secretory and most membrane proteins into the exocytic pathway at the endoplasmic reticulum. Signal sequences can function across kingdoms. However, our attempts at translocating variant surface glycoprotein (VSG) 117, VSG MVAT7, VSG 221 and BiP from Trypanosoma brucei and gp63 from Leishmania chagasi into canine pancreas microsomes failed. On replacing the signal peptide of VSG 117 with that from yeast prepro-alpha-mating factor (ppalphaMF) the chimaeric protein was imported, indicating that the signal sequence of VSG 117 was incompatible with the protein-import machinery of mammalian microsomes. Replacement of the gp63-h-region with a hybrid composed of the N-terminal nine residues from the h-region of gp67 from Autographa californica nuclear polyhedrosis virus and the C-terminal 10 residues from the h-region of gp63 from L. major produced a functional signal peptide. Thus, the h-region of kinetoplastid signal peptides appears to be the subdomain that is non-functional at the mammalian translocon. The calculated biophysical properties and computed discriminant scores (predictive of importability of signal peptides into mammalian microsomes) of the kinetoplastid signal sequences nevertheless are similar to those of ppalphaMF and Escherichia coli beta-lactamase both of which were imported. These signal peptides are the first collection from one biological family that have been found to fail to function across a species barrier. They indicate that signal peptides are not as universally interchangeable as previously believed. Intriguingly, endoplasmic reticulum signal peptides from Leishmania and Crithidia fasciculata are reminiscent of signal peptides from Gram-positive bacteria.
Asunto(s)
Retículo Endoplásmico/metabolismo , Leishmania/química , Metaloendopeptidasas/metabolismo , Señales de Clasificación de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae , Trypanosoma brucei brucei/química , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismo , Secuencia de Aminoácidos , Animales , Perros , Proteínas Fúngicas , Bacterias Grampositivas/química , Metaloendopeptidasas/química , Microsomas/metabolismo , Datos de Secuencia Molecular , Páncreas/ultraestructura , Precursores de Proteínas , Señales de Clasificación de Proteína/química , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la Especie , Glicoproteínas Variantes de Superficie de Trypanosoma/químicaRESUMEN
Glycosyl phosphatidylinositol phospholipase C (GPI-PLC) of Trypanosoma brucei is inhibited by myo-inositol(Ins)-1-O-dodecylphosphonate (VP-602L). Several novel fluoro-substituted analogs of 2-deoxy-myo-Ins-1-O-dedecylphosphonate, among which 2-deoxy-2-fluoro-scyllo-Ins-1-O-dodecylphosphonate (VP-616L) was the most powerful, were shown to be competitive inhibitors of GPI-PLC. VP-616L was 14-fold more active than VP-602L. 2-Deoxy-2-fluoro-myo-Ins-1-O-dodecylphosphonate and 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate were 1.55- and 4.67-fold, respectively, more potent than VP-602L. Methyl 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate did not inhibit GPI-PLC. These observations provide several insights into how GPI-PLC might interact with its substrate at the active site. We surmise that (i) the 2-OH of Ins is probably dispensable for substrate recognition; (ii) an equatorially oriented active site residue might interact with substituents at the 2-position of Ins, and (iii) the negative charge on the phosphoryl at the 1-OH position of Ins might be important for substrate recognition.
Asunto(s)
Inositol/análogos & derivados , Trypanosoma brucei brucei/enzimología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Animales , Relación Dosis-Respuesta a Droga , Flúor , Glicosilfosfatidilinositol Diacilglicerol-Liasa , Inositol/farmacología , Modelos Químicos , Organofosfonatos/farmacología , Compuestos Organofosforados/farmacología , Fosfatidilinositol Diacilglicerol-Liasa , Relación Estructura-Actividad , Fosfolipasas de Tipo C/metabolismoRESUMEN
Recent emergence of microbial resistance to aminoglycoside antibiotics, and the documented cytotoxicity associated with their use, calls for sustained efforts at understanding the effects of the compounds on eukaryotic cells. Using a glycosyl phosphatidylinositol (GPI)-phospholipase C (GPI-PLC) from the protozoan parasite Trypanosoma brucei, we demonstrate that a eukaryotic PLC can be activated 6-fold by aminoglycosides. Neomycin B protected GPI-PLC from a reduction in activity at pH 6.5, and increased the turnover number (kcat) of the enzyme. In structure-activity studies with the neomycin group, 2-deoxy-streptamine was mildly stimulatory; the concentration required to activate GPI-PLC 2-fold (SC200) was 310 microM. Neamine was 150-fold more active (SC200 = 2 microM) than 2-deoxy-streptamine, indicating that a 2,6-dideoxy-2, 6-diaminoglucose substituent at the 4-position of 2-deoxystreptamine plays an important role in activation of GPI-PLC. Ribostamycin and neomycin B also had SC200's of 2 microM, implying that the ribose group in ribostamycin is not involved in activation of GPI-PLC. These conclusions were affirmed in studies with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C. A 2, 6-dideoxy-2,6-diaminoglucose substitution at the 4-OH of 2-deoxystreptamine activates the enzyme 17-fold, while a second 2, 6-dideoxy-2,6-diaminoglucose moiety on the ribose ring of ribostamycin provides an additional 3.5-fold stimulation. Possible implications of these observations for the effects of aminoglycosides on eukaryote cells are discussed.
Asunto(s)
Antibacterianos/farmacología , Glucosamina/análogos & derivados , Fosfolipasas de Tipo C/metabolismo , Animales , Bacillus thuringiensis/enzimología , Activación Enzimática , Framicetina/farmacología , Glucosamina/metabolismo , Glicosilfosfatidilinositol Diacilglicerol-Liasa , Concentración de Iones de Hidrógeno , Cinética , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Relación Estructura-Actividad , Trypanosoma brucei brucei/enzimología , Fosfolipasas de Tipo C/químicaRESUMEN
Induction of a glycosylphosphatidylinositol (GPI) deficiency in Trypanosoma cruzi by the heterologous expression of Trypanosoma brucei GPI-phospholipase C (GPI-PLC) results in decreased expression of major surface proteins (N. Garg, R. L. Tarleton, and K. Mensa-Wilmot, J. Biol. Chem. 272:12482-12491, 1997). To further explore the consequences of a GPI deficiency on replication and differentiation of T. cruzi, the in vitro and in vivo behaviors of GPI-PLC-expressing T. cruzi were studied. In comparison to wild-type controls, GPI-deficient T. cruzi epimastigotes exhibited a slight decrease in overall growth potential in culture. In the stationary phase of in vitro growth, GPI-deficient epimastigotes readily converted to metacyclic trypomastigotes and efficiently infected mammalian cells. However, upon conversion to amastigote forms within these host cells, the GPI-deficient parasites exhibited a limited capacity to replicate and subsequently failed to differentiate into trypomastigotes. Mice infected with GPI-deficient parasites showed a substantially lower rate of mortality, decreased tissue parasite burden, and a moderate tissue inflammatory response in comparison to those of mice infected with wild-type parasites. The decreased virulence exhibited by GPI-deficient parasites suggests that inhibition of GPI biosynthesis is a feasible strategy for chemotherapy of infections by T. cruzi and possibly other intracellular protozoan parasites.
Asunto(s)
Glicosilfosfatidilinositoles/deficiencia , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/patogenicidad , Animales , Enfermedad de Chagas/parasitología , Femenino , Glicosilfosfatidilinositol Diacilglicerol-Liasa , Ratones , Ratones Endogámicos C3H , Fosfatidilinositol Diacilglicerol-Liasa , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismo , Virulencia/genéticaRESUMEN
Glycosylphosphatidylinositols (GPIs) are membrane anchors for cell surface proteins of several major protozoan parasites of humans, including Trypanosoma cruzi, the causative agent of Chagas' disease. To investigate the general role of GPIs in T. cruzi, we generated GPI-deficient parasites by heterologous expression of T. brucei GPI-phospholipase C. Putative protein-GPI intermediates were depleted, causing the biochemical equivalent of a dominant-negative loss of function mutation in the GPI pathway. Cell surface expression of major GPI-anchored proteins was diminished in GPI-deficient T. cruzi. Four proteins that are normally GPI-anchored in T. cruzi exhibited different fates during the GPI shortage; Ssp-4 and p75 were secreted prematurely, while protease gp50/55 and p60 were degraded intracellularly. These observations demonstrate that secretion and intracellular degradation of GPI-anchored proteins may occur in the same genetic background during a GPI deficiency. We postulate that the interaction between a protein-GPI transamidase and the COOH-terminal GPI signal sequence plays a pivotal role in determining the fate of these proteins. At a nonpermissive GPI deficiency, T. cruzi amastigotes inside mammalian cells replicated their single kinetoplast but failed at mitosis. Hence, in these protozoans, GPIs appear to be essential for nuclear division, but not for mitochondrial duplication.
Asunto(s)
Glicosilfosfatidilinositoles/química , Proteínas Protozoarias/química , Animales , División Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Glicosilfosfatidilinositoles/deficiencia , Glicosilfosfatidilinositoles/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma cruziRESUMEN
Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus is inhibited by myo-inositol-1-O-dodecylphosphonate (Ins-1-O-dodecylphosphonate) (Morris, J. C., Ping-Sheng, L., Shen, T. Y., and Mensa-Wilmot, K.(1995) J. Biol. Chem. 270, 2517-2524). A set of novel fluorinated 2-deoxy-Ins-1-O-dodecylphosphonates were tested against PI-PLC, with potent competitive inhibition by 2-deoxy-2-fluoro-scyllo-Ins-1-O-dodecylphosphonate (VP-616L) (Xi(50) = 0.09). 2-Deoxy-2-fluoro-myo-Ins-1-O-dodecylphosphonate and 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate were 8.3-fold and 4.8-fold less effective, respectively, than VP-616L. Methyl 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate was inactive. Also, a hundredfold less PI-PLC is required to cleave a glycosylphosphatidylinositol (GPI) than is needed to cleave PI. Implied in these observations are the following: (i) in powerful inhibitors an active site residue probably interacts with the equatorially oriented fluoro substituent; (ii) substrate recognition requires a negative charge on the phosphoryl at the Ins-1 position, and (iii) a GPI is better substrate than PI, for PI-PLC. Aminoglycoside antibiotics kanamycin A, gentamycin, and G418 stimulated PI-PLC cleavage of the GPI anchor of variant surface glycoprotein (VSG) from Trypanosoma brucei 2- to 4-fold. G418, which appears to act on the enzyme.substrate complex, increased kcat and Km 6.4-fold and 9.9-fold, respectively. PI-PLC was activated by G418 even in the presence of the inhibitor VP-616L. In control experiments, the lectin concanavalin A (ConA), which probably acts by substrate sequestration, inhibited both PI-PLC (Xi(50) = 0.00025) and GPI-specific phospholipase D (Xi(50) = 0.00018). G418 failed to activate PI-PLC when ConA was present. These observations indicate that G418 is an allosteric activator of Bacillus cereus PI-PLC. Since G418 stimulates a purified enzyme that is not involved in aminoglycoside metabolism, we propose that binding of aminoglycosides to cellular proteins could contribute to the development of the nephrotoxicity associated with the use of these aminoglycoside antibiotics.
Asunto(s)
Antibacterianos/farmacología , Activación Enzimática/efectos de los fármacos , Gentamicinas/farmacología , Glicosilfosfatidilinositoles/metabolismo , Inositol/análogos & derivados , Organofosfonatos , Hidrolasas Diéster Fosfóricas/metabolismo , Regulación Alostérica , Animales , Bacillus cereus/enzimología , Unión Competitiva , Bovinos , Células Cultivadas , Concanavalina A/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Inositol/farmacología , Cinética , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Relación Estructura-Actividad , Especificidad por Sustrato , Fosfolipasas de Tipo C/antagonistas & inhibidoresAsunto(s)
ADN Ribosómico/genética , Biosíntesis de Proteínas , Ribosomas/metabolismo , Fosfatasa Alcalina/genética , Animales , Secuencia de Bases , Sitios de Unión , Cartilla de ADN/genética , ADN Bacteriano/genética , ADN Complementario/genética , ADN Protozoario/genética , Escherichia coli/enzimología , Escherichia coli/genética , Técnicas In Vitro , Operón Lac , Datos de Secuencia Molecular , Fosfatidilinositol Diacilglicerol-Liasa , Hidrolasas Diéster Fosfóricas/genética , Conejos , Reticulocitos/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genéticaRESUMEN
Glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosoma brucei and phosphatidylinositol phospholipase C (PI-PLC) from Bacillus sp. both cleave glycosylphosphatidylinositols (GPIs). However, phosphatidylinositol, which is efficiently cleaved by PI-PLC, is a very poor substrate for GPI-PLC. We examined GPI-PLC substrate requirements using glycoinositol analogs of GPI components as potential inhibitors. Glucosaminyl (alpha 1-->6)-D-myo-inositol (GlcN(alpha 1-->6)Ins), GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate, GlcN(alpha 1-->6)-2-deoxy-Ins, and GlcN(alpha 1-->6)Ins 1-dodecyl phosphonate inhibited GPI-PLC. GlcN(alpha 1-->6)Ins was as effective as Man-(alpha 1-->4)GlcN(alpha 1-->6)Ins; we surmise that GlcN(alpha 1-->6)Ins is the crucial glycan motif for GPI-PLC recognition. Inhibition by GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate suggests product inhibition since GPIs cleaved by GPI-PLC possess a GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate at the terminus of the residual glycan. The effectiveness of GlcN(alpha 1-->6)-2-deoxy-Ins indicates that the D-myo-inositol (Ins) 2-hydroxyl is not required for substrate recognition, although it is probably essential for catalysis. GlcN(alpha 1-->6)-2-deoxy-L-myo-inositol, unlike GlcN(alpha 1-->6)-2- deoxy-Ins, had no effect on GPI-PLC; hence, GPI-PLC can distinguish between the two enantiomers of Ins. Surprisingly, GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate was not a potent inhibitor of Bacillus cereus PI-PLC, and GlcN(alpha 1-->6)Ins had no effect on the enzyme. However, both GlcN(alpha 1-->6)Ins 1-phosphate and GlcN(alpha 1-->6)Ins 1-dodecyl phosphonate were competitive inhibitors of PI-PLC. These observations suggest an important role for a phosphoryl group at the Ins 1-position in PI-PLC recognition of GPIs. Other studies indicate that abstraction of a proton from the Ins 2-hydroxyl is not an early event in PI-PLC cleavage of GPIs. Furthermore, both GlcN(alpha 1-->6)-2-deoxy-Ins 1-phosphate and GlcN(alpha 1-->6)-2-deoxy-L- myo-inositol inhibited PI-PLC without affecting GPI-PLC. Last, the aminoglycoside G418 stimulated PI-PLC, but had no effect on GPI-PLC. Thus, these enzymes represent mechanistic subclasses of GPI phospholipases C, distinguishable by their sensitivity to GlcN(alpha 1-->6)Ins derivatives and aminoglycosides. Possible allosteric regulation of PI-PLC by GlcN(alpha 1-->6)Ins analogs is discussed.
Asunto(s)
Hidrolasas Diéster Fosfóricas/metabolismo , Polisacáridos/metabolismo , Trypanosoma brucei brucei/enzimología , Animales , Secuencia de Carbohidratos , Activación Enzimática , Gentamicinas/farmacología , Glicosilfosfatidilinositol Diacilglicerol-Liasa , Glicosilfosfatidilinositoles/metabolismo , Metabolismo de los Lípidos , Datos de Secuencia Molecular , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa CAsunto(s)
Glicosilfosfatidilinositoles/química , Ácidos Mirísticos/metabolismo , Hidrolasas Diéster Fosfóricas/aislamiento & purificación , Hidrolasas Diéster Fosfóricas/metabolismo , Trypanosoma brucei brucei/enzimología , Glicoproteínas Variantes de Superficie de Trypanosoma/química , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Marcaje Isotópico/métodos , Conformación Molecular , Datos de Secuencia Molecular , Ácido Mirístico , Fosfatidilinositol Diacilglicerol-Liasa , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Tritio , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI-PLC, cell-associated gp63 could not be detected in immunoblots. Pulse-chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Leishmania major/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Trypanosoma brucei brucei/enzimología , Animales , Secuencia de Carbohidratos , Glicoesfingolípidos/metabolismo , Glicosilfosfatidilinositol Diacilglicerol-Liasa , Membranas Intracelulares/enzimología , Leishmania major/genética , Leishmania major/crecimiento & desarrollo , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Fenotipo , Fosfatidilinositol Diacilglicerol-Liasa , Hidrolasas Diéster Fosfóricas/genética , TransfecciónRESUMEN
The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide) are glycosylphosphatidylinositol (GPI)-anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associated gp63 could not be detected in immunoblots. gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative protein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmentalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN(1 alpha 6)-myo-inositol-1-phospholipid, is cytosolic. To our knowledge, this represents the first use of a catabolic enzyme, in vivo, to elucidate the topography of biosynthetic pathways. Intriguingly, the phenotype of GPIPLC-expressing L. major, secretion of proteins with GPI addition signals, and depletion of protein-GPI anchor precursors, is similar to that of some protein-GPI mutants in higher eukaryotes. These findings have implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma.
Asunto(s)
Retículo Endoplásmico/metabolismo , Glicoesfingolípidos/metabolismo , Glicosilfosfatidilinositoles/biosíntesis , Leishmania major/metabolismo , Metaloendopeptidasas/metabolismo , Proteínas Protozoarias/biosíntesis , Trypanosoma brucei brucei/metabolismo , Fosfolipasas de Tipo C/biosíntesis , Animales , Compartimento Celular , Citoplasma/metabolismo , Glicosilfosfatidilinositoles/química , Hemoglobinuria Paroxística/metabolismo , Leishmania major/genética , Mamíferos , Linfocitos T/metabolismoRESUMEN
The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide) are glycosylphosphatidylinositol (GPI)-anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associated gp63 could not be detected in immunoblots, gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative protein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmentalalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man (1Ó6)Man(1Ó4)GlcN(1Ó6)-myo-inositol-1-phospholipid, is cystolic. To our knowledge, this represents the first use of a catabolic enzyme, in vivo, to elucidate the topography of biosynthetic pathways. Intriguingly, the phenotype of GPIPLC-expressing L. major, secretion of proteins with GPI addition signals, and depletion of protein-GPI anchor precursors, is similar to that of some protein-GPI mutants in higher eukaryotes. These findings have implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma
Asunto(s)
Humanos , Retículo Endoplásmico , Fosfatidilinositoles/biosíntesis , Glucolípidos/biosíntesis , Hemoglobinuria Paroxística/metabolismo , Leishmania tropica , Trypanosoma brucei brucei , Fosfolipasas de Tipo C , Línea Celular , Fosfatidilinositoles/metabolismo , Glucolípidos/metabolismo , Mamíferos , Glicoproteínas Variantes de Superficie de TrypanosomaRESUMEN
Glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) from Trypanosoma brucei cleaves the glycosyl phosphatidylinositol (GPI) anchor of the trypanosome variant surface glycoprotein (VSG) and other GPI structures. We have expressed this enzyme in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein. We have purified large amounts of GPI-PLC from E. coli membranes, using a single step immunoaffinity technique. The expressed enzyme is identical to its trypanosome counterpart in enzymatic specificity, mobility on SDS-PAGE, and isoelectric point. Recombinant GPI-PLC is a membrane enzyme; it associates with E. coli membranes and, like the T. brucei GPI-PLC, partitions into the detergent phase in Triton X-114 phase separation experiments. The Michaelis constants for the two enzymes are similar (400 nM, with VSG as substrate). The turnover number (kcat, 72 min-1) of the recombinant enzyme (expressed from a. T. brucei rhodesiense WRATat 1.1 cDNA) is about one-tenth that of GPI-PLC from T. brucei brucei (ILTat 1.3).