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1.
J Cell Biol ; 123(5): 1069-79, 1993 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8245118

RESUMO

Kinetoplast DNA (kDNA), the mitochondrial DNA of Crithidia fasciculata and related trypanosomatids, is a network containing approximately 5,000 covalently closed minicircles which are topologically interlocked. kDNA synthesis involves release of covalently closed minicircles from the network, and, after replication of the free minicircles, reattachment of the nicked or gapped progeny minicircles to the network periphery. We have investigated this process by electron microscopy of networks at different stages of replication. The distribution of nicked and closed minicircles is easily detectable either by autoradiography of networks radiolabeled at endogenous nicks by nick translation or by twisting the covalently closed minicircles with intercalating dye. The location of newly synthesized minicircles within the network is determined by autoradiography of network is determined by autoradiography of networks labeled in vivo with a pulse of [3H]thymidine. These studies have clarified structural changes in the network during replication, the timing of repair of nicked minicircles after replication, and the mechanism of division of the network.


Assuntos
Crithidia fasciculata/ultraestrutura , Replicação do DNA , DNA de Cinetoplasto/ultraestrutura , Animais , Autorradiografia , Divisão Celular , Crithidia fasciculata/genética , Crithidia fasciculata/crescimento & desenvolvimento , Crithidia fasciculata/metabolismo , Reparo do DNA , DNA de Cinetoplasto/biossíntese , DNA de Cinetoplasto/efeitos dos fármacos , Marcação por Isótopo , Microscopia Eletrônica , Propídio/farmacologia
2.
J Cell Biol ; 143(4): 911-9, 1998 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-9817750

RESUMO

Kinetoplast DNA (kDNA), the mitochondrial DNA in kinetoplastids, is a network containing several thousand topologically interlocked minicircles. We investigated cell cycle-dependent changes in the localization of kDNA replication enzymes by combining immunofluorescence with either hydroxyurea synchronization or incorporation of fluorescein-dUTP into the endogenous gaps of newly replicated minicircles. We found that while both topoisomerase II and DNA polymerase beta colocalize in two antipodal sites flanking the kDNA during replication, they behave differently at other times. Polymerase beta is not detected by immunofluorescence either during cell division or G1, but is abruptly detected in the antipodal sites at the onset of kDNA replication. In contrast, topoisomerase II is localized to sites at the network edge at all cell cycle stages; usually it is found in two antipodal sites, but during cytokinesis each postscission daughter network is associated with only a single site. During the subsequent G1, topoisomerase accumulates in a second localization site, forming the characteristic antipodal pattern. These data suggest that these sites at the network periphery are permanent components of the mitochondrial architecture that function in kDNA replication.


Assuntos
Crithidia fasciculata/genética , Replicação do DNA/fisiologia , DNA de Cinetoplasto/fisiologia , Animais , Ciclo Celular/fisiologia , Crithidia fasciculata/citologia , Crithidia fasciculata/enzimologia , DNA Polimerase beta/metabolismo , DNA Primase/metabolismo , DNA Topoisomerases Tipo II/metabolismo , DNA de Protozoário/fisiologia
3.
J Cell Biol ; 153(4): 735-44, 2001 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-11352935

RESUMO

Kinetoplast DNA, the mitochondrial DNA of Crithidia fasciculata, is organized into a network containing 5,000 topologically interlocked minicircles. This network, situated within the mitochondrial matrix, is condensed into a disk-shaped structure located near the basal body of the flagellum. Fluorescence in situ hybridization revealed that before their replication, minicircles are released vectorially from the network face nearest the flagellum. Replication initiates in the zone between the flagellar face of the disk and the mitochondrial membrane (we term this region the kinetoflagellar zone [KFZ]). The replicating minicircles then move to two antipodal sites that flank the disk-shaped network. In later stages of replication, the number of free minicircles increases, accumulating transiently in the KFZ. The final replication events, including primer removal, repair of many of the gaps, and reattachment of the progeny minicircles to the network periphery, are thought to take place within the antipodal sites.


Assuntos
DNA de Cinetoplasto/fisiologia , DNA Mitocondrial/fisiologia , Mitocôndrias/genética , Origem de Replicação/fisiologia , Animais , Ciclo Celular/fisiologia , Crithidia fasciculata , DNA de Cinetoplasto/análise , DNA Mitocondrial/análise , DNA de Protozoário/análise , DNA de Protozoário/fisiologia , Dosagem de Genes , Hibridização in Situ Fluorescente
4.
J Cell Biol ; 103(1): 255-63, 1986 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-3722267

RESUMO

After synthesis on membrane-bound ribosomes, the variant surface glycoprotein (VSG) of Trypanosoma brucei is modified by: (a) removal of an N-terminal signal sequence, (b) addition of N-linked oligosaccharides, and (c) replacement of a C-terminal hydrophobic peptide with a complex glycolipid that serves as a membrane anchor. Based on pulse-chase experiments with the variant ILTat-1.3, we now report the kinetics of three subsequent processing reactions. These are: (a) conversion of newly synthesized 56/58-kD polypeptides to mature 59-kD VSG, (b) transport to the cell surface, and (c) transport to a site where VSG is susceptible to endogenous membrane-bound phospholipase C. We found that the t 1/2 of all three of these processes is approximately 15 min. The comparable kinetics of these processes is compatible with the hypotheses that transport of VSG from the site of maturation to the cell surface is rapid and that VSG may not reach a phospholipase C-containing membrane until it arrives on the cell surface. Neither tunicamycin nor monensin blocks transport of VSG, but monensin completely inhibits conversion of 58-kD VSG to the mature 59-kD form. In the presence of tunicamycin, VSG is synthesized as a 54-kD polypeptide that is subsequently processed to a form with a slightly higher Mr. This tunicamycin-resistant processing suggests that modifications unrelated to N-linked oligosaccharides occur. Surprisingly, the rate of VSG transport is reduced, but not abolished, by dropping the chase temperature to as low as 10 degrees C.


Assuntos
Antígenos de Protozoários , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Transporte Biológico , Compartimento Celular , Membrana Celular/metabolismo , Temperatura Baixa , Cinética , Peso Molecular , Monensin/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Tunicamicina/farmacologia , Fosfolipases Tipo C/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma
5.
J Cell Biol ; 153(4): 725-34, 2001 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-11352934

RESUMO

Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase beta, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication.


Assuntos
DNA de Cinetoplasto/fisiologia , DNA Mitocondrial/fisiologia , Proteínas de Ligação a DNA/genética , Animais , Ciclo Celular/fisiologia , Crithidia fasciculata , Proteínas de Ligação a DNA/análise , Técnicas Microbiológicas , Mitocôndrias/química , Mitocôndrias/genética , Proteínas de Protozoários/análise , Proteínas de Protozoários/genética , RNA Mensageiro/análise , Origem de Replicação/fisiologia
6.
J Cell Biol ; 126(3): 631-9, 1994 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8045928

RESUMO

Kinetoplast DNA, the mitochondrial DNA of trypanosomatid parasites, is a network containing several thousand minicircles and a few dozen maxicircles. We compared kinetoplast DNA replication in Trypanosoma brucei and Crithidia fasciculata using fluorescence in situ hybridization and electron microscopy of isolated networks. One difference is in the location of maxicircles in situ. In C. fasciculata, maxicircles are concentrated in discrete foci embedded in the kinetoplast disk; during replication the foci increase in number but remain scattered throughout the disk. In contrast, T. brucei maxicircles generally fill the entire disk. Unlike those in C. fasciculata, T. brucei maxicircles become highly concentrated in the central region of the kinetoplast after replication; then during segregation they redistribute throughout the daughter kinetoplasts. T. brucei and C. fasciculata also differ in the pattern of attachment of newly synthesized minicircles to the network. In C. fasciculata it was known that minicircles are attached at two antipodal sites but subsequently are found uniformly distributed around the network periphery, possibly due to a relative movement of the kinetoplast disk and two protein complexes responsible for minicircle synthesis and attachment. In T. brucei, minicircles appear to be attached at two antipodal sites but then remain concentrated in these two regions. Therefore, the relative movement of the kinetoplast and the two protein complexes may not occur in T. brucei.


Assuntos
Crithidia/genética , Replicação do DNA , DNA de Cinetoplasto/biossíntese , Trypanosoma brucei brucei/genética , Animais , DNA de Cinetoplasto/genética , Hibridização in Situ Fluorescente , Microscopia Eletrônica , Trypanosoma brucei brucei/ultraestrutura
7.
Science ; 288(5463): 140-3, 2000 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-10753118

RESUMO

African trypanosomes, the cause of sleeping sickness, need massive amounts of myristate to remodel glycosyl phosphatidylinositol (GPI) anchors on their surface glycoproteins. However, it has been believed that the parasite is unable to synthesize any fatty acids, and myristate is not abundant in the hosts' bloodstreams. Thus, it has been unclear how trypanosomes meet their myristate requirement. Here we found that they could indeed synthesize fatty acids. The synthetic pathway was unique in that the major product, myristate, was preferentially incorporated into GPIs and not into other lipids. The antibiotic thiolactomycin inhibited myristate synthesis and killed the parasite, making this pathway a potential chemotherapeutic target.


Assuntos
Ácidos Graxos/biossíntese , Glicosilfosfatidilinositóis/metabolismo , Miristatos/metabolismo , Trypanosoma brucei brucei/metabolismo , Acil Coenzima A/metabolismo , Animais , Antibacterianos/farmacologia , Caprilatos/metabolismo , Sistema Livre de Células , Cerulenina/farmacologia , Ácidos Decanoicos/metabolismo , Ácido Graxo Sintases/metabolismo , Lauratos/metabolismo , Frações Subcelulares/enzimologia , Tiofenos/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/crescimento & desenvolvimento
8.
Science ; 238(4823): 81-4, 1987 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-2443973

RESUMO

A group of proteins anchored to the cell by phosphatidylinositol (PI) has recently been identified. The significance of this new class of membrane anchor is unknown; one possibility is that it facilitates release of the molecule by phospholipases. In fact, phospholipase C enzymes specific for the complex carboxyl-terminal glycolipids of these proteins have been isolated from African trypanosomes and from hepatocyte plasma membranes. This study reports the discovery of a glycan-PI-specific phospholipase D in human serum that cleaves both the membrane form of the variant surface glycoprotein of African trypanosomes and its glycolipid precursor, but not phosphatidylethanolamine, phosphatidylcholine, or phosphatidylinositol. Decay-accelerating factor, another PI-anchored molecule, is also cleaved by the enzyme and converted from a hydrophobic to a soluble protein. The enzyme is Ca2+-dependent, heat labile, and not affected by the inhibitor of serine proteases, phenylmethylsulfonylfluoride. Its function is not known, but the present findings indicate that it participates in the metabolism of glycolipid-anchored membrane proteins.


Assuntos
Glicolipídeos/metabolismo , Fosfatidilinositóis/metabolismo , Fosfolipase D/sangue , Fosfolipases/sangue , Antígenos CD55 , Humanos , Proteínas de Membrana/metabolismo , Fosfolipase D/antagonistas & inibidores , Solubilidade , Especificidade por Substrato , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo
9.
Science ; 252(5014): 1851-4, 1991 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-1829548

RESUMO

Trypanosoma brucei, the protozoan parasite responsible for African sleeping sickness, evades the host immune response through the process of antigenic variation. The variant antigen, known as the variant surface glycoprotein (VSG), is anchored to the cell surface by a glycosyl phosphatidylinositol (GPI) structure that contains myristate (n-tetradecanoate) as its only fatty acid component. The utilization of heteroatom-containing analogs of myristate was studied both in a cell-free system and in vivo. Results indicated that the specificity of fatty acid incorporation depends on chain length rather than on hydrophobicity. One analog, 10-(propoxy)decanoic acid, was highly toxic to trypanosomes in culture although it is nontoxic to mammalian cells.


Assuntos
Ácidos Mirísticos/metabolismo , Ácidos Mirísticos/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Acil Coenzima A/metabolismo , Animais , Sistema Livre de Células , Glicolipídeos/metabolismo , Glicosilfosfatidilinositóis , Cinética , Camundongos , Ácido Mirístico , Fosfatidilinositóis/metabolismo , Relação Estrutura-Atividade , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/ultraestrutura
10.
Mol Cell Biol ; 15(12): 6794-803, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8524245

RESUMO

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is composed of several thousand minicircles and a few dozen maxicircles, all of which are topologically interlocked in a giant network. We have studied the replication of maxicircle DNA, using electron microscopy to analyze replication intermediates from both Crithidia fasciculata and Trypanosoma brucei. Replication intermediates were stabilized against branch migration by introducing DNA interstrand cross-links in vivo with 4,5',8-trimethylpsoralen and UV radiation. Electron microscopy of individual maxicircles resulting from a topoisomerase II decatenation of kinetoplast DNA networks revealed intact maxicircle theta structures. Analysis of maxicircle DNA linearized by restriction enzyme cleavage revealed branched replication intermediates derived from theta structures. Measurements of the linearized branched molecules in both parasites indicate that replication initiates in the variable region (a noncoding segment characterized by repetitive sequences) and proceeds unidirectionally, clockwise on the standard map.


Assuntos
Crithidia fasciculata/genética , Replicação do DNA , DNA de Cinetoplasto/biossíntese , Trypanosoma brucei brucei/genética , Animais , Sequência de Bases , Crithidia fasciculata/metabolismo , Reagentes de Ligações Cruzadas , DNA Topoisomerases Tipo II/metabolismo , DNA de Cinetoplasto/isolamento & purificação , DNA de Cinetoplasto/ultraestrutura , Microscopia Eletrônica , Dados de Sequência Molecular , Mapeamento por Restrição , Especificidade da Espécie , Trioxsaleno/farmacologia , Trypanosoma brucei brucei/metabolismo
11.
Mol Cell Biol ; 9(8): 3212-7, 1989 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-2552285

RESUMO

Kinetoplast DNA, the mitochondrial DNA in trypanosomes, is a giant network containing topologically interlocked minicircles. Replication occurs on free minicircles that have been detached from the network. In this paper, we report studies on the synthesis and processing of the minicircle L and H strands. Analysis of free minicircles from Trypanosoma equiperdum by two-dimensional agarose gel electrophoresis indicated that elongating L strands are present on theta structures. Hybridization studies indicated that L-strand elongation is continuous and unidirectional, starting near nucleotide 805 and proceeding around the entire minicircle. The theta structures segregate into monomeric progeny minicircles, and those with a newly synthesized L strand have a 8-nucleotide gap between nucleotides 805 and 814 (J. M. Ntambi, T. A. Shapiro, K. A. Ryan, and P. T. Englund, J. Biol. Chem. 261:11890-11895, 1986). These molecules are reattached to the network, where repair of the gap takes place. Of the molecules labeled during a 10-min pulse with [3H]thymidine, gap filling occurred on half within about 15 min and on virtually all by 60 min; however, there was no detectable covalent closure of the newly synthesized L strand by 60 min.


Assuntos
Replicação do DNA , DNA Circular/metabolismo , DNA Mitocondrial/metabolismo , Trypanosoma/genética , Animais , DNA/análise , DNA/metabolismo , Reparo do DNA , DNA Circular/biossíntese , DNA de Cinetoplasto , DNA Mitocondrial/biossíntese , Eletroforese em Gel Bidimensional , Cinética
12.
Mol Cell Biol ; 10(2): 720-6, 1990 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-1688997

RESUMO

The surface of the bloodstream form of the African trypanosome, Trypansoma brucei, is covered with about 10(7) molecules of the variant surface glycoprotein (VSG), a protein tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) membrane anchor. This anchor is cleavable by an endogenous GPI-specific phospholipase C (GPI-PLC). GPI-PLC activity is down regulated when trypanosomes differentiate from the bloodstream form to the procyclic form found in the tsetse fly vector. We have mapped the GPI-PLC locus in the trypanosome genome and have examined the mechanism for this developmental regulation in T. brucei. Southern blot analysis indicates a single-copy gene for GPI-PLC, with two allelic variants distinguishable by two NcoI restriction fragment length polymorphisms. The gene was localized solely to a chromosome in the two-megabase compression region by contour-clamped homogeneous electric field gel electrophoresis. No rearrangement of the GPI-PLC gene occurs during differentiation to procyclic forms, which could potentially silence GPI-PLC gene expression. Enzymological studies give no indication of a diffusible inhibitor of GPI-PLC activity in procyclic forms, and Western immunoblot analysis reveals no detectable GPI-PLC polypeptide in these forms. Therefore, it is highly unlikely that the absence of GPI-PLC activity in procyclic forms is due to posttranslational control. Northern (RNA) blot analysis reveals barely detectable levels of GPI-PLC mRNA in procyclic forms; therefore, regulation of GPI-PLC activity in these forms correlates with the steady-state mRNA level.


Assuntos
Mapeamento Cromossômico , Regulação Enzimológica da Expressão Gênica , Genes , Trypanosoma brucei brucei/genética , Fosfolipases Tipo C/genética , Animais , Northern Blotting , Southern Blotting , Hibridização de Ácido Nucleico , RNA/genética , RNA/isolamento & purificação , Ratos , Ratos Endogâmicos , Mapeamento por Restrição , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/crescimento & desenvolvimento
13.
J Mol Biol ; 304(4): 633-44, 2000 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-11099385

RESUMO

Concanavalin A (Con A) kills procyclic (insect) forms of Trypanosoma brucei by binding to N-glycans on EP-procyclin, a major surface glycosyl phosphatidylinositol (GPI)-anchored protein which is rich in Glu-Pro repeats. We have previously isolated and studied two procyclic mutants (ConA 1-1 and ConA 4-1) that are more resistant than wild-type (WT) to Con A killing. Although both mutants express the same altered oligosaccharides compared to WT cells, ConA 4-1 is considerably more resistant to lectin killing than is ConA 1-1. Thus, we looked for other alterations to account for the differences in sensitivity. Using mass spectrometry, together with chemical and enzymatic treatments, we found that both mutants express types of EP-procyclin that are either poorly expressed or not found at all in WT cells. ConA 1-1 expresses mainly EP1-3, a novel procyclin that contains 18 EP repeats, is partially N-glycosylated, and bears hybrid-type glycans. On the other hand, ConA 4-1 cells express almost exclusively EP2-3, a novel non-glycosylated procyclin isoform with 23 EP repeats and no site for glycosylation. The predominance of EP2-3 in ConA 4-1 cells explains their high resistance to ConA killing. Thus, switching the procyclin repertoire, a process that could be relevant to parasite development in the insect vector, modulates the sensitivity of trypanosomes to cytotoxic lectins.


Assuntos
Concanavalina A/metabolismo , Concanavalina A/toxicidade , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Mutação/genética , Trypanosoma brucei brucei/efeitos dos fármacos , Sequência de Aminoácidos , Amino Açúcares/metabolismo , Animais , Resistência a Medicamentos/genética , Evolução Molecular , Glicosilação/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Hidrólise , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Peptídeos/análise , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Sequências Repetitivas de Aminoácidos , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento
14.
J Mol Biol ; 312(4): 597-607, 2001 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-11575917

RESUMO

Transmission of Trypanosoma brucei by the tsetse fly entails several rounds of differentiation as the parasite migrates through the digestive tract to the salivary glands of its vector. Differentiation of the bloodstream to the procyclic form in the fly midgut is accompanied by the synthesis of a new coat consisting of EP and GPEET procyclins. There are three closely related EP isoforms, two of which (EP1 and EP3) contain N-glycans. To identify the individual EP isoforms that are expressed early during synchronous differentiation in vitro, we exploited the selective extraction of GPI-anchored proteins and mass spectrometry. Unexpectedly, we found that GPEET and all isoforms of EP were coexpressed for a few hours at the onset of differentiation. At this time, the majority of EP1 and EP3 molecules were already glycosylated. Within 24 hours, GPEET became the major surface component, to be replaced in turn by glycosylated forms of EP, principally EP1, at a later phase of development. Transient transfection experiments using reporter genes revealed that each procyclin 3' untranslated region contributes to differential expression as the procyclic form develops. We postulate that programmed expression of other procyclin species will accompany further rounds of differentiation, enabling the parasite to progress through the fly.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Moscas Tsé-Tsé/parasitologia , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Diferenciação Celular , Genes de Protozoários/genética , Genes Reporter/genética , Glicosilfosfatidilinositóis/metabolismo , Espectrometria de Massas , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Protozoários/genética , Análise de Sequência de DNA , Transfecção , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Moscas Tsé-Tsé/anatomia & histologia
15.
Trends Parasitol ; 17(8): 381-7, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11685899

RESUMO

The glycosyl phosphatidylinositol anchor of the trypanosome variant surface glycoprotein contains myristate as its sole fatty acid component. Surprisingly, there does not appear to be enough myristate in either the parasite or its host's bloodstream to sustain myristoylation of the enormous quantity of variant surface glycoprotein produced. Here, we discuss how the trypanosome solves its myristate dilemma. The parasite not only efficiently salvages and processes myristate from the bloodstream, but it also makes myristate de novo using a recently discovered specialized fatty acid synthesis system.


Assuntos
Ácidos Graxos/biossíntese , Ácido Mirístico/metabolismo , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/biossíntese , Animais , Glicosilfosfatidilinositóis/metabolismo , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/crescimento & desenvolvimento
16.
Mol Biochem Parasitol ; 56(2): 311-21, 1992 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1362451

RESUMO

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).


Assuntos
Genes de Protozoários/genética , Glicosilfosfatidilinositóis/metabolismo , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/enzimologia , Fosfolipases Tipo C/metabolismo , Animais , Sequência de Bases , Relação Dose-Resposta a Droga , Escherichia coli/genética , Glutamatos/farmacologia , Ácido Glutâmico , Glicosilfosfatidilinositóis/genética , Isoenzimas , Cinética , Dados de Sequência Molecular , Proteínas de Protozoários/biossíntese , Proteínas Recombinantes/biossíntese , Especificidade por Substrato , Trypanosoma brucei brucei/genética , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/isolamento & purificação
17.
Mol Biochem Parasitol ; 76(1-2): 115-23, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8920000

RESUMO

Linearized free maxicircle DNA, present in detergent lysates of Crithidia fasciculata mitochondria, was thought to be a replication intermediate formed during rolling circle replication of maxicircle DNA. Gel electrophoresis of the linearized free maxicircles indicated that they were slightly larger than the maxicircle genome, raising the possibility of the presence of terminal repetitions (Hajduk, S.L., Klein, V.A. and Englund, P.T. (1984) Cell 36, 483-492). We recently found, however, that maxicircles replicate by a theta-mechanism, and not as rolling circles (Carpenter, L.R. and Englund, P.T. (1995) Mol. Cell Biol. 15, 6794-6803). Given that theta-replication does not easily explain the presence of linearized free maxicircles, we investigated alternative explanations for their existence. We present evidence that this DNA species results from the double-strand cleavage of maxicircles due to detergent denaturation of intracellular topoisomerase II cleavable complexes. As expected for a topoisomerase II cleavage product, the linearized free maxicircle DNA is covalently bound to protein at both 5' ends. In addition, the slightly larger apparent size of linearized free maxicircle DNA or maxicircles linearized by a restriction enzyme can be explained by anomalous electrophoretic migration during conventional or pulsed-field agarose gel electrophoresis. This anomalous migration is presumably due to bends or other unusual structures in the DNA.


Assuntos
Crithidia fasciculata/genética , DNA Topoisomerases Tipo II/metabolismo , DNA Circular/metabolismo , DNA Mitocondrial/química , DNA de Protozoário/química , Animais , Southern Blotting , Replicação do DNA , DNA Circular/química , DNA Circular/isolamento & purificação , Eletroforese em Gel de Campo Pulsado
18.
Mol Biochem Parasitol ; 115(2): 157-64, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11420102

RESUMO

Trypanosoma brucei is the protozoan parasite that causes African sleeping sickness. Its surface is packed with 10(7) copies of the glycosyl phosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG). This GPI anchor is unusual in that it contains two myristates (14:0) in its lipid moiety. This fatty acid specificity is achieved through myristoylation of the GPI precursor, and the acyltransferases involved in the GPI remodeling were presumed to be specific for myristate. However, their specificity had never been fully evaluated. Here we found as expected that the remodeling acyltransferases completely excluded palmitate (16:0) and stearate (18:0) in a cell-free fatty acid remodeling system. In contrast, we found surprisingly that one of these enzymes was permissive to shorter fatty acids such as laurate (12:0) and octanoate (8:0). However, the rates of incorporation of shorter fatty acids were lower than that of myristate at low substrate concentration. Since shorter fatty acids are virtually absent in the parasite and in the host bloodstream, it is unlikely that shorter fatty acids compete effectively with myristate as remodeling substrates under physiological conditions. Even if they were present in small quantities, a recently identified specialized fatty acid synthetase efficiently elongates shorter fatty acids to myristate prior to incorporation into GPIs (Morita et al., Science 288 (2000) 140-3.). Therefore, even though a remodeling acyltransferase is permissive with regard to substrate chain length, the myristate specificity in GPI anchors is very high.


Assuntos
Ácidos Graxos/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Trypanosoma brucei brucei/metabolismo , Aciltransferases/metabolismo , Animais , Ácido Mirístico/metabolismo , Ratos , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/crescimento & desenvolvimento
19.
Mol Biochem Parasitol ; 59(2): 191-200, 1993 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8341318

RESUMO

MCP72 is a mitochondrial hsp70 protein from the trypanosomatid Crithidia fasciculata. An MCP72 cDNA clone was isolated from a C. fasciculata cDNA library by screening with antiserum specific for the homologous protein of Trypanosoma cruzi [9]. The MCP72 cDNA encodes a polypeptide of 663 amino acids which is 84% identical to the Trypanosoma cruzi protein and 56% identical to the Escherichia coli hsp70 protein DnaK. MCP72 is less similar to other hsp70 proteins. Native MCP72 was purified to homogeneity by ATP-agarose affinity chromatography. Comparison of its N-terminal amino acid sequence with that deduced from the cDNA sequence shows that 20 amino acid residues had been cleaved from the N-terminus; this sequence probably represents a mitochondrial import signal which is cleaved during translocation into the mitochondrion. Fluorescence microscopy, using antibodies specific for MCP72, indicates that the protein is concentrated in a region of the mitochondrial matrix which surrounds the kinetoplast.


Assuntos
Crithidia fasciculata/metabolismo , Proteínas de Choque Térmico/biossíntese , Mitocôndrias/metabolismo , Adenosina Trifosfatases/biossíntese , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Crithidia fasciculata/genética , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , Biblioteca Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo
20.
Mol Biochem Parasitol ; 36(3): 263-70, 1989 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-2797062

RESUMO

The variant surface glycoprotein (VSG) of Trypanosoma brucei is covalently linked to a phosphatidylinositol-containing glycolipid which serves as a membrane anchor. We previously identified a molecule, glycolipid A, which appears to be a biosynthetic precursor to the anchor [9]. In this paper we describe a related molecule, glycolipid C, which is similar to glycolipid A but which is more hydrophobic. Chromatographic analyses indicate that the polar head groups in glycolipids A and C are similar or identical. Both glycolipids contain phosphatidylinositol, but the inositol in glycolipid C is modified by a hydrophobic moiety. Since treatment of glycolipid C with mild alkali results in partial conversion to a molecule chromatographically identical to glycolipid A, it is likely that glycolipid C has an alkali-sensitive hydrophobic group, such as a fatty acid, linked to its inositol moiety.


Assuntos
Glicolipídeos/análise , Glicoproteínas de Membrana/análise , Trypanosoma brucei brucei/análise , Animais , Cromatografia em Camada Fina , Glicolipídeos/isolamento & purificação , Fosfatidilinositóis , Solubilidade
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