RESUMO
BACKGROUND: Trypanosoma cruzi is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, T. cruzi GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, T. cruzi genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: In silico analysis of the T. cruzi genome resulted in the identification of 18 genes encoding proteins of the GPI biosynthetic pathway as well as the inositolphosphorylceramide (IPC) synthase gene. Expression of GFP fusions of some of these proteins in T. cruzi epimastigotes showed that they localize in the endoplasmic reticulum (ER). Expression analyses of two genes indicated that they are constitutively expressed in all stages of the parasite life cycle. T. cruzi genes TcDPM1, TcGPI10 and TcGPI12 complement conditional yeast mutants in GPI biosynthesis. Attempts to generate T. cruzi knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding TcGPI8, which encodes the catalytic subunit of the transamidase complex, although we were able to generate single allele knockout mutants, attempts to disrupt both alleles failed, resulting instead in parasites that have undergone genomic recombination and maintained at least one active copy of the gene. CONCLUSIONS/SIGNIFICANCE: Analyses of T. cruzi sequences encoding components of the GPI biosynthetic pathway indicated that they are essential genes involved in key aspects of host-parasite interactions. Complementation assays of yeast mutants with these T. cruzi genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this parasite.
Assuntos
Vias Biossintéticas/genética , Glicosilfosfatidilinositóis/biossíntese , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Biologia Computacional , Retículo Endoplasmático/enzimologia , Deleção de Genes , Perfilação da Expressão Gênica , Genes Essenciais , Genes de Protozoários , Teste de Complementação Genética , Trypanosoma cruzi/enzimologiaRESUMO
The major free glycoinositolphospholipids and protein-linked glycoinositolphospholipids in Trypanosoma cruzi contain ceramide as the lipid moiety. Ceramide was not found in mammalian glycosylphosphatidylinositol (GPI)-anchors. An alkylglycerol, either as a lyso species or acylated has been also found in T. cruzi anchors. However, unlike African trypanosomes, no diacylglycerol was detected in the GPI-anchors. Using a membrane preparation from epimastigotes upon labelling with UDP[3H]GlcNAc we identified [3H]GlcNAcPI as the first step of GPI biosynthesis. Both, alkylacylglycerol (major) and diacylglycerol are constituents of the lipid. Although inositolphosphoceramide is the main inositolphospholipid in epimastigotes, it does not incorporate GlcNAc. The de-N-acetylation step afforded [3H]GlcN(alkylacylglycerol)PI and we also detected the [3H]GlcN(lysoacyl)PI. A new metabolite, phosphoGlcN(lysoacyl)PI, which was formed on long incorporation times, was characterized by chemical and enzymatic degradations. Several [3H]-Man labelled GPI precursors were obtained by in vitro GDP[3H]-Man labelling in the presence of UDPGlcNAc. All of them were sensitive to PI-PLC and to saponification conditions, thus, supporting a glycerolipid structure.
Assuntos
Acetilglucosamina/análogos & derivados , Glicolipídeos/biossíntese , Glicoesfingolipídeos/metabolismo , Fosfolipídeos/biossíntese , Trypanosoma cruzi/metabolismo , Acetilglucosamina/análise , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Animais , Sistema Livre de Células , Cromatografia em Camada Fina , Glicolipídeos/química , Glicosilação , Glicosilfosfatidilinositóis/biossíntese , Glicosilfosfatidilinositóis/química , Metabolismo dos Lipídeos , Lipídeos/química , Manose/metabolismo , Ácido Palmítico/metabolismo , Fosfatidilinositóis/análise , Fosfatidilinositóis/química , Fosfolipídeos/química , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/química , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
Glycosylphosphatidylinositols (GPI) are essential components in the plasma membrane of the protozoan parasite Leishmania mexicana, both as membrane anchors for the major surface macromolecules and as the sole class of free glycolipids. We provide evidence that L.mexicana dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis, is localized to a distinct tubular subdomain of the endoplasmic reticulum (ER), based on the localization of a green fluorescent protein (GFP)-DPMS chimera and subcellular fractionation experiments. This tubular membrane (termed the DPMS tubule) is also enriched in other enzymes involved in GPI biosynthesis, can be specifically stained with the fluorescent lipid, BODIPY-C5-ceramide, and appears to be connected to specific subpellicular microtubules that underlie the plasma membrane. Perturbation of microtubules and DPMS tubule structure in vivo results in the selective accumulation of GPI anchor precursors, but not free GPIs. The DPMS tubule is closely associated morphologically with the single Golgi apparatus in non-dividing and dividing cells, appears to exclude luminal ER resident proteins and is labeled, together with the Golgi apparatus, with another GFP chimera containing the heterologous human Golgi marker beta1,2-N-acetylglucosaminyltransferase-I. The possibility that the DPMS-tubule is a stable transitional ER is discussed.
Assuntos
Retículo Endoplasmático/enzimologia , Glicosilfosfatidilinositóis/biossíntese , Membranas Intracelulares/enzimologia , Leishmania mexicana/enzimologia , Manosiltransferases/metabolismo , Animais , Biomarcadores/análise , Divisão Celular , Fracionamento Celular , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Fluorescência , Glicosilfosfatidilinositóis/metabolismo , Complexo de Golgi/metabolismo , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Leishmania mexicana/citologia , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Mitose , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The cell surface of the parasitic protozoan Leishmania mexicana is coated by glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a GPI-anchored lipophosphoglycan and a class of free GPI glycolipids. To investigate whether the anchor or free GPIs are required for parasite growth we cloned the L.mexicana gene for dolichol-phosphate-mannose synthase (DPMS) and attempted to create DPMS knockout mutants by targeted gene deletion. DPMS catalyzes the formation of dolichol-phosphate mannose, the sugar donor for all mannose additions in the biosynthesis of both the anchor and free GPIs, except for a alpha1-3-linked mannose residue that is added exclusively to the free GPIs and lipophosphoglycan anchor precursors. The requirement for dolichol-phosphate-mannose in other glycosylation pathways in L.mexicana is minimal. Deletion of both alleles of the DPMS gene (lmdpms) consistently resulted in amplification of the lmdpms chromosomal locus unless the promastigotes were first transfected with an episomal copy of lmdpms, indicating that lmdpms, and possibly GPI biosynthesis, is essential for parasite growth. As evidence presented in this and previous studies indicates that neither GPI-anchored glycoproteins nor lipophosphoglycan are required for growth of cultured parasites, it is possible that the abundant and functionally uncharacterized free GPIs are essential membrane components.
Assuntos
Glicolipídeos/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Leishmania mexicana/enzimologia , Manosiltransferases/genética , Animais , Sequência de Carboidratos , Clonagem Molecular , Dolicol Monofosfato Manose/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Glicolipídeos/química , Glicoesfingolipídeos/metabolismo , Glicosilfosfatidilinositóis/biossíntese , Leishmania mexicana/genética , Leishmania mexicana/crescimento & desenvolvimento , Manosiltransferases/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Mapeamento por Restrição , Alinhamento de SequênciaRESUMO
Glycosylphosphatidylinositol (GPI) glycolipids are major cell surface constituents in the Leishmania parasites. Distinct classes of GPI are present as membrane anchors for several surface glycoproteins and an abundant lipophosphoglycan as well as being the major glycolipids (GIPLs) in the plasma membrane. In this study we have identified putative precursors for the protein and lipophosphoglycan anchors and delineated the complete pathway for GIPL biosynthesis in Leishmania mexicana promastigotes. Based on the structural analyses of these GPI intermediates and their kinetics of labeling in vivo and in cell-free systems, we provide evidence that the GIPLs are the products of an independent biosynthetic pathway rather than being excess precursors of the anchor pathways. First, we show that the similar glycan head groups of the GIPL and protein/lipophosphoglycan anchor precursors are assembled on two distinct pools of PI corresponding to 1-O-(C18:0)alkyl-2-stearoyl-PI and 1-O-(C24:0/C26:0)-2-stearoyl-PI, respectively. These PI species account for 20 and 1% of the total PI pool, respectively, indicating a remarkable specificity in their selection. Second, analysis of the flux of intermediates through these pathways in vivo and in a cell-free system suggests that the GIPL and anchor pathways are independently regulated. We also show that GIPL biosynthesis requires fatty acid remodeling, in which the sn-2 stearoyl chains are replaced with myristoyl or lauroyl chains. Fatty acid remodeling is dependent on CoA and ATP and occurs on pre-existing but not on de novo synthesized GIPLs. We suggest that the compartmentalization of different GPI pathways may be important in regulating the species and stage-specific expression of different GPI structures in these parasites.
Assuntos
Glicosilfosfatidilinositóis/biossíntese , Leishmania mexicana/metabolismo , Animais , Ácidos Graxos/metabolismo , Glicolipídeos/metabolismo , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/classificação , Glicosilfosfatidilinositóis/metabolismo , Lipídeos/análise , Lipídeos/química , Manose/metabolismo , Espectrometria de Massas , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Polissacarídeos/químicaRESUMO
Glycoinositolphospholipids (GIPLs) were isolated from promastigotes of the lizard parasites Leishmania adleri by phenol/water extraction. Phosphoinositol oligosaccharides were liberated by mild alkaline hydrolysis, purified by gel filtration and high pH anion exchange chromatography, and characterized by methylation analysis, fast atom bombardment mass spectrometry, and nuclear magnetic resonance spectroscopy. The four major compounds (I-IV) from L. adleri were linked to alkylacyl glycerol, and their glycan moieties had the following structures: Man alpha(1-2)Man alpha(1-6)[Man alpha(1-3)] Man alpha(1-4)GlcN alpha(1-6)Ins-1-PO4 (I), Galp alpha(1-6) Galp alpha(1-3)Galf beta(1-3)Man alpha(1-3)Man alpha(1-4)GlcN alpha(1-6)Ins-1-PO4 (II), Galp alpha(1-3)Galf beta(1-3)Man alpha(1-3) Man alpha(1-4)GlcN alpha(1-6)Ins-1-PO4 (III), Man alpha(1-2)[EtNP(-6)]Man alpha(1-6)[Man alpha(1-3)] Man alpha(1-4)GlcN alpha(1-6)Ins-1-PO4 (IV). These compounds are analogous to the previously characterized GIPLs from New and Old World leishmanial parasites of mammals designated iM4 (identical to compound I), GIPLs 3 and 2 (identical to compounds II and III, respectively), and EPiM4 (identical to compound IV), which is consistent with a close phylogenetic relationship between lizard and mammalian Leishmania. However, in contrast to the mammalian parasites, the abundant surface glycoconjugate known as lipophosphoglycan was either absent or confined to the flagellar pocket region in L. adleri.
Assuntos
Glucanos/química , Glicosilfosfatidilinositóis/química , Leishmania/metabolismo , Lagartos/parasitologia , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia em Gel , Cromatografia por Troca Iônica , Glucanos/isolamento & purificação , Glicosilfosfatidilinositóis/biossíntese , Glicosilfosfatidilinositóis/isolamento & purificação , Leishmania/química , Leishmania/classificação , Espectroscopia de Ressonância Magnética , Mamíferos , Metilação , Dados de Sequência Molecular , Filogenia , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
Mucins are heavily O-glycosylated Thr/Ser/Pro-rich molecules. Given their relevant functions, mucins and their genes have been mainly studied in higher eukaryotes. In the protozoan parasite Trypanosoma cruzi, mucin-like glycoproteins were shown to play an important role in the interaction with the surface of the mammalian cell during the invasion process. We show now that this parasite has a family of putative mucin genes, whose organization resembles the one present in mammalian cells. Different parasite isolates have different sets of genes, as defined by their central domain. Central domains, rich in codons for Thr and/or Ser and Pro residues, are made up of either a variable number of repeat units in tandem or non-repetitive sequences. Conversely, 5'- and 3'-ends from different genes in different isolates have similar sequences, suggesting their common origin. Comparison of deduced amino acid sequences revealed that all members of the family have the same putative signal peptide on the N terminus and a putative sequence for glycophosphatidylinositol anchoring on the C terminus. The deduced molecular mass of the core proteins is small (from 17 to 21 kDa), in agreement with the 1-kilobase size of the mRNA detected. Putative mucin genes in T. cruzi are located on large chromosomal bands of about 1.6-2.2 megabase pairs.
Assuntos
Genes de Protozoários , Mamíferos/genética , Mucinas/genética , Família Multigênica , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Chlorocebus aethiops , Clonagem Molecular , Códon , Glicosilfosfatidilinositóis/biossíntese , Dados de Sequência Molecular , Mucinas/biossíntese , Mucinas/química , Oligodesoxirribonucleotídeos , Sinais Direcionadores de Proteínas/biossíntese , Sinais Direcionadores de Proteínas/química , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA de Protozoário/biossíntese , RNA de Protozoário/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Serina , Especificidade da Espécie , Treonina , Células VeroRESUMO
The variant surface glycoprotein (VSG) of T. brucei is anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor which is unique in that its fatty acids are exclusively myristate (a fourteen carbon saturated fatty acid). We showed that the myristate is added to the GPI precursor in a remodeling reaction involving deacylation and reacylation. We now demonstrate that trypanosomes have a second pathway of myristoylation for GPI anchors that we call "myristate exchange" which is distinct from the fatty acid remodeling pathway. We propose that this is an exchange of [3H]myristate into both sn-1 and sn-2 positions of glycolipid A, which already contains myristate, and have demonstrated this using inhibitors and a variety of other methods. We have partially characterized myristate exchange with respect to specificity and susceptibility to some inhibitors. The apparent Km for myristoyl CoA is 7 nM. This myristate-specific process may represent a proof-reading system to ensure that the fatty acids on VSG are exclusively myristate. Although myristate exchange was first discovered for glycolipid A, we now believe that VSG is the true substrate of this reaction. VSG is efficiently labeled by exchange in the presence of cycloheximide, which prevents anchoring of newly synthesized protein. Although its location is not yet known, we have evidence that exchange does not localize to either the endoplasmic reticulum or the plasma membrane. We will present data indicating that surface VSG may be internalized and undergo myristate exchange.
Assuntos
Glicosilfosfatidilinositóis/biossíntese , Miristatos/metabolismo , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Animais , Sistema Livre de Células , Trypanosoma brucei brucei/químicaRESUMO
The glycosylphosphatidylinositol (GPI) biosynthetic pathway in Trypanosoma brucei bloodstream forms includes the formation of glycolipid C. This molecule is the inositol-acylated form of the GPI anchor precursor, glycolipid A. There is no evidence for the transfer of glycolipid C to protein in vivo and the role of glycolipid C is unclear. In this paper we show that glycolipid C is not synthesised in the presence of phenylmethylsulphonyl fluoride (PMSF) and that glycolipid C is not an obligatory intermediate on the pathway to the formation of glycolipid A. Using pulse-chase experiments we show that glycolipid A and glycolipid C are in a dynamic equilibrium and we suggest that only the forward reaction (glycolipid A conversion to glycolipid C) is inhibited by PMSF.
Assuntos
Glicolipídeos/fisiologia , Glicosilfosfatidilinositóis/biossíntese , Proteínas de Protozoários/biossíntese , Trypanosoma brucei brucei/metabolismo , Animais , Fluoreto de Fenilmetilsulfonil/metabolismoRESUMO
Cloning genes for glycosylphosphatidylinositol (GPI)-anchor biosynthesis is important to further understand its mechanisms and regulation. We have been using expression cloning methods in which a cDNA library was transfected into GPI-anchor-deficient mutant cells. The transfectants which restored surface expression of GPI-anchored proteins were isolated and the plasmids were rescued. In this way we previously cloned cDNAs of genes for complementation classes A and F, and named them PIG-A and PIG-F, respectively. In the present study we have cloned the gene for class B, termed PIG-B. In each case we used different methods. For cloning PIG-A cDNA we used a cDNA library made with an Epstein-Barr-virus-based vector and human class A mutant JY5 which expresses EBNA-1 protein. The EBNA-1 protein allows stable replication of oriP-containing plasmids in the episomal form. For cloning PIG-F cDNA we chose a transient expression method and cotransfected a human T-cell cDNA library made with a vector bearing an origin of replication of polyoma virus with a plasmid bearing polyoma virus large T into the class F murine thymoma mutant. This cotransfection strategy was unsuccessful for cloning PIG-B due to low transfection efficiency of the class B thymoma mutant SIA-b. Thus, we first established large T-expressing SIA-b cells and then transfected them with a cDNA library. PIG-B cDNA restored the surface expression of Thy-1 on SIA-b cells and also synthesis of mature type GPI-anchor precursors in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Clonagem Molecular/métodos , Regulação da Expressão Gênica/genética , Glicosilfosfatidilinositóis/biossíntese , Animais , Células Cultivadas , DNA Complementar/genética , Biblioteca Gênica , Vetores Genéticos , Glicosilfosfatidilinositóis/deficiência , Glicosilfosfatidilinositóis/genética , Hemoglobinúria Paroxística/genética , Hemoglobinúria Paroxística/metabolismo , Herpesvirus Humano 4/genética , Humanos , Mutação/genética , TransfecçãoRESUMO
Most macromolecules on the surface of Leishmania parasites, including the major surface proteins and a complex lipophosphoglycan (LPG) are anchored to the plasma membrane via GPI glycolipids. Free glycoinositol-phospholipids (GIPLs) which are not linked to protein or phosphoglycan are also abundant in the plasma membrane. From structural and metabolic labeling studies it is proposed that most Leishmania species express three distinct pathways of GPI biosynthesis. Some of these pathways (i.e. those involved in the protein and LPG anchor biosynthesis) are down-regulated during the differentiation of the insect (promastigote) stage to the mammalian (amastigote) stage. In contrast, the GIPLs are expressed in high copy number in both developmental stages. Based on analysis of the lipid moieties of the different GPI species it is possible that the pathways of GPI anchor and GIPL biosynthesis are located in different subcellular compartments. The relative flux through the GIPL and LPG biosynthetic pathways has been examined in L. major promastigotes. These studies showed that while the rate of synthesis of the GIPLs and LPG is similar, LPG is shed more rapidly from the plasma membrane and has a higher turnover. The possible metabolic relationship between the GIPL and LPG biosynthetic pathways is discussed.
Assuntos
Glicolipídeos/metabolismo , Glicoesfingolipídeos/metabolismo , Glicosilfosfatidilinositóis/biossíntese , Leishmania major/metabolismo , Fosfatidilinositóis/biossíntese , Proteínas de Protozoários/biossíntese , Animais , Glucosamina/metabolismo , Leishmania major/química , Leishmania major/crescimento & desenvolvimento , Proteínas de Membrana/metabolismoRESUMO
We are using a genetic approach to explore the synthesis and function of glycosylphosphatidylinositol (GPI). We have developed a novel strategy to isolate Saccharomyces cerevisiae mutants blocked in GPI anchoring by screening colonies of mutagenized yeast cells for those that fail to incorporate [3H]inositol into protein. Among our isolates are strains blocked in mannosylation of the GPI-anchorprecursor, and strains defective in the synthesis of N-acetylglucosaminyl phosphatidylinositol (GlcNAc-PI). We have characterized one mutant, gpi1, further. This strain is defective in GlcNAc-PI synthesis and is temperature-sensitive for growth. Completion of the first step in GPI assembly is therefore required for the growth of the unicellular eukaryote S. cerevisiae. We have isolated plasmids that complement the gpi1 mutation from S. cerevisiae genomic DNA-and fission yeast cDNA libraries.
Assuntos
Proteínas Fúngicas/biossíntese , Glicosilfosfatidilinositóis/biossíntese , Mutação/genética , Precursores de Proteínas/biossíntese , Saccharomyces cerevisiae/isolamento & purificação , Temperatura , DNA Complementar/genética , Proteínas Fúngicas/fisiologia , Biblioteca Genômica , Glicosilfosfatidilinositóis/fisiologia , Precursores de Proteínas/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
In Trypanosoma brucei, glycosylphosphatidylinositol (GPI) anchors of proteins and free GPIs with identical structures have been characterized. This identity provides strong presumptive evidence that the free GPIs are in fact precursors of the GPI anchors on proteins. In mammalian tissues, however, rather consistent differences in the structures of free GPIs and GPI anchors are observed. The terminal GPIs produced by the mammalian biosynthetic pathway differ from GPI anchors in being almost exclusively fatty acid acylated on the inositol residue, having a greater number of phosphoethanolamine residues, and perhaps in containing a greater percentage of diacylglycerol components. While in principle these differences could be reconciled by remodeling reactions before or after attachment of GPI anchors, it is possible that some of the mammalian free GPIs play cellular roles other than as anchor precursors. We have approached this question by studying the lifetimes of the last three GPIs on the biosynthetic pathway, denoted H6, H7 and H8, in K562 cells and in a K562 mutant designated class K that is devoid of GPI-anchored proteins. Pulse-chase metabolic labeling with [3H]-mannose indicated that H6 was a precursor of H7 and H8 and that the H8 lifetime was more than one hour in the parental cells and even longer in the mutant. Preliminary data indicated that the majority of each of the three GPIs was localized in the plasma membrane fraction rather than the endoplasmic reticulum. These observations argue that mammalian GPIs are not utilized exclusively as GPI anchor precursors.
Assuntos
Glicosilfosfatidilinositóis/biossíntese , Precursores de Proteínas/biossíntese , Animais , Glicosilfosfatidilinositóis/fisiologia , Mamíferos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Precursores de Proteínas/fisiologia , Células Tumorais CultivadasRESUMO
Glycosylphosphatidylinositol (GPI) anchors are constructed in the endoplasmic reticulum (ER) through the action of at least seven unique enzymes. Using cell-free systems, mainly derived from African trypanosomes, it has been experimentally possible to re-create many aspects of the GPI biosynthetic pathway in vitro and to obtain a series of glycosylated phosphatidylinositol structures that correspond to biosynthetic intermediates. This approach led to the identification of the biosynthetic donors of individual components of the GPI glycan, and the discovery of unusual fatty acid re-modelling reactions in the GPI pathway in trypanosomes. Despite this progress, questions remain concerning the enzymology of the pathway, particularly the topological distribution of the different assembly steps in the ER membrane. In the work described here we have attempted to define the transbilayer orientation of different GPI biosynthetic intermediates in the ER membrane bilayer. The experiments were performed with a microsomal fraction derived from bloodstream-form Trypanosoma brucei, and standard radiolabeling procedures. The orientation of GPIs was probed with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and the jackbean lectin Concanavalin A. Contrary to expectations based on other ER glycosylation reactions, most notably the reactions involved in the dolichol pathway of N-glycosylation, our results suggest that non-inositol-acylated (PI-PLC-sensitive) GPIs are synthesized in the cytoplasmic leaflet of the ER membrane bilayer and that the final reaction product, a phosphoethanolamine-containing GPI, flips into the luminal leaflet for transfer to protein.
Assuntos
Retículo Endoplasmático/metabolismo , Glicosilfosfatidilinositóis/biossíntese , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Concanavalina A/metabolismo , Citosol , Glicolipídeos/metabolismo , Microssomos/metabolismo , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Diester Fosfórico Hidrolases/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
We have used microsomes prepared from murine lymphoma cell lines to investigate the individual reactions by which glycosylphosphatidylinositol (GPI) is synthesized in mammalian cells. Previously, GTP was found to specifically stimulate the second reaction in the pathway, the deacetylation of GlcNAc-PI to GlcN-PI. An additional GPI precursor was detected in incubations with GTP and was found to be GlcN-PI(acyl), the glycolipid proposed to be the third intermediate in mammalian GPI biosynthesis. Investigation into the factors that affect the formation of GlcN-PI(acyl) revealed that, in the presence of GTP, the addition of either CoA or palmitoyl-CoA to the incubation greatly enhanced the amount of this product made. CoA stimulation of this reaction persisted even when ATP was depleted and no formation of acyl-CoA was possible, indicating that the free CoA rather than an acyl-CoA is the actual effector of GlcN-PI acylation. Therefore, we propose that the third reaction in mammalian GPI biosynthesis is catalyzed by a CoA-dependent transacylase rather than an acyl-CoA acyltransferase.
Assuntos
Acetilglucosamina/análogos & derivados , Glicosilfosfatidilinositóis/biossíntese , Fosfatidilinositóis/metabolismo , Precursores de Proteínas/metabolismo , Acetilglucosamina/metabolismo , Acil Coenzima A/metabolismo , Acilação , Animais , Coenzima A/fisiologia , Linfoma/metabolismo , Camundongos , Palmitoil Coenzima A/metabolismo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Retículo Endoplasmático/metabolismo , Glicoesfingolipídeos/metabolismo , Glicosilfosfatidilinositóis/biossíntese , Leishmania major/metabolismo , Metaloendopeptidases/metabolismo , Proteínas de Protozoários/biossíntese , Trypanosoma brucei brucei/metabolismo , Fosfolipases Tipo C/biossíntese , Animais , Compartimento Celular , Citoplasma/metabolismo , Glicosilfosfatidilinositóis/química , Hemoglobinúria Paroxística/metabolismo , Leishmania major/genética , Mamíferos , Linfócitos T/metabolismoRESUMO
To test whether the requirements for GPI-attachment are the same in mammalian cells and parasitic protozoa, we expressed the GPI-linked variant surface glycoprotein (VSG) of Trypanosoma brucei (T. brucei) in COS cells. Although large amounts of VSG were produced, only a small fraction became GPI-linked. This impaired processing is not due to the VSG ectodomain since replacement of the VSG GPI-signal with that of decay accelerating factor (DAF) produced GPI-linked VSG. Further, whereas fusion of the DAF GPI-signal to the COOH-terminus of human growth hormone (hGH) produces GPI-linked hGH, an analogous fusion using the VSG GPI-signal does not, indicating that the VSG GPI-signal functions poorly in mammalian cells. By constructing chimeric VSG-DAF GPI-signals and fusing them to the COOH-terminus of hGH, we show that of the two critical elements that comprise the GPI-signal--the cleavage/attachment site and the hydrophobic domain--the former is responsible for the impaired activity of the VSG GPI-signal in COS cells. To confirm this, we show that the VSG GPI-signal can be converted to a viable signal for mammalian cells by altering the amino acid configuration at the cleavage/attachment site. We also show that when fused to hGH, the putative GPI-signal from the malaria circumsporozoite (CS) protein produces low levels of GPI-anchored hGH, suggesting that the CS protein is indeed GPI-linked, but that the CS protein GPI-signal, like the VSG-signal, functions poorly in COS cells.
Assuntos
Glicosilfosfatidilinositóis/biossíntese , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Sequência de Aminoácidos , Animais , Antígenos/química , Antígenos/metabolismo , Células Cultivadas , Glicosilfosfatidilinositóis/química , Mamíferos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Transdução de Sinais/imunologia , Transdução de Sinais/fisiologia , Glicoproteínas Variantes de Superfície de Trypanosoma/químicaRESUMO
We designed a trap system to isolate different amino acid sequences which could target proteins to the cell surface via GPI anchor transfer. This selection procedure is based on the insertion of various sequences which regenerate a functional GPI anchor signal sequence and therefore provoke re-expression at the surface of a reporter molecule. Using this trap for cell surface targeting sequences, we could show the importance of the defined elements essential for GPI anchor addition. Such a system could be used for an exhaustive analysis of the carboxyl terminus structural requirements for GPI membrane anchoring.
Assuntos
Antígenos de Superfície/metabolismo , Glicosilfosfatidilinositóis/biossíntese , Glicoproteínas de Membrana/metabolismo , Sequência de Aminoácidos , Antígenos de Superfície/química , Células Cultivadas , Citometria de Fluxo , Vetores Genéticos , Humanos , Glicoproteínas de Membrana/química , Peptídeos/metabolismo , Sinais Direcionadores de Proteínas , TransfecçãoRESUMO
The leucocyte surface glycosylphosphatidylinositol (GPI)-anchored membrane proteins are localized within specific membrane microdomains which also contain specific (glyco)lipids and intracellular proteins including protein kinases. These "GPI-domains" are devoid of most abundant transmembrane proteins, but in T-cells they appear to contain small amounts of CD4 and CD8 and in B-cell lines, small amounts of CD10. The existence of these relatively detergent-resistant membrane microdomains explains the signal-transducing ability of GPI-anchored receptors. In addition to the "GPI-microdomains", several other types of analogous very large detergent-resistant complexes/domains appear to exist, such as those containing T-cell receptor, others containing CD45R molecules associated with a protein kinase, and still others composed mainly of several proteins of the tetraspan family. Therefore, we suggest that the leucocyte surface is a mosaic of microdomains of unique composition associated with specific signal-transducing molecules.
Assuntos
Antígenos CD/análise , Antígenos de Superfície/análise , Glicosilfosfatidilinositóis/química , Leucócitos/química , Glicoproteínas de Membrana/análise , Antígenos CD/química , Antígenos de Superfície/química , Linfócitos B/química , Linfócitos B/imunologia , Glicosilfosfatidilinositóis/biossíntese , Humanos , Leucócitos/imunologia , Glicoproteínas de Membrana/química , Linfócitos T/química , Linfócitos T/imunologiaRESUMO
Prions are small proteinaceous particles that transmit scrapie and other fatal encephalopathies of humans and animals, and that appear to be devoid of nucleic acids. The only known--and perhaps the sole--component of the scrapie prion is an abnormal host-encoded protein, the scrapie prion protein PrPSc. The biosynthesis of this pathological protein in the host cell, which is thus of paramount importance to prion replication, is still poorly understood. We are studying the biosynthesis and degradation of the scrapie prion protein PrPSc and of its normal isoform PrPC in scrapie-infected rodent cells in culture. PrPC is anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI) moiety. In scrapie-infected mouse neuroblastoma N2a cells, PrPSc is formed post-translationally, probably from plasma membrane PrPC, in an unknown subcellular compartment that is readily accessible from the plasma membrane. Transport along the secretory pathway is necessary for PrPSc synthesis. In contrast to PrPC, PrPSc accumulates intracellularly, primarily in secondary lysosomes. The subcellular compartment(s) in which PrPSc is formed remain to be determined.