RESUMO
Galectin-3 is the best-characterized member of galectins, an evolutionary conserved family of galactoside-binding proteins that play central roles in infection and immunity, regulating inflammation, cell migration and cell apoptosis. Differentially expressed by cells and tissues with immune privilege, they bind not only to host ligands, but also to glycans expressed by pathogens. In this regard, we have previously shown that human galectin-3 recognizes several genetic lineages of the protozoan parasite Trypanosoma cruzi, the causal agent of Chagas' disease or American trypanosomiasis. Herein we describe a molecular mechanism developed by T. cruzi to proteolytically process galectin-3 that generates a truncated form of the protein lacking its N-terminal domain - required for protein oligomerization - but still conserves a functional carbohydrate recognition domain (CRD). Such processing relies on specific T. cruzi proteases, including Zn-metalloproteases and collagenases, and ultimately conveys profound changes in galectin-3-dependent effects, as chemical inhibition of parasite proteases allows galectin-3 to induce parasite death in vitro. Thus, T. cruzi might have established distinct mechanisms to counteract galectin-3-mediated immunity and microbicide properties. Interestingly, non-pathogenic T. rangeli lacked the ability to cleave galectin-3, suggesting that during evolution two genetically similar organisms have developed different molecular mechanisms that, in the case of T. cruzi, favoured its pathogenicity, highlighting the importance of T. cruzi proteases to avoid immune mechanisms triggered by galectin-3 upon infection. This study provides the first evidence of a novel strategy developed by T. cruzi to abrogate signalling mechanisms associated with galectin-3-dependent innate immunity.
Assuntos
Doença de Chagas/imunologia , Galectina 3/imunologia , Imunidade Inata , Metaloproteases/imunologia , Proteólise , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/imunologia , Proteínas Sanguíneas , Doença de Chagas/patologia , Galectina 3/química , Galectinas , Humanos , Metaloproteases/química , Domínios Proteicos , Proteínas de Protozoários/químicaRESUMO
BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) is an inducible enzyme that suppresses the immune response. The role of IDO as a negative regulator of inflammatory responses has been documented in several experimental autoimmune diseases. OBJECTIVES: To explore the regulation of IDO by immune cells in psoriasis and its relation with disease severity. METHODS: The expression and activity of IDO were assessed by reverse-transcriptase polymerase chain reaction, flow cytometry and high-performance liquid chromatography in peripheral blood of patients with moderate-to-severe plaque-type psoriasis. The ability of immune cells to express IDO in response to inflammatory stimuli was studied. The functional role of IDO expression was evaluated in a regulatory T cell (Treg) differentiation assay, using cocultures of immature monocyte-derived dendritic cells with autologous peripheral CD4+ T cells. RESULTS: Analysis of the kynurenine-to-tryptophan ratio in serum samples indicated higher IDO activity in patients with psoriasis than in healthy controls. However, correlation studies showed lower IDO activity in those patients with higher Psoriasis Area and Severity Index (PASI). Although myeloid dendritic cells from patients with psoriasis expressed higher levels of IDO than those from healthy controls, these cells did not upregulate IDO in response to a combination of tumour necrosis factor-α, interleukin (IL)-1ß and IL-6 cytokines. The defective expression of IDO correlated with PASI. Immature monocyte-derived dendritic cells from patients with psoriasis also expressed low levels of IDO and induced CD4+ Treg differentiation poorly. CONCLUSIONS: Immune cells from patients with psoriasis have a defect in upregulating IDO in response to inflammation associated with the severity of psoriasis.
Assuntos
Citocinas/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Psoríase/enzimologia , Linfócitos T Reguladores/fisiologia , Estudos de Casos e Controles , Diferenciação Celular/imunologia , Células Dendríticas/fisiologia , Combinação de Medicamentos , Humanos , Leucócitos Mononucleares , Lipopolissacarídeos/farmacologia , Psoríase/imunologia , Linfócitos T Reguladores/citologiaRESUMO
We report here the specific interaction between several members of the human galectin family with the three developmental stages of several genetic lineages of the protozoan parasite Trypanosoma cruzi. We provide data of specific and differential binding of human galectin (gal)-1, -3, -4, -7 and -8 to 14 strains of T. cruzi that belong to the six genetic lineages representing the genetic diversity of the parasite. It is shown that galectins preferentially bind forms present in the host, trypomastigotes and amastigotes, compared with the non-infective epimastigote present on the intestinal tract of the vector, reflecting the changes on glycosylation that occur during the metacyclogenesis and amastigogenesis process. Also, it is evidenced that galectin binding to the parasites promotes binding to the host cells and higher infection rates. In addition, evidence is provided indicating that the intracellular amastigotes may take over the cytosolic pool of some galectins when released to the extracellular medium. Finally, by applying unweighted pair group method analysis to the galectin-binding profile to either cell-derived trypomastigotes or amastigotes, we show that the differential-binding profile by the host galectins to the six lineages resembles the clustering based in genetic data. Therefore, the differential-binding profile for the six lineages could have implications in the immunopathology of Chagas' disease, affecting the complex network of immune responses on which galectins mediate, thus providing linkage clues to the notion that different lineages may be related to different clinical forms of the disease.
Assuntos
Galectinas/química , Trypanosoma cruzi/genética , Animais , Sítios de Ligação , Células CACO-2 , Chlorocebus aethiops , Análise por Conglomerados , Interações Hospedeiro-Parasita , Humanos , Ligantes , Mucinas/química , Ligação Proteica , Proteínas de Protozoários/química , Trypanosoma cruzi/imunologia , Células VeroRESUMO
The search for disease-associated T helper 2 (Th2) Leishmania antigens and the induction of a Th1 immune response to them using defined vaccination protocols is a potential strategy to induce protection against Leishmania infection. Leishmania infantum LiP2a and LiP2b acidic ribosomal protein (P proteins) have been described as prominent antigens during human and canine visceral leishmaniasis. In this study we demonstrate that BALB/c mice infected with Leishmania major develop a Th2-like humoral response against Leishmania LiP2a and LiP2b proteins and that the same response is induced in BALB/c mice when the parasite P proteins are immunized as recombinant molecules without adjuvant. The genetic immunization of BALB/c mice with eukaryotic expression plasmids coding for these proteins was unable to redirect the Th2-like response induced by these antigens, and only the co-administration of the recombinant P proteins with CpG oligodeoxynucleotides (CpG ODN) promoted a mixed Th1/Th2 immune response. According to the preponderance of a Th2 or mixed Th1/Th2 responses elicited by the different regimens of immunization tested, no evidence of protection was observed in mice after challenge with L. major. Although alterations of the clinical outcome were not detected in mice presensitized with the P proteins, the enhanced IgG1 and interleukin (IL)-4 response against total Leishmania antigens in these mice may indicate an exacerbation of the disease.
Assuntos
Leishmania major/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Cutânea/prevenção & controle , Proteínas de Protozoários/imunologia , Células Th1/imunologia , Células Th2/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antiprotozoários/biossíntese , Antígenos de Bactérias/imunologia , Antígenos de Protozoários/imunologia , Feminino , Imunoglobulina G/biossíntese , Interleucina-4/biossíntese , Leishmaniose Cutânea/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/imunologia , Proteínas Recombinantes/imunologia , Proteínas Ribossômicas/imunologia , Vacinas de DNA/imunologiaRESUMO
Among the Trichinella TSL-1 antigens, whose antigenicity is generally due mainly to tyvelose-containing epitopes, gp53 is unusual in that its antigenicity is due mainly to protein epitopes. In the present study we mapped two of these epitopes, recognized by monoclonal antibodies (mAbs) that specifically recognize gp53 from all encysting Trichinella species (mAb US9), or gp53 from Trichinella spiralis alone (mAb US5). Based on previously published sequences of this glycoprotein [Mol. Biochem. Parasitol. 72 (1995) 253], in this study, we cloned the full gp53 cDNA from a new strain, Trichinella britovi (ISS 11; AN: ), and from another T. spiralis isolate (ISS 115; AN: ). The gp53 sequence comprised an ORF of 1239bp, coding for 412 amino acids, with 61 nucleotide differences (resulting in 38 residue changes) between the two species. Mapping of US5- and US9-recognized epitopes was undertaken through the construction and expression in the pGEX4T vector of truncated gp53 peptides, and by the construction of peptides derived from the antigenic regions. The epitope recognized by mAb US9 was a linear peptide of 8 residues, 33Met- 40Ser, located in the amino-terminal region, while the corresponding epitope recognized by mAb US5 was a 47-amino acid sequence containing two alpha-helix regions flanked by random coils, 290Thr- 336Lys. Molecular modeling of these peptides seems to indicate that recognition of the US9 epitope depends on the presence of two available hydroxyl groups provided by one methionine and one serine on T. spiralis gp53 (not present on Trichinella pseudospiralis gp53). Additionally, the stability of the US5 epitope seems to depend on correct folding of the 47-amino acid sequence (only present in T. spiralis). The relevance of these findings for understanding the antigenic recognition of Trichinella TSL-1 antigens, and for further studies to investigate possible function(s) of gp53 in Trichinella, is discussed.
Assuntos
Antígenos de Helmintos/imunologia , Epitopos Imunodominantes/imunologia , Trichinella/imunologia , Tirosina Quinase da Agamaglobulinemia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , DNA Complementar/genética , DNA de Helmintos/genética , Larva , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Knockout , Modelos Moleculares , Dados de Sequência Molecular , Fases de Leitura Aberta , Polissacarídeos/química , Polissacarídeos/imunologia , Conformação Proteica , Dobramento de Proteína , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Trichinella/classificação , Trichinella/crescimento & desenvolvimento , Trichinella spiralis/crescimento & desenvolvimento , Trichinella spiralis/imunologiaRESUMO
Potatoes are a cheap and easily available source for the preparation of beta 1,2-xylosidase. The soluble enzyme was purified from potato tubers by ammonium sulfate precipitation, hydrophobic interaction chromatography, affinity gel blue chromatography, ion exchange and size exclusion chromatography yielding a glycoprotein with a molecular weight of 39-40 kDa, an isoelectric point of 5.1 and a typical plant N-glycosylation pattern. The enzyme releases xylose residues beta1,2-linked to the beta-mannose of an N-glycan core, if the 3-position of this mannose is not occupied. It showed an optimal enzymatic activity at pH 4.0-4.5 and at a temperature of 50 degrees C. The activity was reduced in the presence of Ni(2+) and Cu (2+) and slightly increased by the addition of Mn(2+) or Ca(2+). At 37 degrees C the cleavage of xylose from p-nitrophenyl-beta-xylopyranoside or appropriate pyridylaminated N-glycans was proportional to the time of incubation over a period of 8 h and increased with time for at least 24 h. N-Methoxycarbonylpentyl-1,5-dideoxy-1,5-iminoxylitol inhibits the enzyme effectively. Sequencing of the N-terminus showed a high homology to a number of isoforms of patatin, the main protein of potato tubers. This enzyme will be an important tool for the analysis of N-glycans and in the modification of N-glycans for immunological studies.
Assuntos
Solanum tuberosum/enzimologia , Xilosidases/isolamento & purificação , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Temperatura , Xilosidases/metabolismoRESUMO
The sugar binding specificity of the recently described mannose-specific carbohydrate-binding proteins (CBP) isolated to homogeneity from both the epimastigote and trypomastigote stages of the pathogenic protozoa Trypanosoma cruzi has been studied by quantitative hapten inhibition of the biotinylated CBPs to immobilized thyroglobulin using model oligosaccharides. The results clearly show a differential specificity toward high-mannose glycans between the CBPs from the two developmental stages. Thus, the isolated CBP from epimastigotes exhibited stronger affinity for higher mannose oligomers containing the Manalpha1-2Manalpha1-6Manalpha1-6 structure. Its affinity decreased, as did the number of mannose residues on the oligomer or removal of the terminal Manalpha1-2-linked mannose. By contrast the CBP isolated from the trypomastigote stage showed about 400-fold lower avidity than the epimastigote form, and contrary to it, it was slightly more specific toward Man5GlcNAc than Man9GlcNAc. Analysis of the interaction of epimastigote-Man-CBP with its ligands by UV difference spectroscopy indicates the existence of an extended binding site in that protein with a large enthalpic contribution to the binding. The thermodynamic parameters of binding were obtained by isothermal titration calorimetry and been found that the DeltaH values to be in good agreement with the van't Hoff values. The binding reactions are mainly enthalpically driven and exhibit enthalpy-enthropy compensation. In addition, analysis of the high-mannose glycans from different parts of the digestive tract of the reduviid insect vector of T. cruzi suggest a role of the CBP in the retention of the epimastigote stage in the anterior portion of the gut.
Assuntos
Proteínas de Transporte/metabolismo , Manose/metabolismo , Receptores de Superfície Celular , Trypanosoma cruzi/metabolismo , Animais , Calorimetria , Sequência de Carboidratos , Membrana Celular/metabolismo , Dados de Sequência Molecular , Polissacarídeos/metabolismo , Ligação Proteica , Espectrofotometria Ultravioleta , Tireoglobulina/metabolismoRESUMO
Apolipoprotein E externally added to neuroblastoma cells in culture stimulates [35S]sulfate incorporation on cell and extracellular matrix glycosaminoglycans (sGAG). This stimulation is mainly observed for ApoE4 compared to ApoE3. The increase in sulfation is not due to increased synthesis as there is no corresponding increase in the [3H]glucosamine incorporation. Since the presence of ApoE is a risk factor for Alzheimer's disease (AD) and the presence of sGAG could facilitate the assembly of the main components, beta-amyloid and tau proteins, of the aberrant structures found in AD, the present study indicates a possible relation between those factors.
Assuntos
Doença de Alzheimer/metabolismo , Apolipoproteínas E/farmacologia , Glicosaminoglicanos/metabolismo , Neurônios/efeitos dos fármacos , Apolipoproteína E4 , Relação Dose-Resposta a Droga , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Glucosamina/química , Glicosaminoglicanos/biossíntese , Glicosaminoglicanos/química , Humanos , Neurônios/metabolismo , Fatores de Risco , Sulfatos/química , Radioisótopos de Enxofre , Trítio , Células Tumorais CultivadasRESUMO
The cloning and characterization of seven Rab and three Ran/TC4 partial cDNA sequences in both cystic (Trichinella spiralis and T. britovi) and noncystic species (T. pseudospiralis) are reported. These molecules were cloned by rapid amplification of cDNA ends via polymerase chain reaction (RACE-PCR), using cDNA from the aforementioned Trichinella spp. coupled to the AP1 adaptor. As primers, AP1 and 5B (derived from the WDTAGQE sequence of region 2 specific for Rab and Ran proteins) sequences were included in the PCR. The cloned cDNAs were sequenced and characterized by both Southern-blot and Northern-blot analysis. Trichinella spp. Rab- and Ran-like molecules showed divergences in both the nucleotide and the deduced amino acid sequences as compared with the corresponding homologues previously described in other organisms. In addition, differences were observed among the Trichinella species, mainly between the cystic and the noncystic species, in both DNA restriction-enzyme polymorphism and expression of the six GTPases isolated.
Assuntos
Clonagem Molecular , Proteínas de Ligação ao GTP/genética , Proteínas Nucleares/genética , Trichinella/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Southern Blotting , DNA Complementar , DNA de Helmintos/genética , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Trichinella/crescimento & desenvolvimento , Trichinella/metabolismo , Proteína ran de Ligação ao GTPRESUMO
Trypanosoma cruzi is an obligatory intracellular protozoan parasite that causes Chagas' disease in humans. Although a fair amount is known about the biochemistry of certain trypanosomes, very little is known about the enzymic complement of synthesis and processing of glycoproteins and/or functions of the subcellular organelles in this parasite. There have been very few reports on the presence of acid and neutral hydrolases in Trypanosoma cruzi. Here we report the first purification and characterization of a neutral mannosidase from the epimastigote stage of Trypanosoma cruzi. The neutral mannosidase was purified nearly 800-fold with an 8% recovery to apparent homogeneity from a CHAPS extract of epimastigotes by the following procedures: (1) metal affinity chromatography on Co+2-Sepharose, (2) anion exchange, and (3) hydroxylapatite. The purified enzyme has a native molecular weight of 150-160 kDa and is apparently composed of two subunits of 76 kDa. The purified enzyme exhibits a broad pH profile with a maximum at pH 5.9-6.3. It is inhibited by swainsonine (Ki, 0.1 microM), D-mannono-delta-lactam (Ki, 20 microM), kifunensine (Ki, 60 microM) but not significantly by deoxymannojirimycin. The enzyme is activated by Co2+and Ni2+and strongly inhibited by EDTA and Fe2+. The purified enzyme is active against p-nitrophenyl alpha-D-mannoside (km = 87 microM). High-mannose Man9GlcNAc substrate was hydrolyzed by the purified enzyme to Man7GlcNAc at pH 6.1. The purified enzyme does not show activity against alpha1,3- or alpha1,6-linked mannose residues. Antibodies against the recently purified lysosomal alpha-mannosidase from T.cruzi did not react with the neutral mannosidase reported here.
Assuntos
Manosidases/isolamento & purificação , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Carboidratos , Cromatografia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Cinética , Manosidases/química , Manosidases/metabolismo , Dados de Sequência Molecular , Peso Molecular , Oligossacarídeos/química , Conformação Proteica , Especificidade por SubstratoRESUMO
According to current models the inhibitory capacity of I(kappa)B(alpha) would be mediated through the retention of Rel/NF-kappaB proteins in the cytosol. However, I(kappa)B(alpha) has also been detected in the nucleus of cell lines and when overexpressed by transient transfection. To gain better insight into the potential role of nuclear I(kappa)B(alpha) in a physiological context we have analysed its presence in the nucleus of human peripheral blood T lymphocytes (PBL). We demonstrate the nuclear localization of I(kappa)B(alpha) in PBL by different techniques: Western blot, indirect immunofluorescence and electron microscopy. Low levels of nuclear I(kappa)B(alpha) were detected in resting cells whereas a superinduction was obtained after PMA activation. The nuclear pool of I(kappa)B(alpha) showed a higher stability than cytosolic I(kappa)B(alpha) and was partially independent of the resynthesis of the protein. Unexpectedly, the presence of nuclear I(kappa)B(alpha) did not inhibit NF-kappaB binding to DNA and this phenomenon was not due to the presence of IkappaBbeta at the nuclear level. Immunoprecipitation experiments failed to demonstrate an association between nuclear I(kappa)B(alpha) and NF-kappaB proteins. Our results demonstrate that in resting and PMA-activated human PBL, I(kappa)B(alpha) is present in the nucleus in an apparently inactive form unable to disrupt NF-kappaB binding from DNA.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas I-kappa B , Linfócitos T/metabolismo , Células Sanguíneas , Western Blotting , Citosol/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Células Jurkat/metabolismo , Ativação Linfocitária , Microscopia Confocal , Microscopia Eletrônica , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-rel , Acetato de Tetradecanoilforbol/farmacologia , Transcrição GênicaRESUMO
Factor J (FJ) is a complement inhibitor that acts on the classical and the alternative pathways. We demonstrated FJ-cell interactions in fluid phase by flow cytometry experiments using the cell lines Jurkat, K562, JY, and peripheral blood lymphocytes. FJ bound to plastic plates was able to induce in vitro adhesion of these cells with potency equivalent to fibronectin. As evidence for the specificity of this reaction, the adhesion was blocked by MAJ2, an anti-FJ monoclonal antibody, and by soluble FJ. Attachment of the cells required active metabolism and cytoskeletal integrity. The glycosaminoglycans heparin, heparan sulfate, or chondroitin sulfates A, B, and C inhibited to varying degrees the binding of FJ to cells, as did treatment with chondroitinase ABC. In the search for a putative receptor, a protein of 110 kDa was isolated by affinity chromatography, and microsequence analysis identified this protein as nucleolin. Confocal microscopy evidenced the presence of nucleolin in cell membrane by immunofluorescence with monoclonal (D3) and polyclonal anti-nucleolin antibodies in Jurkat cells. The interaction FJ-nucleolin was evidenced by Western blot and enzyme-linked immunosorbent assay. Furthermore, purified nucleolin and D3 inhibited adhesion of Jurkat cells to immobilized FJ, suggesting that the interaction was specific and that nucleolin mediated the binding.
Assuntos
Proteínas de Transporte/fisiologia , Adesão Celular/fisiologia , Proteínas Inativadoras do Complemento/fisiologia , Glicoproteínas/fisiologia , Glicosaminoglicanos/farmacologia , Linfócitos/fisiologia , Fosfoproteínas/fisiologia , Proteínas de Ligação a RNA/fisiologia , Anticorpos Monoclonais/farmacologia , Linfócitos B/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Sulfatos de Condroitina/farmacologia , Cromatografia de Afinidade , Citometria de Fluxo , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Glicosaminoglicanos/fisiologia , Heparina/farmacologia , Heparitina Sulfato/farmacologia , Humanos , Células Jurkat , Células K562 , Cinética , Proteínas Nucleares/fisiologia , Células U937 , NucleolinaRESUMO
The macrolide antibiotic megalomicin (MGM) has been shown to inhibit vesicular transport between the medial- and trans-Golgi, resulting in the undersialylation of cellular proteins (P. Bonay, S. Munro, M. Fresno, and B. Alarcón, J. Biol. Chem. 271:3719-3726, 1996). Due to the effects of MGM on the Golgi and on the replication of enveloped viruses, we decided to test whether it has any antiparasitic activity. The results showed that MGM has potent activity against the epimastigote stage of Trypanosoma cruzi, producing a 50% inhibitory concentration (IC50) of 0.2 microg/ml. Furthermore, MGM was also active against the intracellular replicative, amastigote form of T. cruzi, completely preventing its replication in infected murine LLC/MK2 macrophages at a dose of 5 microg/ml. Although less potent, MGM was also active against Trypanosoma brucei epimastigotes (IC50, 2 microg/ml) and Leishmania donovani and Leishmania major promastigotes (IC50, 3 and 8 microg/ml, respectively). MGM also blocked intracellular replication of the asexual stage of Plasmodium falciparum-infected erythrocytes at 1 microg/ml. Finally, MGM was active in an in vivo model, resulting in the complete protection of BALB/c mice from death caused by acute T. brucei infection and significantly reducing the parasitemia. These results suggest that MGM is a potential drug for the treatment of veterinary and human parasitic diseases.
Assuntos
Antibacterianos/farmacologia , Antiparasitários/farmacologia , Complexo de Golgi/metabolismo , Macrolídeos , Animais , Transporte Biológico/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacosRESUMO
We describe the molecular cloning of the PSA genes from the Leishmania infantum parasite, which show high sequence similarity with the L. major PSA-2 and L. amazonensis GP46/M2 genes. The PSA genes in L. infantum are arrayed in tandem with a repetition unit of 6 kb. A single-size class of PSA mRNA of 4 kb was detected. The characterised L. infantum PSA genes code for a protein lacking the glycosylphosphatidylinositol addition signal described in other Leishmania species due to the presence of a stop codon located upstream from the DNA sequence coding for the signal. The data obtained after immunoprecipitation of PSA indicate that the protein is present as a water-soluble form, but that also a membrane-anchored form can be detected. The amino acid sequence derived from the isolated PSA gene shows that 60% of the deduced protein is formed by 13 leucine-rich repeats, each one of which is 24 amino acids long. The analysis of the consensus sequence of the repeats revealed that the L. infantum PSA as well as the described L. major PSA-2 and L. amazonensis GP46/M2 proteins may be classified as new members of the leucine-rich repeat-containing protein superfamily. The number of leucine-rich motifs, however, varies considerably between the PSA protein from L. infantum and from the other Leishmania species. The PSA protein is a major antigen determinant during L. infantum infections since 87% of the sera from naturally infected dogs recognise the recombinant PSA purified from Escherichia coli.
Assuntos
Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Superfície/genética , Leishmania infantum/genética , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Antígenos de Superfície/química , Sequência de Bases , Clonagem Molecular , Cães , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , SolubilidadeRESUMO
Human protein HC is a member of the lipocalin superfamily with unique properties since it carries a covalently bound fluorescent chromophore mediating the linkage of the major part of protein HC to several plasma proteins, with IgA as the dominating complex partner. Native protein HC displays characteristic absorption and fluorescence spectra similar to those of glycated proteins with advanced glycosylation end products (AGEs). In vitro glycation of protein HC induces the formation of fibril aggregates with a corresponding increase of absorption in the visible region of the spectrum. Boronate-affinity chromatography and a novel galactosyltransferase assay indicate that protein HC is modified with residues of glucose exposed in a terminal non-reducing position which is typical of glycated proteins. The glycation level of several isolated batches of protein HC as measured by both assays was around 35%, which represents the highest level described for human plasma-derived proteins from healthy individuals.
Assuntos
alfa-Globulinas/química , Inibidores de Proteases/química , alfa-Globulinas/metabolismo , alfa-Globulinas/urina , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Colorimetria , Diabetes Mellitus , Galactosiltransferases , Glicosídeo Hidrolases , Glicosilação , Humanos , Inibidores de Proteases/metabolismo , Inibidores de Proteases/urina , Valores de ReferênciaRESUMO
Megalomicin (MGM) has been shown to cause a dilation of the most distal cisternae of the Golgi complex. The effects of MGM on Golgi morphology correlated with an inhibition of protein transport to the trans-Golgi resulting in an accumulation of poorly sialylated glycoproteins. Here we show that the addition of 50 microM MGM caused a rapid swelling of lysosomes in cultured cells and inhibited the degradation of the newly synthesized T cell antigen receptor CD36 subunit. Although MGM did not affect the uptake of fluid phase markers, it prevented their degradation. Interestingly, endocytosed ovalbumin did not colocalize with lysosomes in MGM-treated cells, suggesting an MGM-induced impairment in the delivery to lysosomes. This was confirmed by Percoll density gradients, where the fluid phase marker remained in endosomal fractions, even after long chase times, whereas in control cells the endocytosed marker was located in lysosomes. The effect of MGM was not confined to soluble proteins since it did also inhibit the delivery of the membrane-bound epidermal growth factor receptor to lysosomes. Finally, MGM strongly inhibited the ATP-dependent acidification of lysosomes in vitro, suggesting a possible mechanism for its in vivo activity.
Assuntos
Antibacterianos/farmacologia , Lisossomos/efeitos dos fármacos , Animais , Células COS , Linhagem Celular , Endocitose , Exocitose , Humanos , Rim , Cinética , Lisossomos/fisiologia , Lisossomos/ultraestrutura , Macrolídeos/farmacologia , Ovalbumina/farmacocinética , Polissacarídeos/análise , Ratos , Complexo Receptor-CD3 de Antígeno de Linfócitos T/biossíntese , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
IL-2 and IL-4 induce proliferation of TS1 alpha beta cells. Activation of the zeta isoform of protein kinase C is an important step in IL-2-, but not IL-4-mediated proliferation. In addition, protein kinase C-zeta is implicated in IL-2-mediated actin organization. Given the established involvement of the Rho family of small guanine nucleotide-binding proteins in organization of actin structures, we analyze the possible relationships between Rho and protein kinase C-zeta. Using the Rho-like protein family-specific toxin B from Clostridium difficile, we report in this work that IL-2, but not IL-4, induces a Rho-dependent activation of protein kinase C-zeta. This signaling event is mediated by the activation of phosphatidylinositol 3-kinase. In contrast, IL-4 induces a Rho-independent, phosphatidylinositol 3-kinase-mediated activation of protein kinase C-zeta, but this pathway has no implications in cytoskeleton organization.
Assuntos
Actinas/metabolismo , Proteínas de Bactérias , Proteínas de Ligação ao GTP/fisiologia , Interleucina-2/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Proteína Quinase C/fisiologia , Actinas/efeitos dos fármacos , Actinas/imunologia , Animais , Toxinas Bacterianas/farmacologia , Linhagem Celular , Clostridioides difficile/imunologia , Ativação Enzimática/imunologia , Humanos , Interleucina-4/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Fosfatidilinositol 3-Quinases , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/imunologiaRESUMO
Megalomicin (MGM) is a macrolide antibiotic which has been demonstrated previously to cause an anomalous glycosylation of viral proteins. Here we show that MGM produces profound alterations on Golgi morphology and function. The addition of MGM at 50 microM for 1 h caused a dilation of the Golgi detected by immunofluorescence staining for medial- and trans-Golgi markers. The effect of MGM was clearly more intense on the trans-side of the Golgi, as evidenced in electron microscope preparations. The effect on Golgi morphology was reversible and correlated with an impairment of glycoprotein processing in the trans-Golgi. Thus, although the vesicular stomatitis virus G protein was processed in the presence of MGM to an endoglycosidase H-resistant form, it was poorly sialylated. The sialylation of cellular proteins was also inhibited, resulting in cells with low level of sialylation on the cell surface. However MGM did not inhibit the activities of the galactosyl- or sialyltransferase as measured in vitro. MGM inhibited cis- to medial-, and more strongly, medial- to trans-Golgi transport of vesicular stomatitis virus G protein in an in vitro system, suggesting that the impairment in glycoprotein maturation observed in vivo is the result of intra-Golgi transport inhibition.
Assuntos
Antibacterianos/farmacologia , Complexo de Golgi/efeitos dos fármacos , Glicoproteínas de Membrana , Animais , Células CHO , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Galactosiltransferases/antagonistas & inibidores , Galactosiltransferases/metabolismo , Glicoproteínas/metabolismo , Glicosilação/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Rim , Lectinas , Macrolídeos/isolamento & purificação , Macrolídeos/farmacologia , Microscopia Eletrônica , Microscopia Imunoeletrônica , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Ratos , Sialiltransferases/antagonistas & inibidores , Sialiltransferases/metabolismo , Vírus da Estomatite Vesicular Indiana , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/metabolismoRESUMO
The target for the antitumoral peptidic drug, Kahalalide F, has been studied in cultured cells. In the presence of the compound, the cells became impressively swollen, showing the formation of large vacuoles. The formation of these vacuoles appears to be the consequence of changes in lysosomal membranes. Thus, lysosomes are a target for Kahalalide F action.