RESUMO
Gangliosides are sialic acid-containing glycosphingolipids mainly expressed at the outer leaflet of the plasma membrane. Sialidase NEU3 is a key enzyme in the catabolism of gangliosides with its up-regulation having been observed in human cancer cells. In the case of CME (clathrin-mediated endocytosis), although this has been widely studied, the role of NEU3 and gangliosides in this cellular process has not yet been established. In the present study, we found an increased internalization of Tf (transferrin), the archetypical cargo for CME, in cells expressing complex gangliosides with high levels of sialylation. The ectopic expression of NEU3 led to a drastic decrease in Tf endocytosis, suggesting the participation of gangliosides in this process. However, the reduction in Tf endocytosis caused by NEU3 was still observed in glycosphingolipid-depleted cells, indicating that NEU3 could operate in a way that is independent of its action on gangliosides. Additionally, internalization of α2-macroglobulin and low-density lipoprotein, other typical ligands in CME, was also decreased in NEU3-expressing cells. In contrast, internalization of cholera toxin ß-subunit, which is endocytosed by both clathrin-dependent and clathrin-independent mechanisms, remained unaltered. Kinetic assays revealed that NEU3 caused a reduction in the sorting of endocytosed Tf to early and recycling endosomes, with the Tf binding at the cell surface being also reduced. NEU3-expressing cells showed an altered subcellular distribution of clathrin adaptor AP-2 (adaptor protein 2), but did not reveal any changes in the membrane distribution of clathrin, PtdIns(4,5)P2 or caveolin-1. Overall, these results suggest a specific and novel role of NEU3 in CME.
Assuntos
Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Neuraminidase/fisiologia , Animais , Células CHO , Células COS , Galinhas , Chlorocebus aethiops , Cricetinae , Cricetulus , Humanos , Ligação Proteica/fisiologiaRESUMO
Chagas' disease is a chronic, debilitating and incapacitating illness, caused by the protozoan parasite Trypanosoma cruzi when infective trypomastigotes invade host cells. Although the mechanism of trypomastigotes interaction with mammalian cells has been intensively studied, a final and integrated picture of the signal transduction mechanisms involved still remains to be elucidated. Our group has previously shown that the conserved FLY domain (VTVXNVFLYNR), present in all members of the gp85/trans-sialidase glycoprotein family coating the surface of trypomastigotes, binds to cytokeratin 18 (CK18) on the surface of LLC-MK(2) epithelial cells, and significantly increases parasite entry into mammalian cells. Now it is reported that FLY, present on the surface of trypomastigotes or on latex beads binds to CK18, promotes dephosphorylation and reorganization of CK18 and activation of the ERK1/2 signaling cascade culminating in an increase of approximately 9-fold in the number of parasites/cell. Inhibition of ERK1/2 phosphorylation completely blocks the adhesion of FLY to cells and blocks by 57% the host cell infection by T. cruzi. Taken together our results indicate that the conserved FLY domain is an important tool that trypomastigotes have evolved to specific exploit the host cell machinery and guarantee a successful infection.
Assuntos
Glicoproteínas/química , Glicoproteínas/fisiologia , Neuraminidase/química , Neuraminidase/fisiologia , Proteínas de Protozoários/química , Proteínas de Protozoários/fisiologia , Trypanosoma cruzi/fisiologia , Trypanosoma cruzi/patogenicidade , Sequência de Aminoácidos , Animais , Linhagem Celular , Sequência Conservada , Glicoproteínas/genética , Humanos , Queratina-18/química , Queratina-18/metabolismo , Sistema de Sinalização das MAP Quinases , Neuraminidase/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , VirulênciaRESUMO
Trypanosoma cruzi actively invades mammalian cells by forming parasitophorous vacuoles (PVs). After entry, the parasite has to escape from these vacuoles in order to replicate inside the host cell cytosol. Trans-sialidase (TS), a parasite enzyme that is used to obtain sialic acid from host glycoconjugates, has been implicated in cell invasion and PV exit, but how the enzyme acts in these processes is still unknown. Here we show that trypomastigotes derived from infected mammalian cells express and release 20 times more TS activity than axenic metacyclic trypomastigotes, which correspond to the infective forms derived from the insect vector. Both forms have the same capacity to invade mammalian cells, but cell derived trypomastigotes exit earlier from the vacuole. To test whether high TS expression is responsible for this increased exit from the PV, trypomastigote TS was expressed on the surface of metacyclic forms. Transfected and non-transfected metacyclics attached to and invaded HeLa or CHO cells equally. In contrast, metacyclics expressing TS on the surface escaped earlier from the vacuole than non-transfected metacyclics, or metacyclics expressing TS in their cytoplasm. Sialic acid may act as a barrier, which is removed by surface and/or secreted TS, because all types of parasites escaped earlier from the vacuoles of sialic acid-deficient Lec 2 cells than wild-type CHO cells. In addition, trypomastigotes and metacyclic forms expressing TS differentiated earlier into amastigotes. These results indicate that the increased expression of TS in cell-derived trypomastigotes is responsible for the earlier exit from the PV to the cytoplasm and their subsequent differentiation into amastigotes.
Assuntos
Glicoproteínas/fisiologia , Neuraminidase/fisiologia , Trypanosoma cruzi/enzimologia , Animais , Células CHO , Cricetinae , Citosol/parasitologia , Glicoproteínas/química , Células HeLa , Interações Hospedeiro-Parasita , Humanos , Estágios do Ciclo de Vida , Neuraminidase/química , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/fisiologia , Vacúolos/parasitologiaRESUMO
Strong thrombocytopenia is observed during acute infection with Trypanosoma cruzi, the parasitic protozoan agent of American trypanosomiasis or Chagas' disease. The parasite sheds trans-sialidase, an enzyme able to mobilize the sialyl residues on cell surfaces, which is distributed in blood and is a virulence factor. Since the sialic acid content on the platelet surface is crucial for determining the half-life of platelets in blood, we examined the possible involvement of the parasite-derived enzyme in thrombocytopenia induction. We found that a single intravenous injection of trans-sialidase into naive mice reduced the platelet count by 50%, a transient effect that lasted as long as the enzyme remained in the blood. CD43(-/-) mice were affected to a similar extent. When green fluorescent protein-expressing platelets were treated in vitro with trans-sialidase, their sialic acid content was reduced together with their life span, as determined after transfusion into naive animals. No apparent deleterious effect on the bone marrow was observed. A central role for Kupffer cells in the clearance of trans-sialidase-altered platelets was revealed after phagocyte depletion by administration of clodronate-containing liposomes and splenectomy. Consistent with this, parasite strains known to exhibit more trans-sialidase activity induced heavier thrombocytopenia. Finally, the passive transfer of a trans-sialidase-neutralizing monoclonal antibody to infected animals prevented the clearance of transfused platelets. Results reported here strongly support the hypothesis that the trans-sialidase is the virulence factor that, after depleting the sialic acid content of platelets, induces the accelerated clearance of the platelets that leads to the thrombocytopenia observed during acute Chagas' disease.
Assuntos
Plaquetas/química , Doença de Chagas/complicações , Ácido N-Acetilneuramínico/sangue , Neuraminidase/fisiologia , Trombocitopenia/etiologia , Trypanosoma cruzi/enzimologia , Fatores de Virulência/fisiologia , Doença Aguda , Animais , Antígenos CD/fisiologia , Glicoproteínas , Células de Kupffer/fisiologia , Leucossialina , Camundongos , Camundongos Endogâmicos , Sialoglicoproteínas/fisiologia , Trypanosoma cruzi/patogenicidadeRESUMO
Trans-sialidase is a membrane-bound and shed sialidase from Trypanosoma cruzi, the protozoan parasite responsible for Chagas disease. We investigated the role of soluble trans-sialidase on host CD4+ T cell activation. Trans-sialidase activated naive CD4+ T cells in vivo. Both enzymatically active and inactive recombinant trans-sialidases costimulated CD4+ T cell activation in vitro. Costimulation resulted in increased mitogen-activated protein kinase activation, proliferation, and cytokine synthesis. Furthermore, active and inactive trans-sialidases blocked activation-induced cell death in CD4+ T cells from T. cruzi-infected mice. By flow cytometry, inactive trans-sialidase bound the highly sialylated surface Ag CD43 on host CD4+ T cells. Both costimulatory and antiapoptotic effects of trans-sialidases required CD43 signaling. These results suggest that trans-sialidase family proteins are involved in exacerbated host T lymphocyte responses observed in T. cruzi infection.
Assuntos
Antígenos CD , Linfócitos T CD4-Positivos/imunologia , Ativação Linfocitária/imunologia , Neuraminidase/fisiologia , Sialoglicoproteínas/fisiologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/parasitologia , Células Cultivadas , Ativação Enzimática/imunologia , Glicoproteínas , Injeções Subcutâneas , Interfase/imunologia , Leucossialina , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neuraminidase/administração & dosagem , Neuraminidase/antagonistas & inibidores , Neuraminidase/metabolismo , Sialoglicoproteínas/deficiência , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismoRESUMO
Main primary approaches and new developments in the study of the molecular basis of the adhesive process of Corynebacterium diphtheriae are reviewed along with a discussion of the potential importance of hemagglutinins, exposed sugar residues, hydrophobins and trans-sialidase enzymes as adhesins of strains of the sucrose fermenting and non-fermenting biotypes.
Assuntos
Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/química , Corynebacterium diphtheriae/fisiologia , Corynebacterium diphtheriae/enzimologia , Glicoproteínas/fisiologia , Hemaglutinação , Humanos , Ácido N-Acetilneuramínico/fisiologia , Neuraminidase/fisiologiaAssuntos
Glicoproteínas/fisiologia , Neuraminidase/fisiologia , Trypanosoma cruzi/enzimologia , Animais , Relação Dose-Resposta a Droga , Glicoproteínas/metabolismo , Cloreto de Magnésio/farmacologia , Mucinas/metabolismo , Neuraminidase/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Trypanosoma cruzi/fisiologia , alfa-Galactosidase/imunologiaRESUMO
Serum neuraminidase (NA, sialidase) activity has been demonstrated in acute poststreptococcal glomerulonephritis (APSGN) and implicated in the pathogenesis of the disease. Recent investigations show that neuraminidase-treated leukocytes accumulate preferentially in kidneys; therefore, we were interested in knowing if desialized cells infiltrate the kidney in APSGN. We first tested the capacity of peanut agglutinin lectin (PNA) to detect injected NA-treated leukocytes in the kidney of rats. NA-treated leukocytes were transfused and desialized cells were identified with fluorescein-conjugated peanut lectin (FITC-PNA) in renal tissue. PNA positive cells were identified in rat kidneys 3 hours after injection (glomeruli: 1.67 +/- 0.19 cells/g.c.s.; interstitium: 0.50 +/- 0.12 cells/int). Sections from available renal biopsy material of APSGN (n = 11), other glomerulonephritis (n = 28) and normal kidneys (n = 5) were double-stained with FITC-PNA and with monoclonal antibody to the CD11b molecule, which is expressed on polymorphonuclear and monocytes the main types of infiltrating cells during APSGN. Desialized (FITC-PNA positive) cells were found in the glomeruli (2.17 +/- SEM 0.22 cells per glomerular cross section, g.c.s.) and interstitium (0.61 +/- 0.15 cells per 0.0625 mm2, int) in all biopsies of APSGN. Only in 2 of 28 other glomerulonephritis showed desialized cells. More than 80% of the PNA positive cells in APSGN expressed the CD11b molecule and the infiltration was more intense in early biopsies. In conclusion, desialized leukocytes represent a significant part of the inflammatory infiltrate in APSGN. This finding gives support for a role of NA in the disease and provides clinical validation for a mechanism of renal cellular infiltration suggested by experimental observations.
Assuntos
Glomerulonefrite/metabolismo , Glomérulos Renais/fisiologia , Leucócitos/fisiologia , Neuraminidase/fisiologia , Infecções Estreptocócicas/complicações , Animais , Biópsia , Glomerulonefrite/patologia , Histocitoquímica , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Neuraminidase/farmacologia , Ratos , Ratos Endogâmicos Lew , Estudos Retrospectivos , Fatores de TempoRESUMO
Influenza A viruses exhibit segmented nucleic acid coding for eight different proteins, two of them as glycoproteins exposed on their lipoprotein envelopes, hemagglutinin (HA) and neuraminidase (NA). Hemagglutinin exhibits receptor-binding activity while neuraminidase develops sialidase cleavage activity which acts on cell receptors. Influenza A strains responsible for human, avian, equine and porcine respiratory infections all over the world present antigenically different hemagglutinin (H1 to H14) and neuraminidase (N1 to N9) structures on their surface. The objective of the present investigation was to study the role of N2, N8, and N9, antigenically diverse neuraminidase structures of human (N2) and animal (N8 and N9) influenza viruses, in the receptor-binding process. Receptor-binding activity of N2 and N8 was analyzed by crossed tests using H3N2 and H3N8 antisera and the hemagglutination inhibition test as a model. Hemagglutinating activity of antigenically different N2 and N8 structures was demonstrable and was inhibited by homologous antisera (N2-H3N2, N8-H3N8) but not by heterologous antisera (N2-H3N8,N8-H3N2). This previously demonstrated N9 hemagglutinating activity was analyzed for receptor-binding specificity using hemagglutination tests and NeuAc alpha2,3Gal and NeuAc alpha2,6Gal derivatized erythrocytes. This highly purified N9 strain was obtained from a virus strain isolated from terns by Dr. Peter Colman (CSIRO Division of Biomolecular Engineering, Parkville, Victoria, Australia). It exhibited receptor-binding specificity for NeuAc alpha2,3Gal sequences, a property similar to that observed in hemagglutinins from avian strains. These results indicate the importance of antigenically different neuraminidase structures as alternative agents for developing receptor-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Hemaglutinação por Vírus/fisiologia , Hemaglutininas Virais/fisiologia , Vírus da Influenza A/fisiologia , Neuraminidase/fisiologia , Vírus da Influenza A/enzimologia , Vírus da Influenza A/imunologia , Ligação ProteicaRESUMO
Influenza A viruses exhibit segmented nucleic acid coding for eight different proteins, two of them as glycoproteins exposed on their lipoprotein envelopes, hemagglutinin (HA) and neuraminidase (NA). Hemagglutinin exhibits recptor-binding activity while neuraminidase develps sialidase cleavage activity which acts on cell receptors. Influenza A strains responsible for human, avian, equine and porcine respiratory infections all over the world present antigenically different hemagglutinin (H1 to H14) and neutraminidase (N1 to N9) structures on their surface. The objective of the present investigation was study the role of N2, N8 and N9, anti-genically diverse neuraminidase structures of human (N2) and animal (N8 and N9) influenza viruses, in the receptor-binding process. REceptor-binding activity of N2 and N8 was anlyzed by crossed tests using H3N2 and H3N8 antisera and the hemagglutination inhibition test as a model. Hemangglutinating activity of antigenically different N2 and N8 structures was demonstrable and was inhibited by homologous antisera (N2-H3N2, N8-H3N8) but not by heterologous antisera (N2-H3-N8,N8-H3-N2). This previously demonstrated N9 hemagglutinating activity was analysed for receptor-binding specificity using hemagglutination test and NeuAc alpha2,3Gal and NeuAc alpha2,6Gal derivatized erythrocytes. This highly purified N9 strain was obtained from a virus strain isolated from terns by Dr. Peter Colman (CSIRO Division of Biomolecular Engineering, Parkville, Victoria, Australia)...
Assuntos
Hemaglutininas Virais/fisiologia , Hemaglutinação por Vírus/fisiologia , Neuraminidase/fisiologia , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologiaRESUMO
The present study was conducted to investigate the characteristics of two samples of influenza A/England/42/72 (H3N2) virus, one of them selected by an adsorption-elution technique, to determine the possible existence of virus variants or subpopulations. Based on specificity of virulence-related cell receptor-binding and sialidase activities, this selection technique using human O group erythrocytes revealed the presence of variants within a standard virus sample with diversity for their hemagglutinating and sialidase activities. The standard-like (E1) sample exhibited titers of 4 and 32 HAU (hemagglutinating units in 25 microliters) with human O group and chicken erythrocytes, respectively, while the sample obtained by the adsorption-elution process (E2) exhibited titers of 32 and 4 HAU, respectively, with these same types of erythrocytes. The E2 sample showed higher sialidase activity at pH values between 5.4 and 6.6 with human erythrocytes (128-256 HAU), but the E1 sample did not exhibit significant sialidase activity with either human or chicken erythrocytes. The different pH optima for hemolysis (5.2) and sialidase (5.4-6.6) activities and the higher hemolysis indexes present in samples with sialidase activity inhibited by heating (at 56 degrees C for 30 min) or by treatment with EDTA (dilution in buffer containing 2 mM EDTA, a chelating agent on calcium-dependent sialidase activity) demonstrate the independence of these activities in the selected sample: native E2 (absorbance = 0.18), EDTA-treated native E2 (absorbance = 0.28), heated E2 (absorbance = 0.26), EDTA-treated heated E2 (absorbance = 0.41).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Hemaglutininas Virais/fisiologia , Vírus da Influenza A/classificação , Neuraminidase/fisiologia , Animais , Embrião de Galinha , Variação Genética , Testes de Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas Virais/análise , Hemólise/fisiologia , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Neuraminidase/análise , Fatores de TempoRESUMO
A number of glycoconjugates, including glycolipids and glycoproteins, participate in the process of host-cell invasion by Trypanosoma cruzi and one of the most important carbohydrates involved on this interaction is sialic acid. It is known that parasite trans-sialidase participates with sialic acid in a coordinated fashion in the initial stages of invasion. Given the importance of these sialogycoconjugates, this review sets out various possible biological models for the interaction between the parasite and mammalian cells that possess a sialylated receptor/ligand system
Assuntos
Animais , Sialoglicoproteínas/fisiologia , Trypanosoma cruzi/fisiologia , Ácidos Siálicos/fisiologia , Células Cultivadas/parasitologia , Coração/parasitologia , Neuraminidase/fisiologia , Interações Hospedeiro-Parasita , Trypanosoma cruzi/químicaRESUMO
A number of glycoconjugates, including glycolipids and glycoproteins, participate in the process of host-cell invasion by Trypanosoma cruzi and one of the most important carbohydrates involved on this interaction is sialic acid. It is known that parasite trans-sialidase participates with sialic acid in a coordinated fashion in the initial stages of invasion. Given the importance of these sialoglycoconjugates, this review sets out various possible biological models for the interaction between the parasite and mammalian cells that possess a sialylated receptor/ligand system.