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1.
Biometals ; 27(5): 875-89, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24824995

RESUMEN

Delta-lactoferrin (∆Lf) is a transcription factor belonging to the lactoferrin family, the expression of which inhibits cell proliferation and leads to Skp1 and DcpS gene transactivation. In this study, we showed that ∆Lf expression also induces cell death via apoptosis in HEK 293 and MCF7 cells using a cell viability assay and DNA fragmentation. Western blot analyses showed that apoptosis was caspase-9, 7 and 8 dependent. Proteolytic cleavage of the endonuclease PARP was significantly increased. The levels of expression of Bcl family members were detected by immunochemistry and showed that the Bcl-xl/Bax and Bcl-2/Bax protein ratios were decreased. We determined that the pro-apoptotic effects of ∆Lf are mainly mediated by the activation of the mitochondria-dependent death-signaling pathway. Apoptosis induction by ∆Lf is concomitant with increased cellular levels of Bax protein. Analysis of the Bax promoter region detected a ∆Lf response element located at -155 bp from the transcription start site. Both luciferase reporter gene and chromatin immunoprecipitation assays confirmed that ∆Lf interacts in vitro and in vivo specifically with this sequence. Its deletion, realized using directed mutagenesis, totally abolished ∆Lf transcriptional activity, identifying it as a ∆Lf-responsive element. These results indicate that the Bax gene is a novel ∆Lf target. Moreover we also showed that the O-GlcNAc/P interplay, which controls ∆Lf transcriptional activity, modulates Bax transactivation.


Asunto(s)
Apoptosis/fisiología , Lactoferrina/fisiología , Proteína X Asociada a bcl-2/fisiología , Acilación , Apoptosis/genética , Células HEK293 , Células HeLa , Humanos , Lactoferrina/administración & dosificación , Lactoferrina/genética , Células MCF-7 , Mitocondrias/metabolismo , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Isoformas de Proteínas/administración & dosificación , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Activación Transcripcional , Regulación hacia Arriba , Proteína X Asociada a bcl-2/genética , Receptor fas/genética , Receptor fas/fisiología
2.
Immunobiology ; 217(1): 61-4, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21924517
3.
J Biol Chem ; 286(38): 33678-88, 2011 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-21803773

RESUMEN

We have recently established the fine structure of the glycan backbone of lipooligosaccharides (LOS-I to LOS-IV) isolated from Mycobacterium marinum, a close relative of Mycobacterium tuberculosis. These studies culminated with the description of an unusual terminal N-acylated monosaccharide that confers important biological functions to LOS-IV, such as macrophage activation, that may be relevant to granuloma formation. It was, however, also suggested that the lipid moiety was required for LOSs to exert their immunomodulatory activity. Herein, using highly purified LOSs from M. marinum, we have determined through a combination of mass spectrometric and NMR techniques, the structure and localization of the fatty acids composing the lipid moiety. The occurrence of two distinct polymethyl-branched fatty acids presenting specific localizations is consistent with the presence of two highly related polyketide synthases (Pks5 and Pks5.1) in M. marinum and presumably involved in the synthesis of these fatty acyl chains. In addition, a bioinformatic search permitted us to identify a set of enzymes potentially involved in the biosynthesis or transfer of these lipids to the LOS trehalose unit. These include MMAR_2343, a member of the Pap (polyketide-associated protein) family, that acylates trehalose-based glycolipids in M. marinum. The participation of MMAR_2343 to LOS assembly was demonstrated using a M. marinum mutant carrying a transposon insertion in the MMAR_2343 gene. Disruption of MMAR_2343 resulted in a severe LOS breakdown, indicating that MMAR_2343, hereafter designated PapA4, fulfills the requirements for LOS acylation and assembly.


Asunto(s)
Proteínas Bacterianas/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Lipopolisacáridos/química , Mycobacterium marinum/metabolismo , Acilación , Secuencia de Aminoácidos , Conformación de Carbohidratos , Secuencia de Carbohidratos , Biología Computacional , Cromatografía de Gases y Espectrometría de Masas , Silenciador del Gen , Genes Bacterianos/genética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mycobacterium marinum/genética , Mycobacterium smegmatis/metabolismo , Protones , Alineación de Secuencia , Trehalosa/metabolismo
4.
Biochimie ; 93(3): 593-603, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21167898

RESUMEN

DMBT1 (deleted in malignant brain tumor 1), a human mucin-like glycoprotein, belonging to the scavenger receptor cysteine-rich (SRCR) superfamily, is mainly secreted from mucosal epithelia. It has been shown previously that interaction of hensin, the rabbit ortholog of DMBT1, with galectin 3, a ß-galactoside-binding lectin, induces a terminal differentiation of epithelial cells. In this paper, we have used surface plasmon resonance (SPR), to analyse the binding of galectin 3 to two purified samples of human DMBT1:recombinant DMBT1 produced in CHO cells and DMBT1 isolated from intestinal tissues. Characterization of their glycosylation profile by nano-ESI-Q-TOF tandem mass spectrometry showed significant differences in O-glycans between the two DMBT1 samples. Results obtained by SPR demonstrated that the oligosaccharide side chains of DMBT1 are recognized by the carbohydrate-recognition domain (CRD) of galectin 3 and modification in the pattern of oligosaccharides modulates the binding parameters of DMBT1 with galectin 3. Moreover, using immunohistochemistry on paraffin-embedded colonic tissue sections, we could show a co-localisation of DMBT1 and galectin 3 in human intestine, suggesting a potential physiological interaction.


Asunto(s)
Galectina 3/metabolismo , Oligosacáridos/química , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Animales , Células CHO , Proteínas de Unión al Calcio , Cricetinae , Cricetulus , Proteínas de Unión al ADN , Galectina 3/química , Glicosilación , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Mucosa Intestinal/metabolismo , Cinética , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Resonancia por Plasmón de Superficie , Proteínas Supresoras de Tumor
5.
J Am Chem Soc ; 132(45): 16073-84, 2010 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-20964371

RESUMEN

Although lipo-oligosaccharides (LOSs) are recognized as major parietal components in many mycobacterial species, their involvement in the host-pathogen interactions have been scarcely documented. In particular, the biological implications arising from the high degree of structural species-specificity of these glycolipids remain largely unknown. Growing recognition of the Mycobacterium marinum-Danio rerio as a specific host-pathogen model devoted to the study of the physiopathology of mycobacterial infections prompted us to elucidate the structure-to-function relationships of the elusive end-product, LOS-IV, of the LOS biosynthetic pathway in M. marinum. Combination of physicochemical and molecular modeling methods established that LOS-IV resulted from the differential transfer on the caryophyllose-containing LOS-III of a family of very unusual N-acylated monosaccharides, naturally present as different diastereoisomers. In agreement with the partial loss of pathogenecity previously reported in a LOS-IV-deficient M. marinum mutant, we demonstrated that this terminal monosaccharide conferred to LOS-IV important biological functions, including macrophage activating properties.


Asunto(s)
Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Mycobacterium marinum/inmunología , Antígenos de Superficie/química , Antígenos de Superficie/inmunología , Secuencia de Carbohidratos , Línea Celular Tumoral , Humanos , Activación de Macrófagos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular
6.
Biochem Biophys Res Commun ; 400(3): 403-8, 2010 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-20800577

RESUMEN

A variety of Mycobacterium tuberculosis cell wall components induce expression of matrix metalloproteinase 9 (MMP-9) by monocytic cells and levels of MMP-9 in vivo positively correlate with severity of disease. Toll-like receptor (TLR)2 mediates cellular responses to acylated molecules but can also mediate responsiveness to diverse molecular structures, including non-acylated native viral and bacterial proteins. MPT/B-83 is a cell-associated lipoglycoprotein common to M. tuberculosis and M. bovis and an important antigen during infection of cattle. Since MPB83 is acylated and glycosylated, we investigated whether MPB83 would induce MMP-9 expression via interaction with TLR2, and assessed the contribution of the lipid, glycan and polypeptide components to its activity. Acylated peptide derived from MPB83 stimulated MMP-9 expression by human macrophage cells via interaction with both TLR2 and TLR1, but not TLR4. Lesser induction was found with secreted (non-acylated, but glycosylated) MPB83 protein purified from culture of M. bovis. Stimulation of cells with MPB83 induced TNF-α production which acted to upregulate MMP-9 expression. Surprisingly, recombinant MPB83 protein devoid of any post-translational modification also induced MMP-9 expression. Direct interaction of RecMPB83 with TLR2 was demonstrated by surface plasmon-resonance. MPB83 may act as a virulence factor through TLR2 mediated induction of MMP-9.


Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Metaloproteinasa 9 de la Matriz/biosíntesis , Proteínas de la Membrana/metabolismo , Mycobacterium bovis/patogenicidad , Receptor Toll-Like 1/metabolismo , Receptor Toll-Like 2/metabolismo , Acilación , Anticuerpos Neutralizantes , Línea Celular Tumoral , Humanos , Inhibidores de la Metaloproteinasa de la Matriz , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Mycobacterium bovis/metabolismo , Resonancia por Plasmón de Superficie
7.
J Biol Chem ; 285(43): 32744-32750, 2010 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-20729207

RESUMEN

We showed that the production of tumor necrosis factor (TNF) α by macrophages in response to Toxoplasma gondii glycosylphosphatidylinositols (GPIs) requires the expression of both Toll-like receptors TLR2 and TLR4, but not of their co-receptor CD14. Galectin-3 is a ß-galactoside-binding protein with immune-regulatory effects, which associates with TLR2. We demonstrate here by using the surface plasmon resonance method that the GPIs of T. gondii bind to human galectin-3 with strong affinity and in a dose-dependent manner. The use of a synthetic glycan and of the lipid moiety cleaved from the GPIs shows that both parts are involved in the interaction with galectin-3. GPIs of T. gondii also bind to galectin-1 but with a lower affinity and only through the lipid moiety. At the cellular level, the production of TNF-α induced by T. gondii GPIs in macrophages depends on the expression of galectin-3 but not of galectin-1. This study is the first identification of a galectin-3 ligand of T. gondii origin, and galectin-3 might be a co-receptor presenting the GPIs to the TLRs on macrophages.


Asunto(s)
Galectina 3/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Macrófagos Peritoneales/metabolismo , Toxoplasma/metabolismo , Animales , Chlorocebus aethiops , Galectina 1/genética , Galectina 1/metabolismo , Galectina 3/genética , Humanos , Ratones , Ratones Noqueados , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Células Vero
8.
J Biol Chem ; 284(31): 20975-88, 2009 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-19491094

RESUMEN

Earlier studies have reported a role for lipooligosaccharides (LOSs) in sliding motility, biofilm formation, and infection of host macrophages in Mycobacterium marinum. Although a LOS biosynthetic gene cluster has recently been identified in this species, many structural features of the different LOSs (LOS-I-IV) are still unknown. This clearly hampers assessing the contribution of each LOS in mycobacterial virulence as well as structure-function-based studies of these important cell wall-associated glycolipids. In this study, we have identified an M. marinum isolate, M. marinum 7 (Mma7), which failed to produce LOS-IV but instead accumulated large amounts of LOS-III. Local genomic comparison of the LOS biosynthetic cluster established the presence of a highly disorganized region in Mma7 compared with the standard M strain, characterized by multiple genetic lesions that are likely to be responsible for the defect in LOS-IV production in Mma7. Our results indicate that the glycosyltransferase LosA alone is not sufficient to ensure LOS-IV biosynthesis. The availability of different M. marinum strains allowed us to determine the precise structure of individual LOSs through the combination of mass spectrometric and NMR techniques. In particular, we established the presence of two related 4-C-branched monosaccharides within LOS-II to IV sequences, of which one was never identified before. In addition, we provided evidence that LOSs are capable of inhibiting the secretion of tumor necrosis factor-alpha in lipopolysaccharide-stimulated human macrophages. This unexpected finding suggests that these cell wall-associated glycolipids represent key effectors capable of interfering with the establishment of a pro-inflammatory response.


Asunto(s)
Carbohidratos/química , Pared Celular/metabolismo , Glucolípidos/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Monosacáridos/química , Mycobacterium marinum/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Conformación de Carbohidratos , Carbohidratos/análisis , Diferenciación Celular/efectos de los fármacos , Línea Celular , Pared Celular/efectos de los fármacos , Cromatografía en Capa Delgada , Genoma Bacteriano/genética , Humanos , Lipopolisacáridos/biosíntesis , Lipopolisacáridos/química , Macrófagos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , Monosacáridos/análisis , Familia de Multigenes , Mycobacterium marinum/efectos de los fármacos , Mycobacterium marinum/genética , Mycobacterium marinum/aislamiento & purificación , Análisis de Secuencia
9.
FEBS Lett ; 582(3): 445-50, 2008 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-18201568

RESUMEN

Mycobacterial lipomannan (LM) and lipoarabinomannan (LAM) regulate macrophage activation by interacting with Toll-like receptors (TLRs). The intracellular signalling pathways elicited by these complex molecules are poorly defined. We have demonstrated that LM purified from various mycobacterial species, but not LAM from Mycobacterium kansasii or Mycobacterium bovis BCG, induced expression of the MAP kinase phosphatase 1 (MKP-1) in macrophages. Anti-TLR2 antibodies, as well as specific ERK and p38 MAPK inhibitors, decreased MKP-1 transcription in LM-stimulated cells. These findings suggest that the binding of LM to TLR2 triggers MAPK activation, followed by an up-regulation of MKP-1 expression, which in turn may act as a negative regulator of MAPK activation.


Asunto(s)
Fosfatasa 1 de Especificidad Dual/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Diferenciación Celular , Células Cultivadas , Fosfatasa 1 de Especificidad Dual/metabolismo , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Lipopolisacáridos/metabolismo , Macrófagos/fisiología , Manosa/química , Manosa/metabolismo , Mycobacterium kansasii/química , Fosfatidilinositoles/química , Fosfatidilinositoles/metabolismo , Unión Proteica , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 2/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología
10.
Adv Exp Med Biol ; 606: 163-94, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18183929

RESUMEN

Lactoferrin (Lf) is an iron binding glycoprotein of the transferrin family that is expressed in most biological fluids and is a major component of mammals' innate immune system. Its protective effect ranges from direct antimicrobial activities against a large panel of microorganisms, including bacteria, viruses, fungi, and parasites, to anti-inflammatory and anticancer activities. This plethora of activities is made possible by mechanisms of action implementing not only the capacity of Lf to bind iron but also interactions of Lf with molecular and cellular components of both host and pathogens. This chapter summarizes our current understanding of the Lf structure-function relationships that explain the roles of Lf in host defense.


Asunto(s)
Lactoferrina/química , Lactoferrina/metabolismo , Animales , Antiinfecciosos/farmacología , Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos/farmacología , Humanos , Lactoferrina/genética , Leche Humana/química , Relación Estructura-Actividad
11.
FEBS Lett ; 581(7): 1383-90, 2007 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-17350002

RESUMEN

The mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), regulate host defence mechanisms through their interaction with pattern recognition receptors such as Toll-like receptors (TLRs). We have developed a surface plasmon resonance assay to analyse the molecular basis for the recognition of Mycobacterium kansasii LM or LAM, by immobilized CD14 and LPS-binding protein (LBP) both being capable to promote presentation of bacterial glycolipids to TLRs. The affinity of either LM/LAM was higher to CD14 than to LBP. Kinetic and Scatchard analyses were consistent with a model involving a single class of binding sites. These interactions required the lipidic anchor, but not the carbohydrate domains, of LM or LAM. We also provide evidence that addition of recombinant LBP enhanced the stimulatory effect of LM or LAM on matrix metalloproteinase-9 expression and secretion in macrophages, through a TLR1/TLR2-dependent mechanism.


Asunto(s)
Proteínas de Fase Aguda/metabolismo , Proteínas Portadoras/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/química , Glicoproteínas de Membrana/metabolismo , Mycobacterium kansasii , Proteínas de Fase Aguda/genética , Proteínas Portadoras/genética , Células Cultivadas , Humanos , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Metaloproteinasa 9 de la Matriz/metabolismo , Glicoproteínas de Membrana/genética , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , Receptor Toll-Like 1/inmunología , Receptor Toll-Like 2/inmunología
12.
Biochem Cell Biol ; 84(3): 282-90, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16936798

RESUMEN

The antimicrobial activities of lactoferrin (Lf) depend on its capacity to bind iron and on its direct interaction with the surface of microorganisms. Its protective effect also extends to the regulation of the host response to infections. Depending on the immune status of an individual, Lf can have anti-inflammatory properties that downregulate the immune response and prevent septic shock and damage to tissues. It also acts as a promoter of the activation, differentiation, and (or) proliferation of immune cells. Although most of the anti-inflammatory activities are correlated with the neutralization of proinflammatory molecules by Lf, the promoting activity seems to be related to a direct effect of Lf on immune cells. Although the mechanisms that govern these activities are not clearly defined, and probably differ from cell to cell, several cellular targets and possible mechanisms of action are highlighted. The majority of the molecular targets at the surface of cells are multiligand receptors but, interestingly, most of them have been reported as signaling, endocytosis, and nuclear-targeting molecules. This review focuses on the known and putative mechanisms that allow the immunoregulating effect of Lf in its interactions with immune cells.


Asunto(s)
Sistema Inmunológico/citología , Sistema Inmunológico/inmunología , Lactoferrina/metabolismo , Animales , Humanos , Inmunidad Innata/inmunología , Transducción de Señal
13.
Infect Immun ; 73(10): 7064-8, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16177394

RESUMEN

Lipomannans (LM) from various mycobacterial species were found to induce expression and secretion of the matrix metalloproteinase 9 (MMP-9) both in human macrophage-like differentiated THP-1 cells and in primary human macrophages. Inhibition studies using antireceptor-neutralizing antibodies are indicative of a Toll-like receptor 1 (TLR1)/TLR2- and CD14-dependent signaling mechanism. Moreover, LM was shown to down-regulate transcription of the metalloproteinase inhibitor TIMP-1, a major endogenous MMP-9 regulator.


Asunto(s)
Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Anticuerpos/farmacología , Células Cultivadas , Regulación hacia Abajo , Humanos , Macrófagos/enzimología , Metaloproteinasa 9 de la Matriz/genética , Mycobacterium/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Inhibidor Tisular de Metaloproteinasa-1/genética , Transcripción Genética/efectos de los fármacos
14.
Biometals ; 17(3): 225-9, 2004 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15222469

RESUMEN

Lactoferrin is a member of the transferrin family of iron-binding glycoproteins that is abundantly expressed and secreted from glandular epithelial cells. In secretions, such as milk and fluids of the intestinal tract, lactoferrin is an important component of the first line of host defence. During the inflammatory process, lactoferrin, a prominent component of the secondary granules of neutrophils (PMNs), is released in infected tissues and in blood and then it is rapidly cleared by the liver. In addition to the antimicrobial properties of lactoferrin, a set of studies has focused on its ability to modulate the inflammatory process and the overall immune response. Though many in vitro and in vivo studies report clear regulation of the immune response and protective effect against infection and septic shock by lactoferrin, elucidation of all the cellular and molecular mechanisms of action is far from being achieved. At the cellular level, lactoferrin modulates the migration, maturation and function of immune cells. At the molecular level and in addition to iron binding, interactions of lactoferrin with a plethora of compounds, either soluble or membrane molecules, account for its modulatory properties. This paper reviews our current understanding of the cellular and molecular mechanisms that explain the regulatory properties of lactoferrin in host defence.


Asunto(s)
Antiinflamatorios/metabolismo , Factores Inmunológicos/metabolismo , Lactoferrina/metabolismo , Animales , Antiinfecciosos/metabolismo , Humanos , Sistema Inmunológico/fisiología
15.
Eur J Biochem ; 271(2): 303-17, 2004 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-14717698

RESUMEN

Lactoferrin (Lf), a multifunctional molecule present in mammalian secretions and blood, plays important roles in host defense and cancer. Indeed, Lf has been reported to inhibit the proliferation of cancerous mammary gland epithelial cells and manifest a potent antiviral activity against human immunodeficiency virus and human cytomegalovirus. The Lf-binding sites on the cell surface appear to be proteoglycans and other as yet undefined protein(s). Here, we isolated a Lf-binding 105 kDa molecular mass protein from cell extracts and identified it as human nucleolin. Medium-affinity interactions ( approximately 240 nm) between Lf and purified nucleolin were further illustrated by surface plasmon resonance assays. The interaction of Lf with the cell surface-expressed nucleolin was then demonstrated through competitive binding studies between Lf and the anti-human immunodeficiency virus pseudopeptide, HB-19, which binds specifically surface-expressed nucleolin independently of proteoglycans. Interestingly, binding competition studies between HB-19 and various Lf derivatives in proteoglycan-deficient hamster cells suggested that the nucleolin-binding site is located in both the N- and C-terminal lobes of Lf, whereas the basic N-terminal region is dispensable. On intact cells, Lf co-localizes with surface nucleolin and together they become internalized through vesicles of the recycling/degradation pathway by an active process. Morever, a small proportion of Lf appears to translocate in the nucleus of cells. Finally, the observations that endocytosis of Lf is inhibited by the HB-19 pseudopeptide, and the lack of Lf endocytosis in proteoglycan-deficient cells despite Lf binding, point out that both nucleolin and proteoglycans are implicated in the mechanism of Lf endocytosis.


Asunto(s)
Endocitosis/fisiología , Lactoferrina/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Neoplasias de la Mama/metabolismo , Células CHO , División Celular/efectos de los fármacos , Membrana Celular , Cricetinae , Femenino , VIH-1/efectos de los fármacos , VIH-1/fisiología , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Células Jurkat , Lactoferrina/genética , Microscopía Confocal , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Péptidos , Fosfoproteínas/genética , Conformación Proteica , Proteínas de Unión al ARN/genética , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , Nucleolina
16.
J Immunol ; 171(4): 2014-23, 2003 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-12902506

RESUMEN

Lipoarabinomannans (LAMs) are glycolipids from the mycobacterial cell wall that exhibit various biological activities, including proinflammatory and anti-inflammatory responses. However, little is known about the properties of lipomannans (LMs), considered to be precursors of LAMs. In this study, we provide evidence that LMs purified from Mycobacterium chelonae and a clinical strain of Mycobacterium kansasii stimulated mRNA expression and secretion of TNF-alpha and IL-8 from human macrophage-like differentiated THP-1 cells. In contrast to LMs, LAMs were not able to induce a significant cytokine-inducing effect. The mechanism of activation by LMs was investigated using various Abs raised against surface receptors for multiple bacterial products. The presence of anti-CD14 or anti-Toll-like receptor 2 (TLR2) Abs profoundly affected production of TNF-alpha and IL-8, suggesting that both CD14 and TLR2 participate in the LM-mediated activation process. Furthermore, stimulation of cells was dependent on the presence of the LPS-binding protein, a plasma protein that transfers glycolipids to CD14. Chemical degradation of the arabinan domain of mannose-capped LAM from M. kansasii, which presented no cytokine-eliciting effect, restored the cytokine-inducing activity at a level similar to those of LMs. These results support the hypothesis that the presence of an arabinan in LAMs prevents the interaction of these glycolipids with TLR2/CD14 receptors. In addition, we found that phosphatidylinositol dimannosides isolated from M. kansasii did not induce cytokine secretion. This study suggests that LMs isolated from different mycobacterial species participate in the immunomodulation of the infected host and that the D-mannan core of this glycolipid is essential for this function.


Asunto(s)
Proteínas de Fase Aguda , Interleucina-8/metabolismo , Receptores de Lipopolisacáridos/fisiología , Lipopolisacáridos , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/fisiología , Mycobacterium chelonae/inmunología , Mycobacterium kansasii/inmunología , Receptores de Superficie Celular/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Portadoras/fisiología , Diferenciación Celular/inmunología , Citocinas/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Mycobacterium chelonae/patogenicidad , Mycobacterium kansasii/patogenicidad , Fosfatidilinositoles/fisiología , Polisacáridos/metabolismo , ARN Mensajero/biosíntesis , Relación Estructura-Actividad , Receptor Toll-Like 2 , Receptores Toll-Like , Células Tumorales Cultivadas
17.
J Biol Chem ; 277(34): 30635-48, 2002 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-12063260

RESUMEN

Lipomannan (LM) and lipoarabinomannan (LAM) are major glycolipids present in the mycobacterial cell wall that are able to modulate the host immune response. In this study, we have undertaken the structural determination of these important modulins in Mycobacterium chelonae, a fast growing pathogenic mycobacterial species. One-dimensional and two-dimensional NMR spectra were used to demonstrate that LM and LAM from M. chelonae, designated CheLM and CheLAM, respectively, possess structures that differ from the ones reported earlier in other mycobacterial species. Analysis by gas chromatography/mass spectrometry of the phosphatidyl-myo-inositol anchor, which is thought to play a role in the biological functions of these lipoglycans, pointed to a high degree of heterogeneity based on numerous combinations of acyl groups on the C-1 and C-2 positions of the glycerol moiety. Characterization of the mannan core of CheLM and CheLAM revealed the presence of novel alpha1,3-mannopyranosyl side chains. This motif, which reacted specifically with the lectin from Galanthus nivalis, was found to be unique among a panel of nine mycobacterial species. Then, CheLM and CheLAM were found to be devoid of both the mannooligosaccharide cap present in Mycobacterium tuberculosis and the inositol phosphate cap present in Mycobacterium smegmatis and other fast growing species. Tumor necrosis factor-alpha and interleukin-8 production were assessed from human macrophages with LAM preparations from different species. Our results suggest that the inositol phosphate capping may represent the major cytokine-inducing component of LAMs. This work not only underlines the diversity of LAM structures among various mycobacterial species but also provides new structures that could be useful to dissect the structure-function relationships of these complex molecules.


Asunto(s)
Lipopolisacáridos/química , Mycobacterium chelonae/química , Antígenos CD1/fisiología , Línea Celular , Humanos , Interleucina-8/metabolismo , Lectinas/metabolismo , Lipopolisacáridos/farmacología , Espectroscopía de Resonancia Magnética , Factor de Necrosis Tumoral alfa/metabolismo
18.
Infect Immun ; 70(4): 1860-6, 2002 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-11895948

RESUMEN

Interleukin-8 (IL-8), a C-X-C chemokine bound to endothelium proteoglycans, initiates the activation and selective recruitment of leukocytes at inflammatory foci. We demonstrate that human lactoferrin, an antimicrobial lipopolysaccharide (LPS)-binding protein, decreases both IL-8 mRNA and protein expression induced by the complex Escherichia coli 055:B5 LPS/sCD14 in human umbilical vein endothelial cells. The use of recombinant lactoferrins mutated in the LPS-binding sites indicates that this inhibitory effect is mediated by an interaction of lactoferrin with LPS and CD14s that suppresses the endotoxin biological activity. Furthermore, since dimeric IL-8 and lactoferrin are both proteoglycan-binding molecules, the competition between these proteins for heparin binding was investigated. Lactoferrin strongly inhibited the interaction of radiolabeled IL-8 to immobilized heparin, whereas a lactoferrin variant lacking the amino acid residues essential for heparin binding was not inhibitory. Moreover, this process is specific, since serum transferrin, a glycoprotein whose structure is close to that of lactoferrin, did not prevent the interaction of IL-8 with heparin. These results suggest that the anti-inflammatory properties of lactoferrin during septicemia are related, at least in part, to the regulation of IL-8 production and also to the ability of lactoferrin to compete with chemokines for their binding to proteoglycans.


Asunto(s)
Endotelio Vascular/metabolismo , Interleucina-8/metabolismo , Lactoferrina/farmacología , Lipopolisacáridos/farmacología , Proteoglicanos/metabolismo , Células Cultivadas , Heparina/metabolismo , Humanos , Receptores de Lipopolisacáridos/farmacología , Lipopolisacáridos/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología
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