RESUMO
BACKGROUND: Surfactant protein D (SP-D), a secreted pattern recognition molecule associated with pulmonary innate immunity, has been shown to mediate the clearance of pathogens in multiple ways. However, how SP-D interacts with alveolar macrophages (AMs) and dendritic cells (DCs) during allergen exposure remains unclear. OBJECTIVE: This study was performed to characterize the immunomodulatory effects of SP-D on mite allergen (Dermatophagoides pteronyssinus, Der p)-induced inflammatory signalling in AMs and DCs. METHODS: Murine AM, alveolar macrophage cell line derived from BALB/c mice (MH-S cells), and human monocyte-derived dendritic cells (MDDC) were used as model systems. The production of nitric oxide (NO) and TNF-alpha, expression of surface Toll-like receptors (TLRs), and expression of the C-type lectin receptor known as dendritic cell (DC)-specific ICAM-grabbing non-integrin (DC-SIGN) were measured as a function of pretreatment with SP-D and subsequent exposure to Der p. Der p-dependent cellular activations that were modified by SP-D in these model systems were then identified. RESULTS: Pretreatment of MH-S cells with SP-D reduced Der p-dependent production of NO, TNF-alpha, and the downstream activations of IL-1 receptor-associated kinase, mitogen activated protein kinase (MAPK) kinase, and nuclear factor-kappaB. SP-D interacted with CD14 such that CD14 binding to Der p was inhibited and Der p-induced signalling via TLRs was blocked. DC-SIGN expression was suppressed by Der p in MH-S and MDDC; this down-regulation of DC-SIGN expression was prevented by pretreatment with SP-D. CONCLUSIONS: These results indicated that the inhibition of Der p-induced activation of MH-S and MDDC by SP-D is mediated through suppression of the CD14/TLR signalling pathway and maintenance of DC-SIGN expression, which may protect allergen-induced airway inflammation.
Assuntos
Moléculas de Adesão Celular/metabolismo , Células Dendríticas/imunologia , Dermatophagoides pteronyssinus/imunologia , Lectinas Tipo C/metabolismo , Macrófagos Alveolares/imunologia , Proteína D Associada a Surfactante Pulmonar/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Toll-Like/metabolismo , Alérgenos/imunologia , Animais , Antígenos de Dermatophagoides/imunologia , Moléculas de Adesão Celular/biossíntese , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Humanos , Lectinas Tipo C/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/biossíntese , Proteína D Associada a Surfactante Pulmonar/farmacologia , Receptores de Superfície Celular/biossíntese , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL-10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium-host interaction.
Assuntos
Cápsulas Bacterianas/fisiologia , Lipopolissacarídeos/metabolismo , Manose/metabolismo , Mycobacterium/fisiologia , Animais , Cápsulas Bacterianas/metabolismo , Elementos de DNA Transponíveis/genética , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Eletroforese em Gel de Poliacrilamida , Feminino , Teste de Complementação Genética , Interações Hospedeiro-Patógeno , Humanos , Immunoblotting , Interleucina-10/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Manose/química , Manose/fisiologia , Manosiltransferases/genética , Manosiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Modelos Moleculares , Mutagênese Insercional , Mutação , Mycobacterium/metabolismo , Infecções por Mycobacterium/metabolismo , Infecções por Mycobacterium/microbiologia , Peixe-ZebraRESUMO
Most human peripheral blood gamma delta T lymphocytes respond to hitherto unidentified mycobacterial antigens. Four ligands from Mycobacterium tuberculosis strain H37Rv that stimulated proliferation of a major human gamma delta T cell subset were isolated and partially characterized. One of these ligands, TUBag4, is a 5' triphosphorylated thymidine-containing compound, to which the three other stimulatory molecules are structurally related. These findings support the hypothesis that some gamma delta T cells recognize nonpeptidic ligands.
Assuntos
Antígenos de Bactérias/imunologia , Ativação Linfocitária , Mycobacterium tuberculosis/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Nucleotídeos de Timina/imunologia , Antígenos de Bactérias/química , Antígenos de Bactérias/isolamento & purificação , Células Cultivadas , Cromatografia por Troca Iônica , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Nucleotídeos de Timina/análise , Nucleotídeos de Timina/química , Nucleotídeos de Timina/isolamento & purificaçãoRESUMO
INTRODUCTION: Mycobacterium tuberculosis, the cause of tuberculosis remains a pathogenic organism capable of infecting a large number of individuals and of resisting the immune response of the infected host. The main constituents of this response are the antigen presenting cells such as dendritic cells, macrophages and T lymphocytes. BACKGROUND: Comparative study of the interactions between M. tuberculosis and the antigen presenting cells has shown that dendritic cells do not permit intracellular growth of M. tuberculosis, unlike that seen in macrophages. A hostile intracellular compartment creates a bacteriostatic environment. M. tuberculosis is internalised by binding to a C-type lectin receptor (DC-SIGN). VIEWPOINT: This receptor recognises polysaccharide compounds on the surface of M. tuberculosis. This sugar-lectin bond may compensate for the bond between bacterial compounds and Toll receptors, partially inhibiting the protective inflammatory reaction or compensating for an excessive inflammatory reaction. CONCLUSIONS: This bond encourages both the persistence of quiescent bacteria in the dendritic cells and the reciprocal adaptation of the host and the bacteria over the course of time.
Assuntos
Células Dendríticas/imunologia , Tuberculose/imunologia , Células Apresentadoras de Antígenos/imunologia , Células Dendríticas/microbiologia , Humanos , Lectinas Tipo C/imunologia , Mycobacterium tuberculosis/imunologia , Polissacarídeos Bacterianos/imunologia , Receptores Toll-Like/imunologiaRESUMO
C-mycosides are superficial type-specific glycopeptidolipids of mycobacterial origin. In the present work, we have shown that field desorption mass spectrometry using the cationization method is a useful method for the molecular weight determination of such compounds. Complementary structural information has been obtained by electron-impact mass spectrometry. Combination of both methods has permitted the elucidation of the structure of the C-mycosides of Mycobacterium smegmatis, ATCC 607. This structure is similar to those described elsewhere, but some minor differences are observed in the lipid portion, mainly in the double bond location.
Assuntos
Glicosídeos/isolamento & purificação , Mycobacterium/análise , Fenômenos Químicos , Química , Elétrons , Espectrometria de MassasRESUMO
Lipoarabinomannans are key molecules of the mycobacterial envelopes involved in many steps of tuberculosis immunopathogenesis. Several of the biological activities of lipoarabinomannans are mediated by their ability to bind human C-type lectins, such as the macrophage mannose receptor, the mannose-binding protein and the surfactant proteins A and D. The lipoarabinomannan mannooligosaccharide caps have been demonstrated to be involved in the binding to the lectin carbohydrate recognition domains. We report an original analytical approach, based on capillary electrophoresis monitored by laser-induced fluorescence, allowing the absolute quantification, in nanomole quantities of lipoarabinomannan, of the number of mannooligosaccharide units per lipoarabinomannan molecule. Moreover, this analytical approach was successful for the glycosidic linkage determination of the mannooligosaccharide motifs and has been applied to the comparative analysis of parietal and cellular lipoarabinomannans of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv, H37Ra and Erdman strains. Significant differences were observed in the amounts of the various mannooligosaccharide units between lipoarabinomannans of different strains and between parietal and cellular lipoarabinomannans of the same strain. Nevertheless, no relationship was found between the number of mannooligosaccharide caps and the virulence of the corresponding strain. The results of the present study should help us to gain more understanding of the molecular basis of lipoarabinomannan discrimination in the process of binding to C-type lectins.
Assuntos
Lectinas/metabolismo , Lipopolissacarídeos/análise , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Mycobacterium bovis/química , Mycobacterium tuberculosis/química , Acetilação , Antígenos de Bactérias/análise , Antígenos de Bactérias/química , Configuração de Carboidratos , Eletroforese Capilar/métodos , Fluorescência , Humanos , Lasers , Lectinas/química , Espectroscopia de Ressonância Magnética , Mananas/análise , Mananas/química , Mycobacterium bovis/classificação , Mycobacterium bovis/patogenicidade , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/patogenicidade , Oligossacarídeos/análise , Oligossacarídeos/química , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , VirulênciaRESUMO
A biosynthetic filiation is postulated between the mycobacterial phosphatidyl-myo-inositol mannosides (PIMs), the lipomannans (LMs) and the lipoarabinomannans (LAMs), the major antigens of the envelopes. Moreover, as the PI anchor is thought to play a role in the biological functions of the LAMs, we characterized the lipid moiety of the PI anchor from Mycobacterium bovis BCG cellular LMs. Their structure was investigated along with that of a purified tetra-acylated form of PIM2 (Ac4PIM2). A two-dimensional 1H-31P heteronuclear multiple quantum correlation homonuclear Hartmann-Hahn spectroscopy study of Ac4PIM2 unambiguously localised a fourth fatty acid on the C3 of the myo-Ins beside the fatty acids already described on the C1 and C2 position of the glycerol and on the C6 position of the mannose. This analytical strategy was extended to the structural study of the cellular LM anchor. Using an appropriate solvent system, the one dimensional 31P NMR spectrum exhibited four major resonances typifying the LM populations. These populations differed in number and location of the fatty acids. For one of these populations, we established the presence of an extra fatty acid on the C3 of the myo-Ins of the LM anchor. The fact that both types of molecules have an elaborated anchor in common, indicates that cellular LMs are multimannosylated forms of PIMs. In addition, the LM mannan core structure was analysed by two-dimensional NMR, pointing to a high level of branching by single alpha1-->2 Manp side-chains.
Assuntos
Lipopolissacarídeos/química , Mananas/química , Mycobacterium bovis/química , Fosfatidilinositóis/química , Acilação , Sequência de Carboidratos , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/metabolismo , Espectroscopia de Ressonância Magnética , Mananas/isolamento & purificação , Mananas/metabolismo , Manosídeos/química , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidilinositóis/metabolismo , Isótopos de Fósforo , TrítioRESUMO
We studied the molecular binding specificity of two rabbit polyclonal sera generated against phenolic glycolipid antigens namely PheG1 B and PheG1 B-3 from Mycobacterium bovis BCG. PheG1 B is the well-known mycoside B (2-O-Me-alpha-L-Rhap 1----aglycone), while PheG1 B-3 is a recently found glycolipid (alpha-L-Rhap-(1----3)-2-O-Me-alpha-L-Rhap 1----aglycone). The interaction specificity was mainly explained in terms of the cavity volume of the antibodies paratope. The anti-PheG1 B antibodies paratope fits the 2-O-Me-alpha-L-Rhap ligand, while that of anti-PheG1 B-3 binds the disaccharide moiety of PheG1 B-3, and, with a higher affinity, the monosaccharidic unit localized at the non-reducing end. The B-3 antigen affinity is higher than that of antigen B for their homologous antibodies. This can be explained by the fact that the antibodies against phenolic glycolipid B-3 bind optimally to two sequential glycosyl residues suggesting the presence of two subsites. The immunoglobulin subsite with the major affinity binds the monosaccharidic unit localized at the non-reducing end.
Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Glicolipídeos/imunologia , Mycobacterium bovis/imunologia , Animais , Sítios de Ligação de Anticorpos , Sequência de Carboidratos , Ensaio de Imunoadsorção Enzimática , Soros Imunes , Modelos Moleculares , Dados de Sequência Molecular , Fenóis , CoelhosRESUMO
Commercial aqueous 25% glutaraldehyde solutions contain no stable derivative of this aldehyde, but compounds of variable molecular weight which easily revert to glutaraldehyde. The effect of pH on the reaction of glutarldehyde with amino acids and on the stability of the products under acid conditions, shows the importance of the structure modification of the dialdehyde which occurs when pH increases, and even leads to precipitation in highly alkaline solutions. This precipitate results from aldol condensation of glutaraldehyde molecules. It contains aldehyd groups conjugated with ethylenic double bonds. Such a structure reacts with amino groups to give an imino bond, stabilized by resonance with the ethylenic bond, and does not undergo Michael-type addition reactions. Therefore, glutaraldehyde does not react with proteins under its free form, but as an unsaturated polymer, which gives imino bonds stabilized by conjugation.
Assuntos
Aldeídos , Glutaral , Lisina , Proteínas , Fenômenos Químicos , Química , Cromatografia em Gel , Cromatografia em Camada Fina , Crotonatos , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Propilaminas , Espectrofotometria InfravermelhoRESUMO
The mechanisms and the molecular basis of the mycobacteria virulence remain obscure. However, recent findings provide evidences that, among the glycoconjugates which compose the mycobacterial envelope, lipoglycans including lipoarabinomannan, lipomannan and phosphatidyl-myo-inositol mannosides, are involved in the major steps of the tuberculosis immunopathogenesis. These steps are the mycobacterial phagocytosis process and the macrophage activation via the regulation of the production and secretion of cytokines. In this article, we examine recent observations about comparative structural models of the lipoglycans from the pathogenic Mycobacterium tuberculosis strain, and the vaccinal M. bovis BCG strain, and finally avirulent mycobacteria strains. We also consider the role of the lipoglycans in the mycobacterial phagocytosis process and in the regulation of the macrophage microbicidal activity.
Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/fisiologia , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Mycobacterium tuberculosis/patogenicidade , Tuberculose/microbiologia , Antígenos de Bactérias/imunologia , Citocinas/biossíntese , Humanos , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Mycobacterium bovis/patogenicidade , Mycobacterium tuberculosis/imunologia , Fagocitose , Tuberculose/imunologiaRESUMO
The isolation of a 'palmitone lipid' from Corynebacterium diphtheriae is described. The use of a temporary hydrophobic protecting group allows the obtaining of the lipid in free and pure form. Structural studies by chemical degradation and mass spectrometry allow one to propose structure Ic for this compound, namely 6-(2-tetradecyl 3-keto octadecanoyl)-alpha-D-trehalose. This structure was confirmed by chemical synthesis.
Assuntos
Corynebacterium diphtheriae/análise , Lipídeos/isolamento & purificação , Espectrometria de Massas , Palmitatos/isolamento & purificação , Trealose/análogos & derivados , Trealose/síntese químicaRESUMO
The direct exposure, negative chemical ionisation, chloride-attachment mass spectrometry of trehalose and sucrose gave abundant chloride-attached molecular ions. The same feature was observed when these sugars were subjected to fast-atom bombardment (f.a.b.) in a glycerol matrix containing ammonium chloride. No characteristic fragment ion was found when trehalose was analysed by either method. In contrast, sucrose gave intense chloride-containing fragments, arising by glycosidic cleavage, when analysed by the first method, whereas such cleavage was not detectable by f.a.b.-ammonium chloride analysis. However, the mass-analysed ion kinetic energy (m.i.k.e.) spectra of the (M + Cl)- ions from either trehalose and sucrose, generated under f.a.b.-ammonium chloride conditions, showed glycosidic cleavage reactions in addition to a large loss of HCl. These cleavage reactions might be attributed to SN2-like reactions on the acetal carbon atom and to base-induced eliminations, and they were enhanced by collision-induced dissociations. However, the relative abundance of such glycosidic cleavages from the ionic state would be too weak to explain the presence of the large chloride-containing fragments in the direct exposure spectra of sucrose. Thus, these ions were mainly produced by a thermal cleavage followed by chloride-attachment reactions.
Assuntos
Oligossacarídeos , Cloretos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glicosídeos/análise , Indicadores e ReagentesRESUMO
Phenolic glycolipids were first discovered as cell-wall constituents of M. bovis, M. bovis BCG, M. marinum, and M. kansasii. Recently, such compounds were also isolated from M. leprae and have been shown to be specific-species serological markers. Moreover, they seem to be involved, in the case of lepromatous leprosy, in the stimulation of the suppressor T-cells. The functional activities of these phenolic glycolipids over the immune cells stimulation emphasized the role played by these molecules in the mycobacteria pathogenicity. Phenolic glycolipids have also been found in M. gastri and M. tuberculosis strain Canetti. From a structural point of view, these glycolipids contain the same aglycon moiety mainly assigned to phenolphthiocerol diester while the sugar part structure confers to some of these glycolipids their antigenic specificity. The search of immunoreactive glycolipids and their function analysis remain a challenge for chemists and immunologists for the understanding of the mycobacteria pathogenicity.
Assuntos
Parede Celular/química , Glicolipídeos , Mycobacterium/análise , Animais , Antígenos de Bactérias/imunologia , Sequência de Carboidratos , Saúde Global , Glicolipídeos/química , Glicolipídeos/imunologia , Humanos , Dados de Sequência Molecular , Mycobacterium/imunologia , Mycobacterium/patogenicidade , Infecções por Mycobacterium/epidemiologia , Micobactérias não Tuberculosas/análise , Micobactérias não Tuberculosas/imunologia , Micobactérias não Tuberculosas/patogenicidade , CoelhosRESUMO
A crude phenolic glycolipid extract from Mycobacterium bovis bacille Calmette-Guerin (BCG) was fractionated until homogeneity at the intact level into four phenolic glycolipids called B, B-1, B-2, and B-3 according to their polarity. The apolar one, which is the most abundant was assigned to the well-known mycoside B. The B-2 and B-3 phenolic glycolipids were purified by direct-phase high performance liquid chromatography using a 5 micron Spherisorb column but were only recovered in small amounts (3 mg). A linear gradient of 0-20% methanol in chloroform was used. The B-1, B-2, and B-3 glycolipids were subjected to suitable modern analytical techniques selected for their potential to elucidate the structure at the intact level. Desorption chemical ionization-mass spectrometry allowed the molecular mass of B-3 to be determined as 1652 Da for the major homolog establishing the molecular formula as C103H192O14. Thus, the B-3 polar phenolic glycolipid contained two deoxyhexoses, one molecule of phenolphthiocerol esterified by two molecules of mycocerosic acid. Using two-dimensional 1H NMR (correlated chemical shift and nuclear Overhauser effect spectroscopy) at the intact level the B-3 oligosaccharide structure was determined as an alpha-L-Rhap-(1----3)-2-O-Me-alpha-L-Rhap. This is the first report of a diglycosylated phenolic glycolipid in a nonpathogenic mycobacteria. The disaccharide unit, the antigenic determinant, appears to be characteristic of M. bovis BCG. This polar glycolipid B-3 and the apolar ones, B-1 and B-2, were reactive in enzyme-linked immunosorbent assay against serum from rabbit hyperimmunized with M. bovis BCG.
Assuntos
Glicolipídeos/análise , Mycobacterium bovis/análise , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Glicolipídeos/imunologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Molecular , Mycobacterium bovis/imunologiaRESUMO
An unknown immunogenic glycopeptidolipid, named GPL X-1, was isolated from Mycobacterium xenopi, which is a nontuberculous mycobacterium responsible for pulmonary and disseminated infectious diseases mainly occurring in immunocompromised patients. The glycopeptidolipid was purified until homogeneity, in the native form, by direct phase high performance liquid chromatography. A new route is proposed for the structural elucidation of its unusual lipopeptidic core. The presence of allothreonine (aThr), phenylalanine, and serine in the molecular ratio 1:1:2, respectively, was established by reverse phase high performance liquid chromatography analysis of the phenylthiocarbamyl amino acid derivatives. From the molecular mass (1828 Da) of the native glycopeptidolipid, determined by cesium ion liquid secondary ion mass spectrometry using the amphipathic triethylene glycol monobutyl ether matrix, it was deduced that the tetrapeptide was amidified by a dodecanoic acid. The complete structure, C12-Ser-Ser-Phe-aThr-OCH3, of the lipopeptidic core was established by pyrolysis electron impact-mass spectrometry of the native glycopeptidolipid. To date, this is the first example of a mycobacterial glycopeptidolipid with a C12-tetrapeptidic core containing serine. A novel approach, based on two dimensional 1H,1H correlated spectroscopy analysis of the native and peracetylated GPL X-1, was developed, allowing the structural determination of the monosaccharidic residues with their alkali-labile groups "in situ" on the whole complex molecule. 2-O-Acyl-alpha-L-Rhap, alpha-L-Rhap, 2,4-di-O-acyl-6-deoxy-alpha-L-Glcp, 2,3,4-tri-O-Me-alpha-L-Rhap, and 3-O-Me-6-deoxy-alpha-L-Talp were identified, where Me, Rhap, and Talp are methyl, rhamnopyranosyl, and talopyranosyl, respectively. The latter two were localized at the carbohydrate non-reducing ends, and the C-3's of the remaining monosaccharide residues were found involved in the interglycosidic linkage. The alpha anomeric configurations were inferred from the JC-1,H-1 heteronuclear coupling constants, and the L absolute configurations for all the monosaccharide residues were established by gas chromatography analysis of the trimethylsilyl (+/-)-2-butyl glycosides. Finally, by pyrolysis electron impact mass spectrometry of peracetylated GPL X-1, the following tetrasaccharide appendage structure was proposed: 2,3,4-tri-O-Me-L-Rhap(alpha 1----3)-2-O-lauryl-L-Rhap(alpha 1----3)-L-Rhap- (alpha 1----3)-2,4-di-O-(acetyl,lauryl)-6-deoxy-alpha-L-Glcp. Compared to the oligosaccharidic glycopeptidolipid structures, the particular features of the GPL X-1 tetrasaccharide structure arise from the presence of monosaccharide residues esterified by C12 fatty acids and from the absence of the basal disaccharide core, L-Rhap-(alpha 1----2)-6-deoxy-alpha-L-Talp.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Antígenos de Bactérias/química , Glicoconjugados/química , Glicolipídeos/química , Glicopeptídeos/química , Mycobacterium/análise , Serina/química , Sequência de Carboidratos , Cromatografia Gasosa-Espectrometria de Massas , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mycobacterium/imunologiaRESUMO
A serine-containing glycopeptidolipid antigen isolated from Mycobacterium xenopi typified a new class of mycobacterial glycopeptidolipid antigens devoid of the C-mycoside core structure [Rivière, M., & Puzo, G. (1991) J. Biol. Chem. 266, 9057-9063]. The lipopeptide core assigned to C12-Ser-Ser-Phe-alloThr-OCH3 exhibits three potential sites of glycosylation. The carbohydrate parts are composed of 3-O-methyl-6-deoxy-alpha-L-talopyranosyl and 2,3,4-tri-O-methyl-L- rhamnopyranosyl(alpha 1----3)-2-O-lauroyl-L-rhamnopyranosyl(alpha 1----3)-L- rhamnopyranosyl(alpha 1----3)-2,4-di-O-(acetyl, lauroyl)-6-deoxy-alpha-L-glucopyranosyl appendages. In the present work, the carbohydrate attachment sites were successfully determined by ROESY experiments on the native glycopeptidolipid using chloroform as solvent. From the NOE contacts, we unambiguously established that the acylated serine is glycosylated by the 3-O-methyl-6-deoxy-alpha-L-talopyranosyl appendage while the 2,3,4-tri-O-methyl-L-rhamnopyranosyl(alpha 1----3)-2-O- lauroyl-L-rhamnopyranosyl(alpha 1----3)-L-rhamnopyranosyl(alpha 1----3)-2,4-di- O-(acetyl, lauroyl)-6-deoxy-alpha-L-glucopyranosyl appendage is bound to the C-terminal alloThr-OCH3. From these data, the acetyl and lauroyl residues on the C-2 and C-4 of the basal monosaccharide unit were successfully localized. Furthermore, the "L" absolute configuration for the serines and the phenylalanine residues and the "D" configuration for the allothreonine were established. The primary structure of this novel type of mycobacterial antigen, a serine-containing glycopeptidolipid, has now been fully established.
Assuntos
Aminoácidos/química , Antígenos de Bactérias/química , Glicolipídeos/química , Glicopeptídeos/química , Sequência de Aminoácidos , Sequência de Carboidratos , Carboidratos/química , Glicosilação , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mycobacterium/imunologia , Prótons , Serina/químicaRESUMO
A set of Lipooligosaccharides (LOSs) has previously been characterized in M. gastri W471. The structure of the highly antigenic LOS (LOS-III) was elucidated and this molecule can unambiguously distinguish M. gastri from the opportunistic pathogen M. kansasii. In the present study, the structures of three other M. gastri W471 LOSs were determined by one-dimensional 1H-NMR spectroscopy and gas liquid chromatography. They differ by the number of Xylp units and by the structure of the distal monosaccharide. The two dimensional (2D) NMR approach was successfully applied to the LOS antigen of M. kansasii to locate the acetyl and acyl substituents and to determine the anomeric configuration of the alpha-D-Fucp unit. The molecular specificity of anti-LOS-III antibodies was investigated and the LOS-III epitope was defined as the distal disaccharide: 3,6-dideoxy-4-C-(1,3-dimethoxy-4,5,6,7-tetrahydroxy- heptyl)-alpha-xylohexp-(1-->3)-beta-L-Xylp.
Assuntos
Antígenos de Bactérias/análise , Lipopolissacarídeos/imunologia , Micobactérias não Tuberculosas/imunologia , Reações Antígeno-Anticorpo , Sequência de Carboidratos , Parede Celular/química , Cromatografia Gasosa , Epitopos/imunologia , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura MolecularRESUMO
The following incomplete structure, alpha-X-(1-->3)-[beta-L-Xylp-(1-->4)]6-3-O-Me-alpha-Rhap-(1- ->3)- beta-D-Galp-(1-->3)-beta-D-Glcp-(1-->4)-2-O-acyl-alpha-D-Glcp-(1<-->1)-4 ,6-di-O-acyl-alpha-D-Glcp, was previously established for the antigenic lipo-oligosaccharide typifying Mycobacterium gastri, namely, LOS-III. The partial structure of the distal monosaccharide (X) was assigned as 3,6-dideoxy-4-C-(1,3-di-O-methylpropyl)-alpha-hexopyranose, which corresponds to a new-found monosaccharide in nature [Gilleron M., Vercauteren J., & Puzo G. (1993) J. Biol. Chem. 268, 3168-3179]. This article reports the complete structure of X, which was determined from the FAB-MS and 2D NMR analysis of the peracetylated LOS-III. The comparative analyses of the native and per-O-acetylated LOS-III FAB-MS spectra revealed, for the monosaccharide X, a molecular mass of 370 Da and five hydroxyl groups that could be acetylated. Additionally, the 1D 1H NMR spectrum of the per-O-acetylated LOS-III showed a dramatically increased dispersion of the protons, which resonated between 3 and 4 ppm in the spectrum of the underivatized LOS-III. Thus, thanks to 2D NMR sequences (COSY, HOHAHA, HMQC, HMQC-HOHAHA, and HMBC), the complete assignment of the 1H and 13C signals was achieved. Starting from the quaternary C4 resonance, the spin system of the C-alkyl chain was assigned, allowing us to propose the following structure, 3,6-dideoxy-4-C-(1,3-dimethoxy-4,5,6,7-tetrahydroxyheptyl)-alpha-x ylo- hexopyranose. The xylo configuration was established from the ROESY spectrum.
Assuntos
Antígenos de Bactérias/química , Hexoses/química , Lipopolissacarídeos/química , Mycobacterium/imunologia , Acetilação , Sequência de Carboidratos , Lipopolissacarídeos/imunologia , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
A novel mass spectrometric method has been developed for the detection and identification of dihydrouridine, ribothymidine, 4-thiouridine, and 7-methylguanosine in Escherichia coli tRNAs. The method utilizes (a) Pyrolysis-Electron Impact-Mass Spectrometry (PYEIMS), a procedure which releases the purine and pyrimidine bases from the intact, underivatized tRNA molecule. The mass spectrum exhibits intense peaks for the bases deriving from the common nucleosides in tRNA as well as peaks of much lower intensity at mass values expected for the bases from modified components known to be present in the tRNA; and, (b) Collisional Activation Mass Spectrometry (CAMS), a technique which permits the isolation of a single ion species from a complex mass spectrum. Subsequent fragmentation of that species yields a characteristic collisional activation spectrum. Such analyses of the ion species that were presumed to originate from H2Urd, rThd, 4SUrd, and 7MeGuo in the tRNA were used to define the structure and, thus, the identity of each component. Attributes of the PYEICAMS technique are that (a) precise structural elucidation of minor nucleosides present in tRNAs at the 1 - 4% level is obtained; (b) the high order of sensitivity allows the analysis to be done on microgram amounts of tRNA; and (c) there is no requirement for enzymatic or chemical hydrolysis of the tRNA or for subsequent chromatographic separation methods.
Assuntos
Nucleosídeos/análise , RNA de Transferência/análise , Escherichia coli/análise , Guanosina/análogos & derivados , Guanosina/análise , Espectrometria de Massas/métodos , Tiouridina/análise , Uridina/análogos & derivados , Uridina/análiseRESUMO
The mass spectra of the trimethylsilyl ethers of the four positional isomers of methyl-O-palmitoyl-alpha-D-glucopyranoside have been studied, and the structures of the principal ions assigned by the use of exact mass measurements and deuterium labelling on the aliphatic chain, the trimethylsilyl group and the glucosidic methyl group. The origin of some fragments has been elucidated by analysis of reactions of metastable ions. The mass spectra of the different isomers exhibit major differences, which depend upon the presence or absence of the aliphatic chain. These results indicate that this is a useful method for determining the position of an acyl group in a methyl glucoside. The applicability of this mass spectrometric method in structural determination of unknown acylated sugars is discussed. The structure of a corynomycoloyl-alpha-D-trehalose, isolated from Corynebacterium diphtheriae, has been determined by mass spectrometry as an application of the method. The molecular weight of this compound has been determined by field desorption mass spectrometry (cationization method), and the study of the electron impact spectrum of the trimethylsilyl derivatives clearly demonstrates that the corynomycolic acid is linked by an ester group to position 6 of the trehalose molecule.