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1.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360826

RESUMO

Glycosylation is a complex post-translational modification that conveys functional diversity to glycoconjugates. Cell surface glycosylation mediates several biological activities such as induction of the intracellular signaling pathway and pathogen recognition. Red blood cell (RBC) membrane N-glycans determine blood type and influence cell lifespan. Although several proteomic studies have been carried out, the glycosylation of RBC membrane proteins has not been systematically investigated. This work aims at exploring the human RBC N-glycome by high-sensitivity MALDI-MS techniques to outline a fingerprint of RBC N-glycans. To this purpose, the MALDI-TOF spectra of healthy subjects harboring different blood groups were acquired. Results showed the predominant occurrence of neutral and sialylated complex N-glycans with bisected N-acetylglucosamine and core- and/or antennary fucosylation. In the higher mass region, these species presented with multiple N-acetyllactosamine repeating units. Amongst the detected glycoforms, the presence of glycans bearing ABO(H) antigens allowed us to define a distinctive spectrum for each blood group. For the first time, advanced glycomic techniques have been applied to a comprehensive exploration of human RBC N-glycosylation, providing a new tool for the early detection of distinct glycome changes associated with disease conditions as well as for understanding the molecular recognition of pathogens.


Assuntos
Antígenos de Grupos Sanguíneos/metabolismo , Eritrócitos/metabolismo , Glicômica , Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Glicosilação , Humanos , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
2.
Allergy ; 76(8): 2500-2509, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33583051

RESUMO

PURPOSE: Tear fluid N-Glycome from patients affected with vernal (VKC) and atopic keratoconjunctivitis (AKC) was investigated to identify specific changes in tears and to recognize possible glyco-biomarkers. METHODS: The analysis of the N-glycans was performed using matrix-assisted laser desorption ionization mass spectrometry on single tear samples. Tears from control normal subjects (CTRL), VKC and AKC patients were processed and treated with peptide N-glycosidase F (PNGase F) to deglycosylate N-glycoproteins. Released N-glycans were purified, permethylated, and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and tandem mass spectrometry (MALDI-TOF MS and MALDI-TOF MS/MS). RESULTS: More than 150 complex N-glycans, including highly fucosylated biantennary, triantennary, tetra-antennary, and bisecting species, were observed in our spectra. Three distinct patterns for CTRL, VKC, and AKC patients were identified in terms of relative intensities for some N-glycans structures. Major variations involved bisecting and hyperfucosylated glycoforms. The most intense ions were associated with species at m/z 1907.0 (asialo, agalacto, bisected, biantennary structure-NGA2B) in CTRL MS profiles, at m/z 2605.3 and 2966.5 in VKC, and at m/z 2792.4 in AKC corresponding to a well-known biantennary, disialylated N-glycan. Several peaks were associated with structures bearing one or two Lewis X epitopes. Structures were confirmed by MS/MS analysis. Quantitative differences among the three groups were statistically significant. CONCLUSIONS: Tear MS profiles are rich in specific glycoforms, particularly those with a high fucosylation degree, indicating both core and peripheral decoration. Tear N-glycome analysis provided important information for a better comprehension of VKC and AKC alterations at the molecular level.


Assuntos
Conjuntivite Alérgica , Ceratoconjuntivite , Conjuntivite Alérgica/diagnóstico , Glicômica , Humanos , Polissacarídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Lágrimas
3.
Angew Chem Int Ed Engl ; 60(18): 10023-10031, 2021 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-33522128

RESUMO

Alcaligenes faecalis is the predominant Gram-negative bacterium inhabiting gut-associated lymphoid tissues, Peyer's patches. We previously reported that an A. faecalis lipopolysaccharide (LPS) acted as a weak agonist for Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) receptor as well as a potent inducer of IgA without excessive inflammation, thus suggesting that A. faecalis LPS might be used as a safe adjuvant. In this study, we characterized the structure of both the lipooligosaccharide (LOS) and LPS from A. faecalis. We synthesized three lipid A molecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1- and 4'-phosphates. Hexaacylated A. faecalis lipid A showed moderate agonistic activity towards TLR4-mediated signaling and the ability to elicit a discrete interleukin-6 release in human cell lines and mice. It was thus found to be the active principle of the LOS/LPS and a promising vaccine adjuvant candidate.


Assuntos
Alcaligenes faecalis/química , Lipídeo A/química , Lipopolissacarídeos/química , Animais , Configuração de Carboidratos , Linhagem Celular , Humanos , Interleucina-6/antagonistas & inibidores , Interleucina-6/metabolismo , Lipídeo A/farmacologia , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/farmacologia , Camundongos , Receptor 4 Toll-Like/agonistas
4.
Glycoconj J ; 38(2): 201-211, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32915358

RESUMO

N-glycan analyses may serve uncovering disease-associated biomarkers, as well as for profiling distinctive changes supporting diagnosis of genetic disorders of glycan biosynthesis named congenital disorders of glycosylation (CDG). Strategies based on liquid chromatography (LC) preferentially coupled to electrospray ionization (ESI) - mass spectrometry (MS) have emerged as powerful analytical methods for N-glycan identification and characterization. To enhance detection sensitivity, glycans are commonly labelled with a functional tag prior to LC-MS analysis. Since most derivatization techniques are notoriously time-consuming, some commercial analytical kits have been developed to speed up N-deglycosylation and N-glycan labelling of glycoproteins of pharmaceutical and biological interest such as monoclonal antibodies (mAbs). We exploited the analytical capabilities of RapiFluor-MS (RFMS) to perform, by a slightly modified protocol, a detailed N-glycan characterization of total serum and single serum glycoproteins from specific patients with CDG (MAN1B1-CDG, ALG12-CDG, MOGS-CDG, TMEM199-CDG). This strategy, accomplished by Hydrophilic Interaction Chromatography (HILIC)-UPLC-ESI-MS separation of the RFMS derivatized N-glycans, allowed us to uncover structural details of patients serum released N-glycans, thus extending the current knowledge on glycan profiles in these individual glycosylation diseases. The applied methodology enabled to differentiate in some cases either structural isomers and isomers differing in the linkage type. All the here reported applications demonstrated that RFMS method, coupled to HILIC-UPLC-ESI-MS, represents a sensitive high throughput approach for serum N-glycome analysis and a valuable option for glycan detection and separation particularly for isomeric species.

5.
J Biol Chem ; 295(32): 10969-10987, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32546484

RESUMO

Rhizobia are soil bacteria that form important symbiotic associations with legumes, and rhizobial surface polysaccharides, such as K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS), might be important for symbiosis. Previously, we obtained a mutant of Sinorhizobium fredii HH103, rkpA, that does not produce KPS, a homopolysaccharide of a pseudaminic acid derivative, but whose LPS electrophoretic profile was indistinguishable from that of the WT strain. We also previously demonstrated that the HH103 rkpLMNOPQ operon is responsible for 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-l-glycero-l-manno-nonulosonic acid [Pse5NAc7(3OHBu)] production and is involved in HH103 KPS and LPS biosynthesis and that an HH103 rkpM mutant cannot produce KPS and displays an altered LPS structure. Here, we analyzed the LPS structure of HH103 rkpA, focusing on the carbohydrate portion, and found that it contains a highly heterogeneous lipid A and a peculiar core oligosaccharide composed of an unusually high number of hexuronic acids containing ß-configured Pse5NAc7(3OHBu). This pseudaminic acid derivative, in its α-configuration, was the only structural component of the S. fredii HH103 KPS and, to the best of our knowledge, has never been reported from any other rhizobial LPS. We also show that Pse5NAc7(3OHBu) is the complete or partial epitope for a mAb, NB6-228.22, that can recognize the HH103 LPS, but not those of most of the S. fredii strains tested here. We also show that the LPS from HH103 rkpM is identical to that of HH103 rkpA but devoid of any Pse5NAc7(3OHBu) residues. Notably, this rkpM mutant was severely impaired in symbiosis with its host, Macroptilium atropurpureum.


Assuntos
Lipopolissacarídeos/química , Sinorhizobium fredii/química , Soja/microbiologia , Simbiose , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Superfície/imunologia , Proteínas de Bactérias/genética , Configuração de Carboidratos , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Epitopos/imunologia , Lipopolissacarídeos/imunologia , Espectroscopia de Prótons por Ressonância Magnética , Sinorhizobium fredii/genética , Sinorhizobium fredii/imunologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Açúcares Ácidos/química
6.
Glycoconj J ; 36(6): 461-472, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31529350

RESUMO

Congenital disorders of glycosylation (CDG) are genetic diseases characterized by deficient synthesis (CDG type I) and/or abnormal processing (CDG type II) of glycan moieties linked to protein and lipids. The impact of the molecular defects on protein glycosylation and in turn on the clinical phenotypes of patients with CDG is not yet understood. ALG12-CDG is due to deficiency of ALG12 α1,6-mannosyltransferase that adds the eighth mannose residue on the dolichol-PP-oligosaccharide precursor in the endoplasmic reticulum. ALG12-CDG is a severe multisystem disease associated with low to deficient serum immunoglobulins and recurrent infections. We thoroughly investigated the glycophenotype in a patient with novel ALG12 variants and immunodeficiency. We analyzed serum native transferrin, as first line test for CDG and we profiled serum IgG and total serum N-glycans by a combination of consolidated (N-glycan analysis by MALDI MS) and innovative mass spectrometry-based protocols, such as GlycoWorks RapiFluor N-glycan analysis coupled with LC-ESI MS. Intact serum transferrin showed, as expected for a CDG type I defect, underoccupancy of N-glycosylation sites. Surprisingly, total serum proteins and IgG N-glycans showed some specific changes, consisting in accumulating amounts of definite high-mannose and hybrid structures. As a whole, ALG12-CDG behaves as a dual CDG (CDG-I and II defects) and it is associated with distinct, abnormal glycosylation of total serum and IgG N-glycans. Glycan profiling of target glycoproteins may endorse the molecular defect unraveling the complex clinical phenotype of CDG patients.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Deficiência de IgG/genética , Imunoglobulinas/genética , Manosiltransferases/genética , Criança , Pré-Escolar , Defeitos Congênitos da Glicosilação/sangue , Defeitos Congênitos da Glicosilação/patologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Feminino , Glicoproteínas/sangue , Glicosilação , Humanos , Deficiência de IgG/sangue , Deficiência de IgG/metabolismo , Deficiência de IgG/patologia , Imunoglobulinas/sangue , Imunoglobulinas/deficiência , Lactente , Masculino , Manosiltransferases/sangue , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Polissacarídeos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transferrina/genética , Transferrina/metabolismo , Sequenciamento Completo do Exoma
7.
Methods Mol Biol ; 2044: 255-272, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31432418

RESUMO

CSF diagnostics has proved to be a formidable testing ground for N-glycoproteomic analysis of neurological diseases. To characterize specific N-glycan profiles of CSF in early and advanced phases of Alzheimer's disease, as well as in lysosomal storage disorders such as Tay-Sachs disease, we set up in our lab a robust and feasible protocol by coupling bioanalytical methods and mass spectrometry analysis.Starting from a few microliters of CSF, after protein denaturation, reduction, and alkylation, N-glycans are released from glycoproteins using the peptide-N-glycosidase F (PNGase F) and purified. The analysis of permethylated N-glycans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF MS/MS allowed us to identify specific glyco-structures and also to distinguish between isobaric N-glycans.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Glicoproteínas/líquido cefalorraquidiano , Glicoproteínas/química , Polissacarídeos/líquido cefalorraquidiano , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Doença de Tay-Sachs/líquido cefalorraquidiano , Idoso , Gangliosídeo G(M2)/metabolismo , Humanos , Íons/química , Polissacarídeos/análise , Polissacarídeos/isolamento & purificação
8.
Artigo em Inglês | MEDLINE | ID: mdl-29535976

RESUMO

Toll-like receptor 4 (TLR4) initiates immune response against Gram-negative bacteria upon specific recognition of lipid A moiety of lipopolysaccharide (LPS), the major component of their cell wall. Some natural differences between LPS variants in their ability to interact with TLR4 may lead to either insufficient activation that may not prevent bacterial growth, or excessive activation which may lead to septic shock. In this study we evaluated the biological activity of LPS isolated from pathogenic strain of Campylobacter jejuni, the most widespread bacterial cause of foodborne diarrhea in humans. With the help of hydrophobic chromatography and MALDI-TOF mass spectrometry we showed that LPS from a C. jejuni strain O2A consists of both hexaacyl and tetraacyl forms. Since such hypoacylation can result in a reduced immune response in humans, we assessed the activity of LPS from C. jejuni in mouse macrophages by measuring its capacity to activate TLR4-mediated proinflammatory cytokine and chemokine production, as well as NFκB-dependent reporter gene transcription. Our data support the hypothesis that LPS acylation correlates with its bioactivity.


Assuntos
Campylobacter jejuni/imunologia , Campylobacter jejuni/metabolismo , Doenças Transmitidas por Alimentos/microbiologia , Lipopolissacarídeos/farmacologia , Receptor 4 Toll-Like/efeitos dos fármacos , Receptor 4 Toll-Like/imunologia , Animais , Campylobacter jejuni/patogenicidade , Citocinas/metabolismo , Fator Regulador 3 de Interferon/genética , Interleucina-1beta/metabolismo , Interleucina-6 , Lipídeo A/imunologia , Lipídeo A/isolamento & purificação , Lipídeo A/farmacologia , Lipopolissacarídeos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno , Receptor 4 Toll-Like/genética , Fator de Necrose Tumoral alfa/metabolismo
9.
Methods Mol Biol ; 1750: 75-91, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29512066

RESUMO

In this chapter, we present the methodology currently applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. N-glycans are released from denatured carboxymethylated glycoproteins by digestion with peptide-N-glycosidase F (PNGase F) and purified using both C18 Sep-Pak® and porous graphitized carbon (PGC) HyperSep™ Hypercarb™ solid-phase extraction (SPE) cartridges. The glycan pool is subsequently permethylated to increase mass spectrometry sensitivity. Molecular assignments are performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis considering either the protein N-linked glycosylation pathway or MALDI TOF MS/MS data. Each stage has been optimized to obtain high-quality mass spectra in reflector mode with an optimal signal-to-noise ratio up to m/z 4800. This method has been successfully adopted to associate specific N-glycome profiles to the early and the advanced phases of Alzheimer's disease.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Glicômica/métodos , Extração em Fase Sólida/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Glicoproteínas/líquido cefalorraquidiano , Glicosilação , Humanos , Polissacarídeos/líquido cefalorraquidiano
10.
J Bacteriol ; 200(2)2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29109183

RESUMO

In Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane (OM), preventing the entry of toxic molecules, such as detergents and antibiotics. LPS is transported from the inner membrane (IM) to the OM by the Lpt multiprotein machinery. Defects in LPS transport compromise LPS assembly at the OM and result in increased antibiotic sensitivity. LptA is a key component of the Lpt machine that interacts with the IM protein LptC and chaperones LPS through the periplasm. We report here the construction of lptA41, a quadruple mutant in four conserved amino acids potentially involved in LPS or LptC binding. Although viable, the mutant displays increased sensitivity to several antibiotics (bacitracin, rifampin, and novobiocin) and the detergent SDS, suggesting that lptA41 affects LPS transport. Indeed, lptA41 is defective in Lpt complex assembly, and its lipid A carries modifications diagnostic of LPS transport defects. We also selected and characterized two phenotypic bacitracin-resistant suppressors of lptA41 One mutant, in which only bacitracin sensitivity is suppressed, harbors a small in-frame deletion in mlaA, which codes for an OM lipoprotein involved in maintaining OM asymmetry by reducing accumulation of phospholipids in the outer leaflet. The other mutant, in which bacitracin, rifampin, and SDS sensitivity is suppressed, harbors an additional amino acid substitution in LptA41 and a nonsense mutation in opgH, encoding a glycosyltransferase involved in periplasmic membrane-derived oligosaccharide synthesis. Characterization of the suppressor mutants highlights different strategies adopted by the cell to overcome OM defects caused by impaired LPS transport.IMPORTANCE Lipopolysaccharide (LPS) is the major constituent of the outer membrane (OM) of most Gram-negative bacteria, forming a barrier against antibiotics. LPS is synthesized at the inner membrane (IM), transported across the periplasm, and assembled at the OM by the multiprotein Lpt complex. LptA is the periplasmic component of the Lpt complex, which bridges IM and OM and ferries LPS across the periplasm. How the cell coordinates the processes involved in OM biogenesis is not completely understood. We generated a mutant partially defective in lptA that exhibited increased sensitivity to antibiotics and selected for suppressors of the mutant. The analysis of two independent suppressors revealed different strategies adopted by the cell to overcome defects in LPS biogenesis.


Assuntos
Proteínas de Transporte/genética , Permeabilidade da Membrana Celular , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Lipopolissacarídeos/metabolismo , Supressão Genética , Substituição de Aminoácidos , Bacitracina/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Transporte/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Glicosiltransferases/genética , Lipídeo A/metabolismo , Proteínas de Membrana/metabolismo , Rifampina/farmacologia , Dodecilsulfato de Sódio/farmacologia
11.
Front Microbiol ; 8: 1821, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28983292

RESUMO

In rhizobium strains, the lipid A is modified by the addition of a very long-chain fatty acid (VLCFA) shown to play an important role in rigidification of the outer membrane, thereby facilitating their dual life cycle, outside and inside the plant. In Bradyrhizobium strains, the lipid A is more complex with the presence of at least two VLCFAs, one covalently linked to a hopanoid molecule, but the importance of these modifications is not well-understood. In this study, we identified a cluster of VLCFA genes in the photosynthetic Bradyrhizobium strain ORS278, which nodulates Aeschynomene plants in a Nod factor-independent process. We tried to mutate the different genes of the VLCFA gene cluster to prevent the synthesis of the VLCFAs, but only one mutant in the lpxXL gene encoding an acyltransferase was obtained. Structural analysis of the lipid A showed that LpxXL is involved in the transfer of the C26:25OH VLCFA to the lipid A but not in the one of the C30:29OH VLCFA which harbors the hopanoid molecule. Despite maintaining the second VLCFA, the ability of the mutant to cope with various stresses (low pH, high temperature, high osmolarity, and antimicrobial peptides) and to establish an efficient nitrogen-fixing symbiosis was drastically reduced. In parallel, we investigated whether the BRADO0045 gene, which encodes a putative acyltransferase displaying a weak identity with the apo-lipoprotein N-acyltransferase Lnt, could be involved in the transfer of the C30:29OH VLCFA to the lipid A. Although the mutant exhibited phenotypes similar to the lpxXL mutant, no difference in the lipid A structure was observed from that in the wild-type strain, indicating that this gene is not involved in the modification of lipid A. Our results advance our knowledge of the biosynthesis pathway and the role of VLCFAs-modified lipid A in free-living and symbiotic states of Bradyrhizobium strains.

12.
ChemistryOpen ; 6(4): 541-553, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28794950

RESUMO

The importance of the outer membrane and of its main constituent, lipopolysaccharide, in the symbiosis between rhizobia and leguminous host plants has been well studied. Here, the first complete structural characterization of the entire lipopolysaccharide from an O-chain-deficient Bradyrhizobium ORS285 rfaL mutant is achieved by a combination of chemical analysis, NMR spectroscopy, MALDI MS and MS/MS. The lipid A structure is shown to be consistent with previously reported Bradyrhizobium lipid A, that is, a heterogeneous blend of penta- to hepta-acylated species carrying a nonstoichiometric hopanoid unit and possessing very-long-chain fatty acids ranging from 26:0(25-OH) to 32:0(31-OH). The structure of the core oligosaccharide region, fully characterized for the first time here, is revealed to be a nonphosphorylated linear chain with methylated sugar residues, with a heptose residue exclusively present in the outer core region, and with the presence of two singly substituted 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residues, one of which is located in the outer core region. The lipid A moiety is linked to the core moiety through an uncommon 4-substituted Kdo unit.

13.
Mar Drugs ; 15(7)2017 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-28653982

RESUMO

The structural characterization of the lipopolysaccharide (LPS) from extremophiles has important implications in several biomedical and therapeutic applications. The polyextremophile Gram-negative bacterium Halobacteroideslacunaris TB21, isolated from one of the most extreme habitats on our planet, the deep-sea hypersaline anoxic basin Thetis, represents a fascinating microorganism to investigate in terms of its LPS component. Here we report the elucidation of the full structure of the R-type LPS isolated from H. lacunaris TB21 that was attained through a multi-technique approach comprising chemical analyses, NMR spectroscopy, and Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry. Furthermore, cellular immunology studies were executed on the pure R-LPS revealing a very interesting effect on human innate immunity as an inhibitor of the toxic Escherichia coli LPS.


Assuntos
Extremófilos/química , Bactérias Anaeróbias Gram-Negativas/química , Imunidade Inata/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Animais , Linhagem Celular , Escherichia coli/química , Extremófilos/isolamento & purificação , Feminino , Bactérias Anaeróbias Gram-Negativas/isolamento & purificação , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/isolamento & purificação , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Água do Mar/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
14.
Chembiochem ; 18(8): 772-781, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28186388

RESUMO

Xanthomonas citri pv. citri is the pathogen responsible for Asiatic citrus canker, one of the most serious citrus diseases worldwide. The lipopolysaccharide (LPS) molecule has been demonstrated to be involved in X. citri pv. citri virulence. Despite enormous progress in investigations of the molecular mechanisms for bacterial pathogenicity, determination of the detailed LPS structure-activity relationship is limited, as the current knowledge is mainly based on structural determination of one X. citri pv. citri strain. As X. citri pv. citri strains are distinguished into three main pathogenicity groups, we characterized the full structure of the LPS from two pathotypes that differ in their host-range specificity. This revealed an intriguing difference in LPS O-chain structure. We also tested the LPSs and isolated lipid A moieties for their ability to act as microbe-associated molecular patterns in Arabidopsis thaliana. Both LPS/lipid As induced ROS accumulation, but no difference was observed between the two pathotypes.


Assuntos
Lipopolissacarídeos/química , Fatores de Virulência/química , Xanthomonas/fisiologia , Arabidopsis/imunologia , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Imunidade Inata , Lipídeo A/química , Lipopolissacarídeos/imunologia , Estrutura Molecular , Espectroscopia de Prótons por Ressonância Magnética , Espécies Reativas de Oxigênio/metabolismo , Virulência , Fatores de Virulência/imunologia , Xanthomonas/classificação , Xanthomonas/imunologia
15.
Chemistry ; 23(15): 3637-3647, 2017 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-28004420

RESUMO

The search for novel lipid A analogues from any biological source that can act as antagonists, displaying inhibitory activity towards the production of pro-inflammatory cytokines, or as immunomodulators in mammals, is a very topical issue. To this aim, the structure and immunological properties of the lipopolysaccharide lipid A from the purple nonsulfur bacterium Rhodopseudomonas palustris strain BisA53 have been determined. This lipid A displays a unique structural feature, with a non-phosphorylated skeleton made up of the tetrasaccharide Manp-α-(1→4)-GlcpN3N-ß-1→6-GlcpN3N-α-(1→1)-α-GalpA, and four primary amide-linked 14:0(3-OH) and, as secondary O-acyl substituents, a 16:0 and the very long-chain fatty acid 26:0(25-OAc), appended on the GlcpN3N units. This lipid A architecture is definitely rare, so far identified only in the genus Bradyrhizobium. Immunological tests on both murine bone-marrow-derived and human monocyte-derived macrophages revealed an extremely low immunostimulant capability of this LPS lipid A.


Assuntos
Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Lipídeo A/química , Lipídeo A/farmacologia , Rodopseudomonas/química , Animais , Células Cultivadas , Humanos , Imunidade Inata/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Espectroscopia de Ressonância Magnética , Camundongos Endogâmicos C57BL , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
16.
Carbohydr Res ; 427: 38-43, 2016 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-27085742

RESUMO

The structure of the lipooligosaccharide (LOS) from the rice pathogen Xanthomonas oryzae pv. oryzae has been elucidated. The characterization of the core oligosaccharide structure was obtained by the employment of two chemical degradation protocols and by analysis of the products via NMR spectroscopy. The structure of the lipid A portion was achieved by MALDI mass spectrometry analysis on purified lipid A. The LOS from Xanthomonas oryzae pv. oryzae revealed to possess the same core structure of Xanthomonas campestris pv. campestris and interesting novel features on its lipid A domain. The evaluation of the biological activity of both LOS and isolated lipid A was also executed.


Assuntos
Lipopolissacarídeos/química , Xanthomonas/metabolismo , Lipopolissacarídeos/isolamento & purificação , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Oryza/microbiologia , Doenças das Plantas/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Xanthomonas/química
17.
Angew Chem Int Ed Engl ; 55(2): 654-8, 2016 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-26582281

RESUMO

N-glycosylation is a fundamental modification of proteins and exists in the three domains of life and in some viruses, including the chloroviruses, for which a new type of core N-glycan is herein described. This N-glycan core structure, common to all chloroviruses, is a pentasaccharide with a ß-glucose linked to an asparagine residue which is not located in the typical sequon N-X-T/S. The glucose is linked to a terminal xylose unit and a hyperbranched fucose, which is in turn substituted with a terminal galactose and a second xylose residue. The third position of the fucose unit is always linked to a rhamnose, which is a semiconserved element because its absolute configuration is virus-dependent. Additional decorations occur on this core N-glycan and represent a molecular signature for each chlorovirus.


Assuntos
Phycodnaviridae/química , Polissacarídeos/química , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
18.
J Proteomics ; 131: 29-37, 2016 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-26455811

RESUMO

This work aims at exploring the human CSF (Cerebrospinal fluid) N-glycome by MALDI MS techniques, in order to assess specific glycosylation pattern(s) in patients with Alzheimer's disease (n:24) and in subjects with mild cognitive impairment (MCI) (n:11), these last as potential AD patients at a pre-dementia stage. For comparison, 21 healthy controls were studied. We identified a group of AD and MCI subjects (about 40-50% of the studied sample) showing significant alteration of CSF N-glycome profiling, consisting of a decrease in the overall sialylation degree and an increase in species bearing bisecting GlcNAc. Noteworthy, all the MCI patients that converted to AD within the clinical follow-up, had an abnormal CSF glycosylation profile. Based on the studied cohort, CSF glycosylation changes may occur before an AD clinical onset. Previous studies specifically focused on the key role of glycosyltransferase GnT-III on AD-pathogenesis, addressing the patho-mechanism to specific sugar modification of BACE-1 glycoprotein with bisecting GlcNAc. Our patients addressed protein N-glycosylation changes at an early phase of the whole biomolecular misregulation on AD, pointing to CSF N-glycome analyses as promising tool to enhance early detection of AD and also suggesting alternative therapeutics target molecules, such as specific glyco-enzymes.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico , Diagnóstico Precoce , Glicoproteínas/líquido cefalorraquidiano , Proteoma/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Idoso , Biomarcadores/líquido cefalorraquidiano , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Espectrometria de Massas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
19.
Autism Res ; 9(4): 423-8, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26286102

RESUMO

Protein N-glycosylation consists in the synthesis and processing of the oligosaccharide moiety (N-glycan) linked to a protein and it serves several functions for the proper central nervous system (CNS) development and function. Previous experimental and clinical studies have shown the importance of proper glycoprotein sialylation for the synaptic function and the occurrence of autism spectrum disorders (ASD) in the presence of sialylation deficiency in the CNS. Late-onset Tay Sachs disease (LOTSD) is a lysosomal disorder caused by mutations in the HEXA gene resulting in GM2-ganglioside storage in the CNS. It is characterized by progressive neurological impairment and high co-occurrence of psychiatric disturbances. We studied the N-glycome profile of the cerebrospinal fluid (CSF) in a 14 year-old patient with GM2-gangliosidosis (LOTSD). At the age of 4, the patient presented regressive autism fulfilling criteria for childhood disintegrative disorder (CDD). A CSF sample was obtained in the course of diagnostic work-up for the suspicion of an underlying neurodegenerative disorder. We found definite changes of CSF N-glycans due to a dramatic decrease of sialylated biantennary and triantennary structures and an increase of asialo-core fucosylated bisected N-glycans. No changes of total plasma N-glycans were found. Herein findings highlight possible relationships between the early onset psychiatric disturbance featuring CDD in the patient and defective protein sialylation in the CNS. In conclusion, the study first shows aberrant N-glycan structures of CSF proteins in LOTSD; unveils possible pathomechanisms of GM2-gangliosidosis; supports existing relationships between neuropsychiatric disorders and unproper protein glycosylation in the CNS.


Assuntos
Transtornos Globais do Desenvolvimento Infantil/líquido cefalorraquidiano , Transtornos Globais do Desenvolvimento Infantil/complicações , Gangliosidoses GM2/líquido cefalorraquidiano , Gangliosidoses GM2/complicações , Polissacarídeos/líquido cefalorraquidiano , Adolescente , Transtornos Globais do Desenvolvimento Infantil/psicologia , Pré-Escolar , Feminino , Gangliosidoses GM2/psicologia , Glicosilação , Humanos , Espectrometria de Massas
20.
Front Immunol ; 6: 595, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26635809

RESUMO

Toll-like receptor 4 (TLR4) is required for activation of innate immunity upon recognition of lipopolysaccharide (LPS) of Gram-negative bacteria. The ability of TLR4 to respond to a particular LPS species is important since insufficient activation may not prevent bacterial growth while excessive immune reaction may lead to immunopathology associated with sepsis. Here, we investigated the biological activity of LPS from Burkholderia mallei that causes glanders, and from the two well-known opportunistic pathogens Acinetobacter baumannii and Pseudomonas aeruginosa (causative agents of nosocomial infections). For each bacterial strain, R-form LPS preparations were purified by hydrophobic chromatography and the chemical structure of lipid A, an LPS structural component, was elucidated by HR-MALDI-TOF mass spectrometry. The biological activity of LPS samples was evaluated by their ability to induce production of proinflammatory cytokines, such as IL-6 and TNF, by bone marrow-derived macrophages. Our results demonstrate direct correlation between the biological activity of LPS from these pathogenic bacteria and the extent of their lipid A acylation.

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