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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.
Orphanet J Rare Dis ; 16(1): 307, 2021 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-34246313

RESUMO

BACKGROUND: SLC39A8, a gene located on chromosome 4q24, encodes for the manganese (Mn) transporter ZIP8 and its detrimental variants cause a type 2 congenital disorder of glycosylation (CDG). The common SLC39A8 missense variant A391T is associated with increased risk for multiple neurological and systemic disorders and with decreased serum Mn. Patients with SLC39A8-CDG present with different clinical and neuroradiological features linked to variable transferrin glycosylation profile. Galactose and Mn supplementation therapy results in the biochemical and clinical amelioration of treated patients. RESULTS: Here, we report clinical manifestations, neuroradiological features and glycophenotypes associated with novel SLC39A8 variants (c.1048G > A; p.Gly350Arg and c.131C > G; p.Ser44Trp) in two siblings of the same Italian family. Furthermore, we describe a third patient with overlapping clinical features harbouring the homozygous missense variant A391T. The clinical phenotype of the three patients was characterized by severe developmental disability, dystonic postural pattern and dyskinesia with a more severe progression of the disease in the two affected siblings. Neuroimaging showed a Leigh syndrome-like pattern involving the basal ganglia, thalami and white matter. In the two siblings, atrophic cerebral and cerebellum changes consistent with SLC39A8-CDG were detected as well. Serum transferrin isoelectric focusing (IEF) yielded variable results with slight increase of trisialotransferrin isoforms or even normal pattern. MALDI-MS showed the presence of hypogalactosylated transferrin N-glycans, spontaneously decreasing during the disease course, only in one affected sibling. Total serum N-glycome depicted a distinct pattern for the three patients, with increased levels of undergalactosylated and undersialylated precursors of fully sialylated biantennary glycans, including the monosialo-monogalacto-biantennary species A2G1S1. CONCLUSIONS: Clinical, MRI and glycosylation features of patients are consistent with SLC39A8-CDG. We document two novel variants associated with Leigh syndrome-like disease presentation of SLC39A8-CDG. We show, for the first time, a severe neurological phenotype overlapping with that described for SLC39A8-CDG in association with the homozygous A391T missense variant. We observed a spontaneous amelioration of transferrin N-glycome, highlighting the efficacy of MS-based serum glycomics as auxiliary tool for the diagnosis and clinical management of therapy response in patients with SLC39A8-CDG. Further studies are needed to analyse more in depth the influence of SLC39A8 variants, including the common missense variant, on the expression and function of ZIP8 protein, and their impact on clinical, biochemical and neuroradiological features.


Assuntos
Defeitos Congênitos da Glicosilação , Doença de Leigh , Defeitos Congênitos da Glicosilação/genética , Glicosilação , Humanos , Manganês , Polissacarídeos
3.
Glycoconj J ; 38(2): 191-200, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33644825

RESUMO

Human ALG2 encodes an α 1,3mannosyltransferase that catalyzes the first steps in the synthesis of N-glycans in the endoplasmic reticulum. Variants in ALG2cause a congenital disorder of glycosylation (CDG) known as ALG2-CDG. Up to date, nine ALG2-CDG patients have been reported worldwide. ALG2-CDG is a rare autosomal recessive inherited disorder characterized by neurological involvement, convulsive syndrome of unknown origin, axial hypotonia, and mental and motor regression. In this study, we used MALDI-TOF MS to define both total serum protein and transferrin (Tf) N-glycan phenotypes in three ALG2-CDG patients carrying a c.752G > T, p.Arg251Leu ALG2 missense variant in homozygous state, as determined by exome sequencing. Comparing it to control samples, we have observed Tf under-occupancy of glycosylation site(s) typical of a defective N-glycan assembly and the occurrence of oligomannose and hybrid type N-glycans. Moreover, we have observed a slight oligomannose accumulation in total serum glyco-profiles. The increased heterogeneity of serum N-glycome in the studied patients suggests a marginal disarrangement of the glycan processing in ALG2-CDG. Previous studies reported on slightly increased concentrations of abnormal serum N-glycans in CDG-I due to defects in the mannosylation steps of dolichol-linked oligosaccharide biosynthesis. This preliminary work aims at considering serum N-glycan accumulation of high mannosylated glycoforms, such as oligomannose and hybrid type N-glycans, as potential diagnostic signals for ALG2-CDG patients.

4.
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
5.
Cerebellum ; 20(4): 596-605, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33619652

RESUMO

We aimed to identify clinical, molecular and radiological correlates of activities of daily living (ADL) in patients with cerebellar atrophy caused by PMM2 mutations (PMM2-CDG), the most frequent congenital disorder of glycosylation. Twenty-six PMM2-CDG patients (12 males; mean age 13 ± 11.1 years) underwent a standardized assessment to measure ADL, ataxia (brief ataxia rating scale, BARS) and phenotype severity (Nijmegen CDG rating scale, NCRS). MRI biometry of the cerebellum and the brainstem were performed in 23 patients (11 males; aged 5 months-18 years) and 19 control subjects with equal gender and age distributions. The average total ADL score was 15.3 ± 8.5 (range 3-32 out of 36 indicating severe functional disability), representing variable functional outcome in PMM2-CDG patients. Total ADL scores were significantly correlated with NCRS (r2 = 0.55, p < 0.001) and BARS scores (r2 = 0.764; p < 0.001). Severe intellectual disability, peripheral neuropathy, and severe PMM2 variants were all significantly associated with worse functional outcome. Higher ADL scores were significantly associated with decreased diameters of cerebellar vermis (r2 = 0.347; p = 0.004), hemispheres (r2 = 0.436; p = 0.005), and brainstem, particularly the mid-pons (r2 = 0.64; p < 0.001) representing the major radiological predictor of functional disability score in multivariate regression analysis. We show that cerebellar syndrome severity, cognitive level, peripheral neuropathy, and genotype correlate with ADL used to quantify disease-related deficits in PMM2-CDG. Brainstem involvement should be regarded among functional outcome predictors in patients with cerebellar atrophy caused by PMM2-CDG.

6.
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
7.
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.

8.
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
9.
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
10.
Carbohydr Res ; 481: 43-51, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31228656

RESUMO

One of the strategies adopted for the development of a bivalent conjugate vaccine against invasive nontyphoidal Salmonella consists of linking the O-antigen component of S. Typhimurium and S. Entertidis lipopolysaccharides to the carrier protein CRM197, a non-toxic variant of diphtheria toxin. The conjugation reaction uses the reducing end residue 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) of the core to which the O-antigen chain is bound (OAg-core). OAg-core chains are cleaved from the lipid A directly in the fermentation broth by mild acid treatment. Kdo has been reported to undergo structural changes under these conditions and therefore the Kdo at the reducing end was thoroughly analysed to verify its structural integrity. For this purpose, low molecular mass OAg-core chains extracted from S. Typhimurium wild type bacteria and core oligosaccharides extracted from S. Typhimurium bacteria mutated not to produce O-antigen repeats were characterized by GLC-MS, MALDI-TOF-MS and NMR spectroscopy. Moreover, a combination of 1H-1H and 1H-13C experiments confirmed the linkage positions, sequence and structure of the octasaccharide core with 5-linked Kdo present at the reducing end in its native structure: α-GlcpNAc-(1→2)-α-Glcp-(1→2)-α-Galp-(1→3)-[α-Galp-(1→6)]-α-Glcp-(1→3)-[α-Hepp-(1→7)]-α-Hepp-(1→3)-α-Hepp-(1→5)-Kdo.


Assuntos
Antígenos O/química , Salmonella typhimurium/química , Salmonella typhimurium/imunologia , Açúcares Ácidos/química , Vacinas Conjugadas/química , Antígenos O/imunologia , Oxirredução , Vacinas Conjugadas/imunologia
11.
Adv Exp Med Biol ; 1104: 237-257, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30484252

RESUMO

The capsid of Paramecium bursaria chlorella virus (PBCV-1) contains a heavily glycosylated major capsid protein, Vp54. The capsid protein contains four glycans, each N-linked to Asn. The glycan structures are unusual in many aspects: (1) they are attached by a ß-glucose linkage, which is rare in nature; (2) they are highly branched and consist of 8-10 neutral monosaccharides; (3) all four glycoforms contain a dimethylated rhamnose as the capping residue of the main chain, a hyper-branched fucose residue and two rhamnose residues ''with opposite absolute configurations; (4) the four glycoforms differ by the nonstoichiometric presence of two monosaccharides, L-arabinose and D-mannose ; (5) the N-glycans from all of the chloroviruses have a strictly conserved core structure; and (6) these glycans do not resemble any structures previously reported in the three domains of life.The structures of these N-glycoforms remained elusive for years because initial attempts to solve their structures used tools developed for eukaryotic-like systems, which we now know are not suitable for this noncanonical glycosylation pattern. This chapter summarizes the methods used to solve the chlorovirus complex glycan structures with the hope that these methodologies can be used by scientists facing similar problems.


Assuntos
Proteínas do Capsídeo/química , Chlorella/virologia , Glicosilação , Phycodnaviridae/química , Polissacarídeos/química
12.
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
13.
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
14.
J Biol Chem ; 292(47): 19226-19237, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-28972189

RESUMO

Lipopolysaccharide, the outer cell-wall component of Gram-negative bacteria, has been shown to be important for symbiotic associations. We recently reported that the lipopolysaccharide O-antigen of Burkholderia enhances the initial colonization of the midgut of the bean bug, Riptortus pedestris However, the midgut-colonizing Burkholderia symbionts lack the O-antigen but display the core oligosaccharide on the cell surface. In this study, we investigated the role of the core oligosaccharide, which directly interacts with the host midgut, in the Riptortus-Burkholderia symbiosis. To this end, we generated the core oligosaccharide mutant strains, ΔwabS, ΔwabO, ΔwaaF, and ΔwaaC, and determined the chemical structures of their oligosaccharides, which exhibited different compositions. The symbiotic properties of these mutant strains were compared with those of the wild-type and O-antigen-deficient ΔwbiG strains. Upon introduction into Riptortus via the oral route, the core oligosaccharide mutant strains exhibited different rates of colonization of the insect midgut. The symbiont titers in fifth-instar insects revealed significantly reduced population sizes of the inner core oligosaccharide mutant strains ΔwaaF and ΔwaaC These two strains also negatively affected host growth rate and fitness. Furthermore, R. pedestris individuals colonized with the ΔwaaF and ΔwaaC strains were vulnerable to septic bacterial challenge, similar to insects without a Burkholderia symbiont. Taken together, these results suggest that the core oligosaccharide from Burkholderia symbionts plays a critical role in maintaining a proper symbiont population and in supporting the beneficial effects of the symbiont on its host in the Riptortus-Burkholderia symbiosis.


Assuntos
Burkholderia/fisiologia , Trato Gastrointestinal/crescimento & desenvolvimento , Heterópteros/crescimento & desenvolvimento , Oligossacarídeos/metabolismo , Simbiose/fisiologia , Animais , Burkholderia/genética , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Heterópteros/genética , Heterópteros/microbiologia , Mutação , Antígenos O/metabolismo
15.
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.

16.
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.

17.
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
18.
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
19.
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
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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