ABSTRACT
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.
Subject(s)
Glycine max/microbiology , Lipopolysaccharides/chemistry , Sinorhizobium fredii/chemistry , Symbiosis , Antibodies, Monoclonal/immunology , Antigens, Bacterial/immunology , Antigens, Surface/immunology , Bacterial Proteins/genetics , Carbohydrate Conformation , Carbon-13 Magnetic Resonance Spectroscopy , Epitopes/immunology , Lipopolysaccharides/immunology , Proton Magnetic Resonance Spectroscopy , Sinorhizobium fredii/genetics , Sinorhizobium fredii/immunology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Sugar Acids/chemistryABSTRACT
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.
Subject(s)
Conjunctivitis, Allergic , Keratoconjunctivitis , Conjunctivitis, Allergic/diagnosis , Glycomics , Humans , Polysaccharides , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass Spectrometry , TearsABSTRACT
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.
Subject(s)
Congenital Disorders of Glycosylation/blood , Polysaccharides/blood , Polysaccharides/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Blood Chemical Analysis/methods , Chromatography, High Pressure Liquid/methods , Humans , Isomerism , Mannosidases/deficiency , Membrane Proteins/deficiency , alpha-Glucosidases/metabolismABSTRACT
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.
Subject(s)
Blood Group Antigens/metabolism , Erythrocytes/metabolism , Glycomics , Polysaccharides/analysis , Protein Processing, Post-Translational , Glycosylation , Humans , Proteomics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Alcaligenes faecalis is the predominant Gram-negative bacterium inhabiting gut-associated lymphoid tissues, Peyer's patches. We previously reported that an A.â faecalis lipopolysaccharide (LPS) acted as a weak agonist for Toll-like receptorâ 4 (TLR4)/myeloid differentiation factor-2 (MD-2) receptor as well as a potent inducer of IgA without excessive inflammation, thus suggesting that A.â 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.â faecalis. We synthesized three lipidâ A molecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1- and 4'-phosphates. Hexaacylated A.â faecalis lipidâ 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.
Subject(s)
Alcaligenes faecalis/chemistry , Lipid A/chemistry , Lipopolysaccharides/chemistry , Animals , Carbohydrate Conformation , Cell Line , Humans , Interleukin-6/antagonists & inhibitors , Interleukin-6/metabolism , Lipid A/pharmacology , Lipopolysaccharides/isolation & purification , Lipopolysaccharides/pharmacology , Mice , Toll-Like Receptor 4/agonistsABSTRACT
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.
Subject(s)
Congenital Disorders of Glycosylation/genetics , IgG Deficiency/genetics , Immunoglobulins/genetics , Mannosyltransferases/genetics , Child , Child, Preschool , Congenital Disorders of Glycosylation/blood , Congenital Disorders of Glycosylation/pathology , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Female , Glycoproteins/blood , Glycosylation , Humans , IgG Deficiency/blood , IgG Deficiency/metabolism , IgG Deficiency/pathology , Immunoglobulins/blood , Immunoglobulins/deficiency , Infant , Male , Mannosyltransferases/blood , Oligosaccharides/genetics , Oligosaccharides/metabolism , Polysaccharides/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transferrin/genetics , Transferrin/metabolism , Exome SequencingABSTRACT
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.
Subject(s)
Carrier Proteins/genetics , Cell Membrane Permeability , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Lipopolysaccharides/metabolism , Suppression, Genetic , Amino Acid Substitution , Bacitracin/pharmacology , Bacterial Outer Membrane Proteins/genetics , Carrier Proteins/metabolism , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli Proteins/metabolism , Glycosyltransferases/genetics , Lipid A/metabolism , Membrane Proteins/metabolism , Rifampin/pharmacology , Sodium Dodecyl Sulfate/pharmacologyABSTRACT
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.â 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.â citri pv. citri strain. As X.â 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 lipidâ A moieties for their ability to act as microbe-associated molecular patterns in Arabidopsis thaliana. Both LPS/lipidâ As induced ROS accumulation, but no difference was observed between the two pathotypes.
Subject(s)
Lipopolysaccharides/chemistry , Virulence Factors/chemistry , Xanthomonas/physiology , Arabidopsis/immunology , Arabidopsis/metabolism , Arabidopsis/microbiology , Immunity, Innate , Lipid A/chemistry , Lipopolysaccharides/immunology , Molecular Structure , Proton Magnetic Resonance Spectroscopy , Reactive Oxygen Species/metabolism , Virulence , Virulence Factors/immunology , Xanthomonas/classification , Xanthomonas/immunologyABSTRACT
The search for novel lipidâ 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 lipidâ A from the purple nonsulfur bacterium Rhodopseudomonas palustris strain BisA53 have been determined. This lipidâ A displays a unique structural feature, with a non-phosphorylated skeleton made up of the tetrasaccharide Manp-α-(1â4)-GlcpN3N-ß-1â6-GlcpN3N-α-(1â1)-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 lipidâ 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 lipidâ A.
Subject(s)
Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/pharmacology , Lipid A/chemistry , Lipid A/pharmacology , Rhodopseudomonas/chemistry , Animals , Cells, Cultured , Humans , Immunity, Innate/drug effects , Macrophages/drug effects , Macrophages/immunology , Magnetic Resonance Spectroscopy , Mice, Inbred C57BL , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
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.
Subject(s)
Extremophiles/chemistry , Gram-Negative Anaerobic Bacteria/chemistry , Immunity, Innate/drug effects , Lipopolysaccharides/pharmacology , Animals , Cell Line , Escherichia coli/chemistry , Extremophiles/isolation & purification , Female , Gram-Negative Anaerobic Bacteria/isolation & purification , Humans , Lipopolysaccharides/chemistry , Lipopolysaccharides/isolation & purification , Magnetic Resonance Spectroscopy , Mice , Mice, Inbred C57BL , Seawater/microbiology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The major capsid protein Vp54 from the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains four Asn-linked glycans. The structure of the four N-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. Vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, PBCV-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) the glycans are attached to the protein by a ß-glucose linkage; (iii) the Asn-linked glycans are not located in a typical N-X-(T/S) consensus site; and (iv) the process probably occurs in the cytoplasm. The four glycoforms share a common core structure, and the differences are related to the nonstoichiometric presence of two monosaccharides. The most abundant glycoform consists of nine neutral monosaccharide residues, organized in a highly branched fashion. Among the most distinctive features of the glycoforms are (i) a dimethylated rhamnose as the capping residue of the main chain, (ii) a hyperbranched fucose unit, and (iii) two rhamnose residues with opposite absolute configurations. These glycoforms differ from what has been reported so far in the three domains of life. Considering that chloroviruses and other members of the family Phycodnaviridae may have a long evolutionary history, we suggest that the chlorovirus glycosylation pathway is ancient, possibly existing before the development of the endoplasmic reticulum and Golgi pathway, and involves still unexplored mechanisms.
Subject(s)
Capsid Proteins/chemistry , Chlorella/virology , DNA Ligases/chemistry , Oligosaccharides/chemistry , Paramecium/microbiology , Viral Proteins/chemistry , Capsid Proteins/isolation & purification , Capsid Proteins/metabolism , DNA Ligases/metabolism , Gas Chromatography-Mass Spectrometry , Magnetic Resonance Spectroscopy , Monosaccharides/chemistry , Oligosaccharides/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Viral Proteins/metabolismABSTRACT
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.
Subject(s)
Phycodnaviridae/chemistry , Polysaccharides/chemistry , Proton Magnetic Resonance Spectroscopy , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
In this chapter, we will present a high-throughput method applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. This methodology is based on a commercial equipment developed by WATERS™ to speed up N-deglycosylation and N-glycan labeling of glycoproteins of pharmaceutical and biological interest such as monoclonal antibodies. This analytical kit is sold under the trade name of RapiFluor-MS (RFMS). We have slightly modified the methodology, increasing the glycosylation time and using a high-resolution mass analyzer for the analysis of CSF N-glycans, thus obtaining a high-throughput method (up to 96 samples simultaneously), mass accuracy better than 5 ppm, and the ability to separate and identify isomers.
Subject(s)
Alzheimer Disease , Glycomics , Humans , Chromatography, High Pressure Liquid , Glycomics/methods , Alzheimer Disease/cerebrospinal fluid , Glycosylation , Glycoproteins/chemistry , Polysaccharides/chemistryABSTRACT
In this chapter, we will 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 (AD).
Subject(s)
Glycomics , Tandem Mass Spectrometry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Glycomics/methods , Glycoproteins/chemistry , Glycosylation , Polysaccharides/chemistryABSTRACT
Mucopolysaccharidoses (MPS) are lysosomal storage diseases caused by defects in catabolism of glycosaminoglycans. MPS I, II, III and VII are associated with lysosomal accumulation of heparan sulphate and manifest with neurological deterioration. Most of these neurological MPS currently lack effective treatments. Here, we report that, compared to controls, neuraminidase 1 (NEU1) activity is drastically reduced in brain tissues of neurological MPS patients and in mouse models of MPS I, II, IIIA, IIIB and IIIC, but not of other neurological lysosomal disorders not presenting with heparan sulphate storage. We further show that accumulated heparan sulphate disrupts the lysosomal multienzyme complex of NEU1 with cathepsin A (CTSA), ß-galactosidase (GLB1) and glucosamine-6-sulfate sulfatase (GALNS) necessary to maintain enzyme activity, and that NEU1 deficiency is linked to partial deficiencies of GLB1 and GALNS in cortical tissues and iPSC-derived cortical neurons of neurological MPS patients. Increased sialylation of N-linked glycans in brain samples of human MPS III patients and MPS IIIC mice implicated insufficient processing of brain N-linked sialylated glycans, except for polysialic acid, which was reduced in the brains of MPS IIIC mice. Correction of NEU1 activity in MPS IIIC mice by lentiviral gene transfer ameliorated previously identified hallmarks of the disease, including memory impairment, behavioural traits, and reduced levels of the excitatory synapse markers VGLUT1 and PSD95. Overexpression of NEU1 also restored levels of VGLUT1-/PSD95-positive puncta in cortical neurons derived from iPSC of an MPS IIIA patient. Together, our data demonstrate that heparan sulphate-induced secondary NEU1 deficiency and aberrant sialylation of glycoproteins implicated in synaptogenesis, memory, and behaviour constitute a novel pathological pathway in neurological MPS spectrum crucially contributing to CNS pathology.
ABSTRACT
This is the first report of the chemical and biological properties of the lipooligosaccharide (LOS) endotoxin isolated from Burkholderia dolosa IST4208, an isolate recovered from a cystic fibrosis (CF) patient in a Portuguese CF center. B. dolosa is a member of the Burkholderia cepacia complex, a group of closely related species that are highly problematic and opportunistic pathogens in CF. B. dolosa infection leads to accelerated loss of lung function and decreased survival. The structural determination of its endotoxin was achieved using a combination of chemistry and spectroscopy, and has revealed a novel endotoxin structure. The purified LOS was tested for its immunostimulatory activity on human HEK 293 cells expressing TLR-4, MD-2, and CD-14. In these assays, the LOS showed strong proinflammatory activity.
Subject(s)
Burkholderia cepacia complex/metabolism , Cystic Fibrosis/microbiology , Endotoxins/chemistry , Animals , Burkholderia cepacia complex/isolation & purification , Cytokines/metabolism , Endotoxins/isolation & purification , Endotoxins/pharmacology , Female , HEK293 Cells , Humans , Lipopolysaccharide Receptors/genetics , Lipopolysaccharide Receptors/metabolism , Lipopolysaccharides/chemistry , Lipopolysaccharides/isolation & purification , Lipopolysaccharides/pharmacology , Lymphocyte Antigen 96/genetics , Lymphocyte Antigen 96/metabolism , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , TransfectionABSTRACT
MUC1 protein overexpressed in human epithelial carcinoma is a target in development of novel anticancer vaccines. Multiple units of immunodominant B-cell epitope PDTRP MUC1 core sequence were conjugated to calix[4,8]arene platforms containing TLR2 ligand, to produce two novel anticancer self-adjuvant vaccine candidates. The immunogenicity of the synthetic constructs was investigated by immunization of mice in vivo. ELISA assay evidenced that the vaccine candidates stimulate anti MUC1 IgG antibody production (major for the octavalent construct) and no additive effect but a multivalency effect was observed when compared to an analogous monovalent. Octa- and tetravalent constructs lacking in PDTRP peptide moieties did not show anti MUC1 IgG antibody production in mice. The antibodies induced by the synthesized constructs are able to recognize the MUC1 structures present on MCF7 tumor cells. The results display that calixarenes are convenient platforms for building multicomponent self-adjuvant vaccine constructs promising as immunotherapeutic anticancer agents.
Subject(s)
Adjuvants, Immunologic/chemistry , Calixarenes/chemistry , Cancer Vaccines/chemistry , Immunodominant Epitopes/chemistry , Mucin-1/chemistry , Neoplasms/prevention & control , Adjuvants, Immunologic/therapeutic use , Animals , Antibody Formation , Calixarenes/therapeutic use , Cancer Vaccines/therapeutic use , Cell Line, Tumor , Female , Humans , Immunodominant Epitopes/therapeutic use , Immunoglobulin G/immunology , Mice , Mice, Inbred BALB C , Mucin-1/immunology , Mucin-1/therapeutic use , Neoplasms/immunology , Oligopeptides/chemistry , Oligopeptides/therapeutic use , Toll-Like Receptor 2/chemistryABSTRACT
Currently, the use of probiotic strains and their products represents a promising innovative approach as an antagonist treatment against many human diseases. Previous studies showed that a strain of Limosilactobacillus fermentum (LAC92), previously defined as Lactobacillus fermentum, exhibited a suitable amensalistic property. The present study aimed to purify the active components from LAC92 to evaluate the biological properties of soluble peptidoglycan fragments (SPFs). The cell-free supernatant (CFS) and bacterial cells were separated after 48 h of growth in MRS medium broth and treated for isolation of SPFs. Antimicrobial activity and proliferation analysis on the human cell line HTC116 were performed using technologies such as xCELLigence, count and viability, and clonogenic analysis. MALDI-MS investigation and docking analysis were performed to determine the molecular structure and hypothetical mode of action, respectively. Our results showed that the antimicrobial activity was mainly due to SPFs. Moreover, the results obtained when investigating the SPF effect on the cell line HCT116 showed substantial preliminary evidence, suggesting their significant cytostatic and quite antiproliferative properties. Although MALDI was unable to identify the molecular structure, it was subsequently revealed by analysis of the bacterial genome. The amino acid structure is called peptide 92. Furthermore, we confirmed by molecular docking studies the interaction of peptide 92 with MDM2 protein, the negative regulator of p53. This study showed that SPFs from the LAC92 strain exerted anticancer effects on the human colon cancer HCT116 cell line via antiproliferation and inducing apoptosis. These findings indicated that this probiotic strain might be a potential candidate for applications in functional products in the future. Further examination is needed to understand the specific advantages of this probiotic strain and improve its functional features to confirm these data. Moreover, deeper research on peptide 92 could increase our knowledge and help us understand if it will be possible to apply to specific diseases such as CRC.
ABSTRACT
Sialidosis is an ultra-rare multisystemic lysosomal disease caused by mutations in the neuraminidase 1 (NEU1) gene. The severe type II form of the disease manifests with a prenatal/infantile or juvenile onset, bone abnormalities, severe neuropathology, and visceromegaly. A subset of these patients present with nephrosialidosis, characterized by abrupt onset of fulminant glomerular nephropathy. We studied the pathophysiological mechanism of the disease in 2 NEU1-deficient mouse models, a constitutive Neu1-knockout, Neu1ΔEx3, and a conditional phagocyte-specific knockout, Neu1Cx3cr1ΔEx3. Mice of both strains exhibited terminal urinary retention and severe kidney damage with elevated urinary albumin levels, loss of nephrons, renal fibrosis, presence of storage vacuoles, and dysmorphic mitochondria in the intraglomerular and tubular cells. Glycoprotein sialylation in glomeruli, proximal distal tubules, and distal tubules was drastically increased, including that of an endocytic reabsorption receptor megalin. The pool of megalin bearing O-linked glycans with terminal galactose residues, essential for protein targeting and activity, was reduced to below detection levels. Megalin levels were severely reduced, and the protein was directed to lysosomes instead of the apical membrane. Together, our results demonstrated that desialylation by NEU1 plays a crucial role in processing and cellular trafficking of megalin and that NEU1 deficiency in sialidosis impairs megalin-mediated protein reabsorption.
Subject(s)
Kidney Diseases , Mucolipidoses , Animals , Humans , Mice , Kidney Diseases/metabolism , Kidney Glomerulus/metabolism , Kidney Tubules, Proximal/metabolism , Low Density Lipoprotein Receptor-Related Protein-2/genetics , Mucolipidoses/genetics , Mucolipidoses/pathology , Neuraminidase/geneticsABSTRACT
Glycosylation is a fundamental post-translational modification of proteins that boosts their structural diversity providing subtle and specialized biological properties and functions. All those genetic diseases due to a defective glycan biosynthesis and attachment to the nascent glycoproteins fall within the wide area of congenital disorders of glycosylation (CDG), mostly causing multisystem involvement. In the present paper, we detailed the unique serum N-glycosylation of a CDG-candidate patient with an unexplained neurological phenotype and liver adenomatosis harboring a recurrent pathogenic HNF1α variant. Serum transferrin isoelectric focusing showed a surprising N-glycosylation pattern consisting on hyposialylation, as well as remarkable hypersialylation. Mass spectrometry-based glycomic analyses of individual serum glycoproteins enabled to unveil hypersialylated complex N-glycans comprising up to two sialic acids per antenna. Further advanced MS analysis showed the additional sialic acid is bonded through an α2-6 linkage to the peripheral N-acetylglucosamine residue.