Publications by authors named "Yann Guerardel"

137 Publications

Macrobrachium rosenbergii nodavirus virus-like particles attach to fucosylated glycans in the gills of the giant freshwater prawn.

Cell Microbiol 2020 12 15;22(12):e13258. Epub 2020 Sep 15.

Department of Anatomy, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand.

The Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white-tail disease (WTD) in many species of shrimp and prawn, has been shown to infect hemocytes and tissues such as the gills and muscles. However, little is known about the host surface molecules to which MrNV attach to initiate infection. Therefore, the present study investigated the role of glycans as binding molecules for virus attachment in susceptible tissues such as the gills. We established that MrNV in their virus-like particle (MrNV-VLP) form exhibited strong binding to gill tissues and lysates, which was highly reduced by the glycan-reducing periodate and PNGase F. The broad, fucose-binding Aleuria Aurantia lectin (AAL) highly reduced MrNV-VLPs binding to gill tissue sections and lysates, and efficiently disrupted the specific interactions between the VLPs and gill glycoproteins. Furthermore, mass spectroscopy revealed the existence of unique fucosylated LacdiNAc-extended N-linked and O-linked glycans in the gill tissues, whereas beta-elimination experiments showed that MrNV-VLPs demonstrated a binding preference for N-glycans. Therefore, the results from this study highly suggested that MrNV-VLPs preferentially attach to fucosylated N-glycans in the susceptible gill tissues, and these findings could lead to the development of strategies that target virus-host surface glycan interactions to reduce MrNV infections.
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http://dx.doi.org/10.1111/cmi.13258DOI Listing
December 2020

-Methylation of the Glycopeptidolipid Acyl Chain Defines Surface Hydrophobicity of and Macrophage Invasion.

ACS Infect Dis 2020 10 14;6(10):2756-2770. Epub 2020 Sep 14.

Centre National de la Recherche Scientifique UMR 9004, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, 1919 route de Mende, 34293 Montpellier, France.

, an emerging pathogen responsible for severe lung infections in cystic fibrosis patients, displays either smooth (S) or rough (R) morphotypes. The S-to-R transition is associated with reduced levels of glycopeptidolipid (GPL) production and is correlated with increased pathogenicity in animal and human hosts. While the structure of GPL is well established, its biosynthetic pathway is incomplete. In addition, the biological functions of the distinct structural parts of this complex lipid remain elusive. Herein, the gene encoding a putative -methyltransferase was deleted in the S variant. Subsequent biochemical and structural analyses demonstrated that methoxylation of the fatty acyl chain of GPL was abrogated in the mutant, and this defect was rescued upon complementation with a functional gene. In contrast, the introduction of derivatives mutated at residues essential for methyltransferase activity failed to complement GPL defects, indicating that encodes an -methyltransferase. Unexpectedly, phenotypic analyses showed that was more hydrophilic than its parental progenitor, as demonstrated by hexadecane-aqueous buffer partitioning and atomic force microscopy experiments with hydrophobic probes. Importantly, the invasion rate of THP-1 macrophages by was reduced by 50% when compared to the wild-type strain. Together, these results indicate that Fmt -methylates the lipid moiety of GPL and plays a substantial role in conditioning the surface hydrophobicity of as well as in the early steps of the interaction between the bacilli and macrophages.
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http://dx.doi.org/10.1021/acsinfecdis.0c00490DOI Listing
October 2020

The Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis.

mBio 2020 08 18;11(4). Epub 2020 Aug 18.

University Lille, CNRS, INRAE, Centrale Lille, UMR 8207-UMET-Unité Matériaux et Transformations, Lille, France.

The crust is the outermost spore layer of most strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens and We demonstrated that the , , , , and genes of are required for Leg biosynthesis during sporulation, while the gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge. species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in adhesion and persistence in the food industry.
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http://dx.doi.org/10.1128/mBio.01153-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439481PMC
August 2020

Complete Structure of the Enterococcal Polysaccharide Antigen (EPA) of Vancomycin-Resistant Enterococcus faecalis V583 Reveals that EPA Decorations Are Teichoic Acids Covalently Linked to a Rhamnopolysaccharide Backbone.

mBio 2020 04 28;11(2). Epub 2020 Apr 28.

Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas, France

All enterococci produce a complex polysaccharide called the nterococcal olysaccharide ntigen (EPA). This polymer is required for normal cell growth and division and for resistance to cephalosporins and plays a critical role in host-pathogen interaction. The EPA contributes to host colonization and is essential for virulence, conferring resistance to phagocytosis during the infection. Recent studies revealed that the "decorations" of the EPA polymer, encoded by genetic loci that are variable between isolates, underpin the biological activity of this surface polysaccharide. In this work, we investigated the structure of the EPA polymer produced by the high-risk enterococcal clonal complex V583. We analyzed purified EPA from the wild-type strain and a mutant lacking decorations and elucidated the structure of the EPA backbone and decorations. We showed that the rhamnan backbone of EPA is composed of a hexasaccharide repeat unit of C2- and C3-linked rhamnan chains, partially substituted in the C3 position by α-glucose (α-Glc) and in the C2 position by β--acetylglucosamine (β-GlcNAc). The so-called "EPA decorations" consist of phosphopolysaccharide chains corresponding to teichoic acids covalently bound to the rhamnan backbone. The elucidation of the complete EPA structure allowed us to propose a biosynthetic pathway, a first essential step toward the design of antimicrobials targeting the synthesis of this virulence factor. Enterococci are opportunistic pathogens responsible for hospital- and community-acquired infections. All enterococci produce a surface polysaccharide called EPA (nterococcal olysaccharide ntigen) required for biofilm formation, antibiotic resistance, and pathogenesis. Despite the critical role of EPA in cell growth and division and as a major virulence factor, no information is available on its structure. Here, we report the complete structure of the EPA polymer produced by the model strain V583. We describe the structure of the EPA backbone, made of a rhamnan hexasaccharide substituted by Glc and GlcNAc residues, and show that teichoic acids are covalently bound to this rhamnan chain, forming the so-called "EPA decorations" essential for host colonization and pathogenesis. This report represents a key step in efforts to identify the structural properties of EPA that are essential for its biological activity and to identify novel targets to develop preventive and therapeutic approaches against enterococci.
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http://dx.doi.org/10.1128/mBio.00277-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188991PMC
April 2020

Mycobacterium bovis BCG infection alters the macrophage N-glycome.

Mol Omics 2020 08 9;16(4):345-354. Epub 2020 Apr 9.

Univ. Lille, CNRS UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, 59 000 Lille, France.

Macrophage glycosylation is essential to initiate the host-immune defense but may also be targeted by pathogens to promote infection. Indeed, the alteration of the cell-surface glycosylation status may affect the binding of lectins involved in cell activation and adhesion. Herein, we demonstrate that infection by M. bovis BCG induces the remodeling of the N-glycomes of both human primary blood monocyte-derived macrophages (MDM) and macrophage-cell line THP1. MALDI-MS based N-glycomic analysis established that mycobacterial infection induced increased synthesis of biantennary and multifucosylated complex type N-glycans. In contrast, infection of macrophages by M. bovis BCG did not modify the glycosphingolipids composition of macrophages. Further nano-LC-MS glycotope-centric analysis of total N-glycans demonstrated that the increased fucosylation was due to an increased expression of the Le (Galβ1-4[Fucα1-3]GlcNAc) epitope, also known as stage-specific embryonic antigen-1. Modification of the surface expression of Le was further confirmed in both MDM and THP-1 cells by FACS analysis using an α1,3-linked fucose specific lectin. Activation with the mycobacterial lipopeptide Pam3Lp19, an agonist of toll-like receptor 2, did not modify the overall fucosylation pattern, which suggests that the infection process is required to modify surface glycosylation. These results pave the way toward the understanding of infection-triggered cell-surface remodeling of macrophages.
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http://dx.doi.org/10.1039/c9mo00173eDOI Listing
August 2020

Deletion of the Zinc Transporter Lipoprotein AdcAII Causes Hyperencapsulation of Streptococcus pneumoniae Associated with Distinct Alleles of the Type I Restriction-Modification System.

mBio 2020 03 31;11(2). Epub 2020 Mar 31.

Centre for Inflammation and Tissue Repair, Department of Medicine, Royal Free and University College Medical School, Rayne Institute, London, United Kingdom.

The capsule is the dominant virulence factor, yet how variation in capsule thickness is regulated is poorly understood. Here, we describe an unexpected relationship between mutation of , which encodes a zinc uptake lipoprotein, and capsule thickness. Partial deletion of in three of five capsular serotypes frequently resulted in a mucoid phenotype that biochemical analysis and electron microscopy of the D39 mutants confirmed was caused by markedly increased capsule thickness. Compared to D39, the hyperencapsulated mutant strain was more resistant to complement-mediated neutrophil killing and was hypervirulent in mouse models of invasive infection. Transcriptome analysis of D39 and the mutant identified major differences in transcription of the Sp_0505-0508 locus, which encodes an SpnD39III (ST5556II) type I restriction-modification system and allelic variation of which correlates with capsule thickness. A PCR assay demonstrated close linkage of the SpnD39IIIC and F alleles with the hyperencapsulated strains. However, transformation of with fixed SpnD39III alleles associated with normal capsule thickness did not revert the hyperencapsulated phenotype. Half of hyperencapsulated strains contained the same single nucleotide polymorphism in the capsule locus gene , which is required for the initiation of capsule synthesis. These results provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identified an unexpected linkage between capsule thickness and mutation of Further investigation will be needed to characterize how mutation of affects SpnD39III (ST5556II) allele dominance and results in the hyperencapsulated phenotype. The capsule affects multiple interactions with the host including contributing to colonization and immune evasion. During infection, the capsule thickness varies, but the mechanisms regulating this are poorly understood. We have identified an unsuspected relationship between mutation of , a gene that encodes a zinc uptake lipoprotein, and capsule thickness. Mutation of resulted in a striking hyperencapsulated phenotype, increased resistance to complement-mediated neutrophil killing, and increased virulence in mouse models of infection. Transcriptome and PCR analysis linked the hyperencapsulated phenotype of the strain to specific alleles of the SpnD39III (ST5556II) type I restriction-modification system, a system which has previously been shown to affect capsule thickness. Our data provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identify an unexpected link between capsule thickness and , further investigation of which could further characterize mechanisms of capsule regulation.
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http://dx.doi.org/10.1128/mBio.00445-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157770PMC
March 2020

Structure-Based Design and Synthesis of Piperidinol-Containing Molecules as New Inhibitors.

ChemistryOpen 2020 03 20;9(3):351-365. Epub 2020 Mar 20.

Univ. Lille, CNRS UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle 59000 Lille France.

Non-tuberculous mycobacterium (NTM) infections, such as those caused by , are increasing globally. Due to their intrinsic drug resistance, pulmonary infections are often difficult to cure using standard chemotherapy. We previously demonstrated that a piperidinol derivative, named PIPD1, is an efficient molecule both against and , the agent of tuberculosis, by targeting the mycolic acid transporter MmpL3. These results prompted us to design and synthesize a series of piperidinol derivatives and to determine the biological activity against . Structure-activity relationship (SAR) studies pointed toward specific sites on the scaffold that can tolerate slight modifications. Overall, these results identified FMD-88 as a new promising active analogue against . Also, we determined the pharmacokinetics properties of PIPD1 and showed that intraperitoneal administration of this compound resulted in promising serum concentration and an elimination half-life of 3.2 hours.
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http://dx.doi.org/10.1002/open.202000042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7083170PMC
March 2020

The endogenous galactofuranosidase GlfH1 hydrolyzes mycobacterial arabinogalactan.

J Biol Chem 2020 04 27;295(15):5110-5123. Epub 2020 Feb 27.

Univ. Lille, CNRS, UMR8576 - UGSF - Unit[c33c]zpi;● de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France

Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus adapts its cell wall structure and composition to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes, but no reports exist supporting this assumption. Herein, using recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we found that the mycobacterial GlfH1 (Rv3096) protein exhibits exo-β-d-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal β-(1,5) and β-(1,6)-Galf linkages. The characterization of this galactosidase represents a first step toward understanding the remodeling of mycobacterial AG.
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http://dx.doi.org/10.1074/jbc.RA119.011817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152746PMC
April 2020

Profiling of -acetylated Gangliosides Expressed in Neuroectoderm Derived Cells.

Int J Mol Sci 2020 Jan 6;21(1). Epub 2020 Jan 6.

Univ. Lille, CNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France.

The expression and biological functions of oncofetal markers GD2 and GD3 were extensively studied in neuroectoderm-derived cancers in order to characterize their potential as therapeutic targets. Using immunological approaches, we previously identified GD3, GD2, and AcGD2 expression in breast cancer (BC) cell lines. However, antibodies specific for -acetylated gangliosides are not exempt of limitations, as they only provide information on the expression of a limited set of -acetylated ganglioside species. Consequently, the aim of the present study was to use structural approaches in order to apprehend ganglioside diversity in melanoma, neuroblastoma, and breast cancer cells, focusing on -acetylated species that are usually lost under alkaline conditions and require specific analytical procedures. We used purification and extraction methods that preserve the -acetyl modification for the analysis of native gangliosides by MALDI-TOF. We identified the expression of GM1, GM2, GM3, GD2, GD3, GT2, and GT3 in SK-Mel28 (melanoma), LAN-1 (neuroblastoma), Hs 578T, SUM 159PT, MDA-MB-231, MCF-7 (BC), and BC cell lines over-expressing GD3 synthase. Among -acetylated gangliosides, we characterized the expression of AcGM1, AcGD3, AcGD2, AcGT2, and AcGT3. Furthermore, the experimental procedure allowed us to clearly identify the position of the sialic acid residue that carries the -acetyl group on b- and c-series gangliosides by MS/MS fragmentation. These results show that ganglioside -acetylation occurs on both inner and terminal sialic acid residue in a cell type-dependent manner, suggesting different -acetylation pathways for gangliosides. They also highlight the limitation of immuno-detection for the complete identification of -acetylated ganglioside profiles in cancer cells.
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http://dx.doi.org/10.3390/ijms21010370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981417PMC
January 2020

Involvement of ST6Gal I-mediated α2,6 sialylation in myoblast proliferation and differentiation.

FEBS Open Bio 2020 01 10;10(1):56-69. Epub 2019 Dec 10.

PEIRENE, EA 7500, Glycosylation and Cell Differentiation, University of Limoges, France.

Myogenesis is a physiological process which involves the proliferation of myoblasts and their differentiation into multinucleated myotubes, which constitute the future muscle fibers. Commitment of myoblasts to differentiation is regulated by the balance between the myogenic factors Pax7 and MyoD. The formation of myotubes requires the presence of glycans, especially N-glycans, on the cell surface. We examined here the involvement of α2,6 sialylation during murine myoblastic C2C12 cell differentiation by generating a st6gal1-knockdown C2C12 cell line; these cells exhibit reduced proliferative potential and precocious differentiation due to the low expression of Pax7. The earlier fusion of st6gal1-knockdown cells leads to a high fusion index and a drop in reserve cells (Pax7 /MyoD ). In st6gal1-knockdown cells, the Notch pathway is inactivated; consequently, Pax7 expression is virtually abolished, leading to impairment of the proliferation rate. All these results indicate that the decrease in α2,6 sialylation of N-glycans favors the differentiation of most cells and provokes a significant loss of reserve cells.
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http://dx.doi.org/10.1002/2211-5463.12745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6943236PMC
January 2020

A piperidinol-containing molecule is active against by inhibiting the mycolic acid flippase activity of MmpL3.

J Biol Chem 2019 11 27;294(46):17512-17523. Epub 2019 Sep 27.

Centre National de la Recherche Scientifique UMR9004, Institut de Recherche en Infectiologie de Montpellier, Université de Montpellier, 34293 Montpellier, France

, the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat because of the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against Herein, we show that PIPD1 and related analogs exert bactericidal activity against the strain mc6230 and also against a panel of multidrug-resistant and extensively drug-resistant clinical isolates of , suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of single-nucleotide polymorphisms in , encoding an inner membrane-associated mycolic acid flippase in Results from functional assays with spheroplasts derived from a strain lacking the endogenous gene but harboring the homolog indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate across the inner membrane without altering the proton motive force. Using a predictive structural model of MmpL3 from , docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria.
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http://dx.doi.org/10.1074/jbc.RA119.010135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873192PMC
November 2019

Interaction of a Densovirus with Glycans of the Peritrophic Matrix Mediates Oral Infection of the Lepidopteran Pest .

Viruses 2019 09 17;11(9). Epub 2019 Sep 17.

Diversité des Génomes et Interactions Microorganismes Insectes (DGIMI), Univ Montpellier, INRA, 34095 Montpellier, France.

The success of oral infection by viruses depends on their capacity to overcome the gut epithelial barrier of their host to crossing over apical, mucous extracellular matrices. As orally transmitted viruses, densoviruses, are also challenged by the complexity of the insect gut barriers, more specifically by the chitinous peritrophic matrix, that lines and protects the midgut epithelium; how capsids stick to and cross these barriers to reach their final cell destination where replication goes has been poorly studied in insects. Here, we analyzed the early interaction of the densovirus (JcDV) with the midgut barriers of caterpillars from the pest . Using combination of imaging, biochemical, proteomic and transcriptomic analyses, we examined in vitro, ex vivo and in vivo the early interaction of the capsids with the peritrophic matrix and the consequence of early oral infection on the overall gut function. We show that the JcDV particle rapidly adheres to the peritrophic matrix through interaction with different glycans including chitin and glycoproteins, and that these interactions are necessary for oral infection. Proteomic analyses of JcDV binding proteins of the peritrophic matrix revealed mucins and non-mucins proteins including enzymes already known to act as receptors for several insect pathogens. In addition, we show that JcDV early infection results in an arrest of -Acetylglucosamine secretion and a disruption in the integrity of the peritrophic matrix, which may help viral particles to pass through. Finally, JcDV early infection induces changes in midgut genes expression favoring an increased metabolism including an increased translational activity. These dysregulations probably participate to the overall dysfunction of the gut barrier in the early steps of viral pathogenesis. A better understanding of early steps of densovirus infection process is crucial to build biocontrol strategies against major insect pests.
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http://dx.doi.org/10.3390/v11090870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783882PMC
September 2019

OGT Controls the Expression and the Glycosylation of E-cadherin, and Affects Glycosphingolipid Structures in Human Colon Cell Lines.

Proteomics 2019 11 20;19(21-22):e1800452. Epub 2019 Aug 20.

Université de Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, F-59000, Lille, France.

Colorectal cancer (CRC) affects both women and men living in societies with a high sedentary lifestyle. Amongst the phenotypic changes exhibited by tumor cells, a wide range of glycosylation has been reported for colon cancer-derived cell lines and CRC tissues. These aberrant modifications affect different aspects of glycosylation, including an increase in core fucosylation and GlcNAc branching on N-glycans, alteration of O-glycans, upregulated sialylation, and O-GlcNAcylation. Although O-GlcNAcylation and complex glycosylations differ in many aspects, sparse evidences report on the interference of O-GlcNAcylation with complex glycosylation. Nevertheless, this relationship is still a matter of debate. Combining different approaches on three human colon cell lines (HT29, HCT116 and CCD841CoN), it is herein reported that silencing O-GlcNAc transferase (OGT, the sole enzyme driving O-GlcNAcylation), only slightly affects overall N- and O-glycosylation patterns. Interestingly, silencing of OGT in HT29 cells upregulates E-cadherin (a major actor of epithelial-to-mesenchymal transition) and changes its glycosylation. On the other hand, OGT silencing perturbs biosynthesis of glycosphingolipids resulting in a decrease in gangliosides and an increase in globosides. Together, these results provide novel insights regarding the selective regulation of complex glycosylations by O-GlcNAcylation in colon cancer cells.
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http://dx.doi.org/10.1002/pmic.201800452DOI Listing
November 2019

Simple Protocol to Purify Cell Wall Polysaccharide from Gram-Positive Bacteria and Assess Its Structural Integrity.

Methods Mol Biol 2019 ;1954:37-45

Univ. Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France.

Cell wall polysaccharides (CWPS), which are usually covalently bound to the peptidoglycan and are closely associated with the cell wall, are considered as ubiquitous components of the cell envelope of gram-positive bacteria and play an important role as mediators of bacterial interactions with the environment. Here, we describe a simple method for purifying CWPS by extraction of bacterial cells with consecutive acid treatments. Purified CWPS are obtained by gel-filtration chromatography following treatment with HF. We also provide the methodology to easily assess the integrity of CWPS using high-resolution magic-angle spinning (HR-MAS) NMR.
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http://dx.doi.org/10.1007/978-1-4939-9154-9_4DOI Listing
July 2019

Evaluation of Mass Spectrometry-Based Detection of Panfungal Serum Disaccharide for Diagnosis of Invasive Fungal Infections: Results from a Collaborative Study Involving Six European Clinical Centers.

J Clin Microbiol 2019 05 26;57(5). Epub 2019 Apr 26.

Laboratoire de Parasitologie Mycologie, CHU Lille, Université Lille, INSERM U995-LIRIC (Lille Inflammation Research International Centre), Lille, France

A mass spectrometry (MS) method that detects a serum disaccharide (DS) (MS-DS) was recently described for the diagnosis of invasive fungal infections (IFI). We carried out a European collaborative study to evaluate this assay. Patients with the following IFI were selected according to the availability of sera obtained at about the time that IFI was documented: invasive candidiasis (IC;  = 26 patients), invasive aspergillosis (IA;  = 19), and mucormycosis (MM;  = 23). Control sera originated from 20 neutropenic patients and 20 patients with bacteremia. MS-DS was carried out in blind manner for the diagnosis of IFI. A diagnosis of IC or IA was confirmed by detection of mannan (Man) or galactomannan (GM), respectively, associated with detection of (1,3)-β-d-glucan (BDG) in both infections. MM was detected by quantitative real-time PCR (qPCR). All tests discriminated sera from patients with IC from sera from control subjects with bacteremia ( ≤ 0.0009). For IC, the MS-DS sensitivity and specificity were 51% and 87%, respectively. MS-DS complemented the high specificity of Man monitoring. All tests discriminated sera from IA patients from sera from neutropenic controls ( ≤ 0.0009). For IA, MS-DS sensitivity and specificity were 64% and 95%, respectively. Only 13/36 serum samples from patients with MM were concordant by MS-DS and qPCR (6 were positive, and 7 were negative); 14 were positive by MS-DS alone. qPCR and MS-DS made a similar contribution to the diagnosis of MM. In patients undergoing long-term monitoring, the persistent circulation of serum disaccharide was observed, whereas DNA was detected only for a short period after initiation of treatment. MS-DS has an important role to play in the early diagnosis of IFI. Its panfungal nature and complementarity with other tests may justify its use in the management of IFI.
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http://dx.doi.org/10.1128/JCM.01867-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498025PMC
May 2019

Novel Zebrafish Mono-α2,8-sialyltransferase (ST8Sia VIII): An Evolutionary Perspective of α2,8-Sialylation.

Int J Mol Sci 2019 Jan 31;20(3). Epub 2019 Jan 31.

Université de Lille, CNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France.

The mammalian mono-α2,8-sialyltransferase ST8Sia VI has been shown to catalyze the transfer of a unique sialic acid residues onto core 1 -glycans leading to the formation of di-sialylated -glycosylproteins and to a lesser extent to diSia motifs onto glycolipids like GD1a. Previous studies also reported the identification of an orthologue of the gene in the zebrafish genome. Trying to get insights into the biosynthesis and function of the oligo-sialylated glycoproteins during zebrafish development, we cloned and studied this fish α2,8-sialyltransferase homologue. In situ hybridization experiments demonstrate that expression of this gene is always detectable during zebrafish development both in the central nervous system and in non-neuronal tissues. Intriguingly, using biochemical approaches and the newly developed in vitro MicroPlate Sialyltransferase Assay (MPSA), we found that the zebrafish recombinant enzyme does not synthetize diSia motifs on glycoproteins or glycolipids as the human homologue does. Using comparative genomics and molecular phylogeny approaches, we show in this work that the human ST8Sia VI orthologue has disappeared in the ray-finned fish and that the homologue described in fish correspond to a new subfamily of α2,8-sialyltransferase named ST8Sia VIII that was not maintained in Chondrichtyes and Sarcopterygii.
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http://dx.doi.org/10.3390/ijms20030622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6387029PMC
January 2019

Structure and biological activities of a hexosamine-rich cell wall polysaccharide isolated from the probiotic Lactobacillus farciminis.

Glycoconj J 2019 02 12;36(1):39-55. Epub 2019 Jan 12.

Toxalim (Research Centre in Food Toxicology), INRA, ENVT, INP-Purpan, UPS, Université de Toulouse, Toulouse, France.

Lactobacillus farciminis CIP 103136 is a bacterial strain with recognized probiotic properties. However, the mechanisms underlying such properties have only been partially elucidated. In this study, we isolated and purified a cell-wall associated polysaccharide (CWPS), and evaluated its biological role in vitro. The structure of CWPS and responses from stimulation of (i) human macrophage-like THP-1 cells, (ii) human embryonal kidney (HEK293) cells stably transfected with Toll-like receptors (TLR2 or TLR4) and (iii) human colonocyte-like T84 intestinal epithelial cells, upon exposure to CWPS were studied. The structure of the purified CWPS from L. farciminis CIP 103136 was analyzed by nuclear magnetic resonance (NMR), MALDI-TOF-TOF MS, and methylation analyses in its native form and following Smith degradation. It was shown to be a novel branched polysaccharide, composed of linear backbone of trisaccharide repeating units of: [→6αGlcpNAc1 → 4βManpNAc1 → 4βGlcpNAc1→] highly substituted with single residues of αGlcp, αGalp and αGlcpNAc. Subsequently, the lack of pro- or anti-inflammatory properties of CWPS was established on macrophage-like THP-1 cells. In addition, CWPS failed to modulate cell signaling pathways dependent of TLR2 and TLR4 in transfected HEK-cells. Finally, in T84 cells, CWPS neither influenced intestinal barrier integrity under basal conditions nor prevented TNF-α/IFN-γ cytokine-mediated epithelium impairment.
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http://dx.doi.org/10.1007/s10719-018-09854-yDOI Listing
February 2019

GORAB scaffolds COPI at the trans-Golgi for efficient enzyme recycling and correct protein glycosylation.

Nat Commun 2019 01 10;10(1):127. Epub 2019 Jan 10.

School of Biology, Faculty of Biology, Medicine and Health, University of Manchester, The Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK.

COPI is a key mediator of protein trafficking within the secretory pathway. COPI is recruited to the membrane primarily through binding to Arf GTPases, upon which it undergoes assembly to form coated transport intermediates responsible for trafficking numerous proteins, including Golgi-resident enzymes. Here, we identify GORAB, the protein mutated in the skin and bone disorder gerodermia osteodysplastica, as a component of the COPI machinery. GORAB forms stable domains at the trans-Golgi that, via interactions with the COPI-binding protein Scyl1, promote COPI recruitment to these domains. Pathogenic GORAB mutations perturb Scyl1 binding or GORAB assembly into domains, indicating the importance of these interactions. Loss of GORAB causes impairment of COPI-mediated retrieval of trans-Golgi enzymes, resulting in a deficit in glycosylation of secretory cargo proteins. Our results therefore identify GORAB as a COPI scaffolding factor, and support the view that defective protein glycosylation is a major disease mechanism in gerodermia osteodysplastica.
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http://dx.doi.org/10.1038/s41467-018-08044-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328613PMC
January 2019

Identification of 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac) as main O-acetylated sialic acid species of GD2 in breast cancer cells.

Glycoconj J 2019 02 5;36(1):79-90. Epub 2019 Jan 5.

CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, University Lille, F-59000, Lille, France.

Mainly restricted to the nervous system in healthy adults, complex gangliosides such as GD3 and GD2 have been shown to be involved in aggressiveness and metastasis of neuro-ectoderm derived tumors such as melanoma and neuroblastoma. Interestingly, O-acetylated forms of GD2, not expressed in human peripheral nerve fibers, are highly expressed in GD2+ tumor cells. Very little information is known regarding the expression of O-acetylated disialogangliosides in breast cancer (BC) cell lines. Here, we analyzed the expression of GD2, GD3 and their O-acetylated forms O-acetyl-GD2 (OAcGD2) and O-acetyl-GD3 (OAcGD3) in BC cells. We used Hs 578T and SUM159PT cell lines, as well as cell clones over-expressing GD3 synthase derived from MDA-MB-231 and MCF-7. Using flow cytometry and immunocytochemistry/confocal microscopy, we report that BC cells express b-series gangliosides GD3 and GD2, as well as significant amounts of OAcGD2. However, OAcGD3 expression was not detected in these cells. O-acetylation of gangliosides isolated from BC cells was examined by LC-MS analysis of sialic acid DMB-derivatives. We report that the main acetylated form of sialic acid expressed in BC gangliosides is 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac). These results highlight a close interrelationship between Neu5,9Ac and OAcGD2 expression, and suggest that OAcGD2 is synthetized from GD2 and not from OAcGD3 in BC cells.
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http://dx.doi.org/10.1007/s10719-018-09856-wDOI Listing
February 2019

Systems glycomics of adult zebrafish identifies organ-specific sialylation and glycosylation patterns.

Nat Commun 2018 11 7;9(1):4647. Epub 2018 Nov 7.

Université de Lille, CNRS, UMR 8576 - UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F- 59000, Lille, France.

The emergence of zebrafish Danio rerio as a versatile model organism provides the unique opportunity to monitor the functions of glycosylation throughout vertebrate embryogenesis, providing insights into human diseases caused by glycosylation defects. Using a combination of chemical modifications, enzymatic digestion and mass spectrometry analyses, we establish here the precise glycomic profiles of eight individual zebrafish organs and demonstrate that the protein glycosylation and glycosphingolipid expression patterns exhibits exquisite specificity. Concomitant expression screening of a wide array of enzymes involved in the synthesis and transfer of sialic acids shows that the presence of organ-specific sialylation motifs correlates with the localized activity of the corresponding glycan biosynthesis pathways. These findings provide a basis for the rational design of zebrafish lines expressing desired glycosylation profiles.
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http://dx.doi.org/10.1038/s41467-018-06950-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220181PMC
November 2018

The Many Ways by Which -GlcNAcylation May Orchestrate the Diversity of Complex Glycosylations.

Molecules 2018 Nov 2;23(11). Epub 2018 Nov 2.

Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, CNRS, UMR 8576, UGSF, 59000 Lille, France.

Unlike complex glycosylations, -GlcNAcylation consists of the addition of a single -acetylglucosamine unit to serine and threonine residues of target proteins, and is confined within the nucleocytoplasmic and mitochondrial compartments. Nevertheless, a number of clues tend to show that -GlcNAcylation is a pivotal regulatory element of its complex counterparts. In this perspective, we gather the evidence reported to date regarding this connection. We propose different levels of regulation that encompass the competition for the nucleotide sugar UDP-GlcNAc, and that control the wide class of glycosylation enzymes via their expression, catalytic activity, and trafficking. We sought to better envision that nutrient fluxes control the elaboration of glycans, not only at the level of their structure composition, but also through sweet regulating actors.
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http://dx.doi.org/10.3390/molecules23112858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6278486PMC
November 2018

MmpL8 controls virulence and production of a previously unknown glycolipid family.

Proc Natl Acad Sci U S A 2018 10 9;115(43):E10147-E10156. Epub 2018 Oct 9.

Université de Versailles Saint Quentin en Yvelines, INSERM UMR1173, 78000 Versailles, France;

is a peculiar rapid-growing (RGM) capable of surviving within eukaryotic cells thanks to an arsenal of virulence genes also found in slow-growing mycobacteria (SGM), such as A screen based on the intracellular survival in amoebae and macrophages (MΦ) of an transposon mutant library revealed the important role of MAB_0855, a yet uncharacterized Mycobacterial membrane protein Large (MmpL). Large-scale comparisons with SGM and RGM genomes uncovered MmpL12 proteins as putative orthologs of MAB_0855 and a locus-scale synteny between the and loci. A KO mutant of the gene, designated herein as , had impaired adhesion to MΦ and displayed a decreased intracellular viability. Despite retaining the ability to block phagosomal acidification, like the WT strain, the mutant was delayed in damaging the phagosomal membrane and in making contact with the cytosol. Virulence attenuation of the mutant was confirmed in vivo by impaired zebrafish killing and a diminished propensity to induce granuloma formation. The previously shown role of MmpL in lipid transport prompted us to investigate the potential lipid substrates of MmpL8 Systematic lipid analysis revealed that MmpL8 was required for the proper expression of a glycolipid entity, a glycosyl diacylated nonadecyl diol (GDND) alcohol comprising different combinations of oleic and stearic acids. This study shows the importance of MmpL8 in modifying interactions between the bacteria and phagocytic cells and in the production of a previously unknown glycolipid family.
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http://dx.doi.org/10.1073/pnas.1812984115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205491PMC
October 2018

MicroPlate Sialyltransferase Assay: A Rapid and Sensitive Assay Based on an Unnatural Sialic Acid Donor and Bioorthogonal Chemistry.

Bioconjug Chem 2018 10 20;29(10):3377-3384. Epub 2018 Sep 20.

Universite Lille , CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, F 59000 Lille , France.

Mammalian sialyltransferases transfer sialic acids onto glycoproteins and glycolipids within the Golgi apparatus. Despite their key role in glycosylation, the study of their enzymatic activities is limited by the lack of appropriate tools. Herein, we developed a quick and sensitive sialyltransferase microplate assay based on the use of the unnatural CMP-SiaNAl donor substrate. In this assay, an appropriate acceptor glycoprotein is coated on the bottom of 96-well plate and the sialyltransferase activity is assessed using CMP-SiaNAl. The alkyne tag of SiaNAl enables subsequent covalent ligation of an azido-biotin probe via CuAAC and an antibiotin-HRP conjugated antibody is then used to quantify the amount of transferred SiaNAl by a colorimetric titration. With this test, we evaluated the kinetic characteristics and substrate preferences of two human sialyltransferases, ST6Gal I and ST3Gal I toward a panel of asialoglycoprotein acceptors, and identified cations that display a sialyltransferase inhibitory effect.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00529DOI Listing
October 2018

Chemical glycomics enrichment: imaging the recycling of sialic acid in living cells.

J Inherit Metab Dis 2018 05 2;41(3):515-523. Epub 2018 Jan 2.

University Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, F-59000, Lille, France.

The development of metabolic oligosaccharide engineering (MOE) over the past two decades enabled the bioimaging studies of glycosylation processes in physio-pathological contexts. Herein, we successfully applied the chemical reporter strategy to image the fate of sialylated glycoconjugates in healthy and sialin-deficient patient fibroblasts. This chemical glycomics enrichment is a powerful tool for tracking sialylated glycoconjugates and probing lysosomal recycling capacities. Thus, such strategies appear fundamental for the characterization of lysosomal storage diseases.
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http://dx.doi.org/10.1007/s10545-017-0118-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959963PMC
May 2018

Investigating the function of Gdt1p in yeast Golgi glycosylation.

Biochim Biophys Acta Gen Subj 2018 Mar 3;1862(3):394-402. Epub 2017 Nov 3.

Univ. Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France; LIA GLYCOLAB4CDG France/Belgium (International Associated Laboratory "Laboratory for the Research on Congenital Disorders of Glycosylation - from cellular mechanisms to cure", France. Electronic address:

The Golgi ion homeostasis is tightly regulated to ensure essential cellular processes such as glycosylation, yet our understanding of this regulation remains incomplete. Gdt1p is a member of the conserved Uncharacterized Protein Family (UPF0016). Our previous work suggested that Gdt1p may function in the Golgi by regulating Golgi Ca/Mn homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdt1Δ mutant cultured in presence of high Ca concentration, as well as the pmr1Δ and gdt1Δ/pmr1Δ strains presented strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination. The addition of Mn confirmed the rescue of these defects. Interestingly, our structural data confirmed that the glycosylation defect in pmr1Δ could also completely be suppressed by the addition of Ca. The use of Pmr1p mutants either defective for Ca or Mn transport or both revealed that the suppression of the observed glycosylation defect in pmr1Δ strains by the intraluminal Golgi Ca requires the activity of Gdt1p. These data support the hypothesis that Gdt1p, in order to sustain the Golgi glycosylation process, imports Mn inside the Golgi lumen when Pmr1p exclusively transports Ca. Our results also reinforce the functional link between Gdt1p and Pmr1p as we highlighted that Gdt1p was a Mn sensitive protein whose abundance was directly dependent on the nature of the ion transported by Pmr1p. Finally, this study demonstrated that the aspartic residues of the two conserved motifs E-x-G-D-[KR], likely constituting the cation binding sites of Gdt1p, play a crucial role in Golgi glycosylation and hence in Mn/Catransport.
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http://dx.doi.org/10.1016/j.bbagen.2017.11.006DOI Listing
March 2018

Glycosylate and move! The glycosyltransferase Maf is involved in bacterial flagella formation.

Environ Microbiol 2018 01 4;20(1):228-240. Epub 2017 Dec 4.

Aix Marseille Univ, CNRS, LCB UMR7283, Marseille 13402, France.

The flagella of various Gram-negative bacteria are decorated with diverse glycan structures, amongst them nonulosonic acids related to the sialic acid family. Although nonulosonic sugar biosynthesis pathways have been dissected in various pathogens, the enzymes transferring the sugars onto flagellin are still poorly characterized. The deletion of genes coding for motility associated factors (Mafs) found in many pathogenic strains systematically gives rise to nonflagellated bacteria lacking specific nonulosonic sugars on the flagellins, therefore, relating Maf function to flagellin glycosylation and bacterial motility. We investigated the role of Maf from our model organism, Magnetospirillum magneticum AMB-1, in the glycosylation and formation of the flagellum. Deletion of the gene amb0685 coding for Maf produced a nonflagellated bacterium where the flagellin was still produced but no longer glycosylated. Our X-ray structure analysis revealed that the central domain of Maf exhibits similarity to sialyltransferases from Campylobacter jejuni. Glycan analysis suggested that the nonulosonic carbohydrate structure transferred is pseudaminic acid or a very close derivative. This work describes the importance of glycosylation in the formation of the bacterial flagellum and provides the first structural model for a member of a new bacterial glycosyltransferase family involved in nonulosonic acids transfer onto flagellins.
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http://dx.doi.org/10.1111/1462-2920.13975DOI Listing
January 2018

Another Brick in the Wall: a Rhamnan Polysaccharide Trapped inside Peptidoglycan of .

mBio 2017 09 12;8(5). Epub 2017 Sep 12.

Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France

Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In , a polysaccharide pellicle (PSP) forms a layer at the cell surface. The PSP structure varies among lactococcal strains; in MG1363, the PSP is composed of repeating hexasaccharide phosphate units. Here, we report the presence of an additional neutral polysaccharide in MG1363 that is a rhamnan composed of α-l-Rha trisaccharide repeating units. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) analysis of whole bacterial cells identified a PSP at the surface of wild-type cells. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan. The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division. Finally, a mutation leading to reduced expression of , encoding a protein of the LytR-CpsA-Psr (LCP) family, was shown to severely affect cell wall structure. In mutant cells, in contrast to wild-type cells, rhamnan was detected by HR-MAS NMR, suggesting that LcpA participates in the attachment of rhamnan to peptidoglycan. In the cell wall of Gram-positive bacteria, the peptidoglycan sacculus is considered the major structural component, maintaining cell shape and integrity. It is decorated with other glycopolymers, including polysaccharides, the roles of which are not fully elucidated. In the ovococcus , a polysaccharide with a different structure between strains forms a layer at the bacterial surface and acts as the receptor for various bacteriophages that typically exhibit a narrow host range. The present report describes the identification of a novel polysaccharide in the cell wall, a rhamnan that is trapped inside the peptidoglycan and covalently bound to it. We propose a model of rhamnan synthesis based on an ABC transporter-dependent pathway. Rhamnan appears as a conserved component of the lactococcal cell wall playing an essential role in growth and division, thus highlighting the importance of polysaccharides in the cell wall integrity of Gram-positive ovococci.
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http://dx.doi.org/10.1128/mBio.01303-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596347PMC
September 2017

BLISS: Shining a light on lignification in plants.

Plant Signal Behav 2017 08 8;12(8):e1359366. Epub 2017 Aug 8.

a Univ. Lille, CNRS , UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, Lille , France.

Lignin is a polyphenolic polymer of the plant cell wall formed by the oxidative polymerization of 3 main monomers called monolignols that give rise to the lignin H-, G- and S-units. Together with cellulose and hemicelluloses, lignin is a major component of plant biomass that is widely exploited by humans in numerous industrial processes. Despite recent advances in our understanding of monolignol biosynthesis, our current understanding of the spatio-temporal regulation of their transport and polymerization is more limited. In a recent publication, we have reported the development of an original Bioorthogonal Labeling Imaging Sequential Strategy (BLISS) that allows us to visualize the simultaneous incorporation dynamics of H and G monolignol reporters into lignifying cell walls of the flax stem. Here, we extend the application of this strategy to other plant organs such as roots and rapidly discuss some of the contributions and perspectives of this new technique for improving our understanding of the lignification process in plants.
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http://dx.doi.org/10.1080/15592324.2017.1359366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5616161PMC
August 2017

Probing the CMP-Sialic Acid Donor Specificity of Two Human β-d-Galactoside Sialyltransferases (ST3Gal I and ST6Gal I) Selectively Acting on O- and N-Glycosylproteins.

Chembiochem 2017 07 22;18(13):1251-1259. Epub 2017 May 22.

Université de Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000, Lille, France.

Sialylation of glycoproteins and glycolipids is catalyzed by sialyltransferases in the Golgi of mammalian cells, whereby sialic acid residues are added at the nonreducing ends of oligosaccharides. Because sialylated glycans play critical roles in a number of human physio-pathological processes, the past two decades have witnessed the development of modified sialic acid derivatives for a better understanding of sialic acid biology and for the development of new therapeutic targets. However, nothing is known about how individual mammalian sialyltransferases tolerate and behave towards these unnatural CMP-sialic acid donors. In this study, we devised several approaches to investigate the donor specificity of the human β-d-galactoside sialyltransferases ST6Gal I and ST3Gal I by using two CMP-sialic acids: CMP-Neu5Ac, and CMP-Neu5N-(4pentynoyl)neuraminic acid (CMP-SiaNAl), an unnatural CMP-sialic acid donor with an extended and functionalized N-acyl moiety.
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http://dx.doi.org/10.1002/cbic.201700024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499661PMC
July 2017