Publications by authors named "Franck Fieschi"

84 Publications

Membrane-Bound Flavocytochrome MsrQ Is a Substrate of the Flavin Reductase Fre in .

ACS Chem Biol 2021 Sep 22. Epub 2021 Sep 22.

CNRS, CEA, IRIG-LCBM, Laboratoire de Chimie et Biologie des Métaux, Univ. Grenoble Alpes, Grenoble 38054, France.

MsrPQ is a new type of methionine sulfoxide reductase (Msr) system found in bacteria. It is specifically involved in the repair of periplasmic methionine residues that are oxidized by hypochlorous acid. MsrP is a periplasmic molybdoenzyme that carries out the Msr activity, whereas MsrQ, an integral membrane-bound hemoprotein, acts as the physiological partner of MsrP to provide electrons for catalysis. Although MsrQ (YedZ) was associated since long with a protein superfamily named FRD (ferric reductase domain), including the eukaryotic NADPH oxidases and STEAP proteins, its biochemical properties are still sparsely documented. Here, we have investigated the cofactor content of the MsrQ and its mechanism of reduction by the flavin reductase Fre. We showed by electron paramagnetic resonance (EPR) spectroscopy that MsrQ contains a single highly anisotropic low-spin (HALS) b-type heme located on the periplasmic side of the membrane. We further demonstrated that MsrQ holds a flavin mononucleotide (FMN) cofactor that occupies the site where a second heme binds in other members of the FDR superfamily on the cytosolic side of the membrane. EPR spectroscopy indicates that the FMN cofactor can accommodate a radical semiquinone species. The cytosolic flavin reductase Fre was previously shown to reduce the MsrQ heme. Here, we demonstrated that Fre uses the FMN MsrQ cofactor as a substrate to catalyze the electron transfer from cytosolic NADH to the heme. Formation of a specific complex between MsrQ and Fre could favor this unprecedented mechanism, which most likely involves transfer of the reduced FMN cofactor from the Fre active site to MsrQ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.1c00613DOI Listing
September 2021

Low-Valent Calix[4]arene Glycoconjugates Based on Hydroxamic Acid Bearing Linkers as Potent Inhibitors in a Model of Ebola Virus Cis-Infection and HCMV-gB-Recombinant Glycoprotein Interaction with MDDC Cells by Blocking DC-SIGN.

J Med Chem 2021 10 15;64(19):14332-14343. Epub 2021 Sep 15.

Laboratoire de Glycochimie des Antimicrobiens et des Agroressources (LG2A-UMR7378-CNRS), Université de Picardie Jules Verne, 10 Rue Baudelocque, Amiens, 80039 Cédex, France.

In addition to a variety of viral-glycoprotein receptors (e.g., heparan sulfate, Niemann-Pick C1, etc.), dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), from the C-type lectin receptor family, plays one of the most important pathogenic functions for a wide range of viruses (e.g., Ebola, human cytomegalovirus (HCMV), HIV-1, severe acute respiratory syndrome coronavirus 2, etc.) that invade host cells before replication; thus, its inhibition represents a relevant extracellular antiviral therapy. We report two novel -Bu-calixarene glycoclusters and , bearing tetrahydroxamic acid groups, which exhibit micromolar inhibition of soluble DC-SIGN binding and provide nanomolar IC inhibition of both DC-SIGN-dependent Jurkat cis-cell infection by viral particle pseudotyped with Ebola virus glycoprotein and the HCMV-gB-recombinant glycoprotein interaction with monocyte-derived dendritic cells expressing DC-SIGN. A unique cooperative involvement of sugar, linker, and calixarene core is likely behind the strong avidity of DC-SIGN for these low-valent systems. We claim herein new promising candidates for the rational development of a large spectrum of antiviral therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.1c00818DOI Listing
October 2021

Controlled density glycodendron microarrays for studying carbohydrate-lectin interactions.

Org Biomol Chem 2021 Sep 13;19(34):7357-7362. Epub 2021 Aug 13.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), CSIC - Universidad de Sevilla, Av. Américo Vespucio 49, 41092 Seville, Spain.

Glycodendron microarrays with defined valency have been constructed by on-chip synthesis on hydrophobic indium tin oxide (ITO) coated glass slides and employed in lectin-carbohydrate binding studies with several plant and human lectins. Glycodendrons presenting sugar epitopes at different valencies were prepared by spotwise strain-promoted azide-alkyne cycloaddition (SPAAC) between immobilised cyclooctyne dendrons and azide functionalised glycans. The non-covalent immobilisation of dendrons on the ITO surface by hydrophobic interaction allowed us to study dendron surface density and SPAAC conversion rate by in situ MALDI-TOF MS analysis. By diluting the dendron surface density we could study how the carbohydrate-lectin interactions became exclusively dependant on the valency of the immobilised glycodendron.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d1ob00872bDOI Listing
September 2021

Lipid bilayer degradation induced by SARS-CoV-2 spike protein as revealed by neutron reflectometry.

Sci Rep 2021 07 21;11(1):14867. Epub 2021 Jul 21.

Institut Laue-Langevin, 71 Avenue des Martyrs, BP 156, 38042, Grenoble, France.

SARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements showed that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-93996-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295359PMC
July 2021

Synthesis, self-assembly and Langerin recognition studies of a resorcinarene-based glycocluster exposing a hyaluronic acid thiodisaccharide mimetic.

Org Biomol Chem 2021 Jul;19(29):6455-6467

Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, C1428EGA Buenos Aires, Argentina. and CONICET-Universidad de Buenos Aires, Centro de Investigaciones en Hidratos de Carbono (CIHIDECAR), C1428EGA Buenos Aires, Argentina.

Herein, we report the synthesis of an octavalent glycocluster exposing a thiodisaccharide mimetic of the repetitive unit of hyaluronic acid, βSGlcA(1 → 3)βSGlcNAc, constructed on a calix[4]resorcinarene scaffold by CuAAC reaction of suitable precursors. This glycocluster showed a strong tendency toward self-aggregation. DOSY-NMR and DLS experiments demonstrated the formation of spherical micelles of d ≅ 6.2 nm, in good agreement. TEM micrographs showed the presence of particles of different sizes, depending on the pH of the starting solution, thus evidencing that the negative charge on the micelle surface due to ionization of the GlcA residues plays an important role in the aggregation process. STD-NMR and DLS experiments provided evidence of the interaction between the synthetic glycocluster and Langerin, a relevant C-type lectin. This interaction was not observed in the STD-NMR experiments performed with the basic disaccharide, providing evidence of a multivalent effect.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d1ob00895aDOI Listing
July 2021

NADPH Oxidases (NOX): An Overview from Discovery, Molecular Mechanisms to Physiology and Pathology.

Antioxidants (Basel) 2021 Jun 1;10(6). Epub 2021 Jun 1.

Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, 38000 Grenoble, France.

The reactive oxygen species (ROS)-producing enzyme NADPH oxidase (NOX) was first identified in the membrane of phagocytic cells. For many years, its only known role was in immune defense, where its ROS production leads to the destruction of pathogens by the immune cells. NOX from phagocytes catalyzes, via one-electron trans-membrane transfer to molecular oxygen, the production of the superoxide anion. Over the years, six human homologs of the catalytic subunit of the phagocyte NADPH oxidase were found: NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2. Together with the NOX2/gp91 component present in the phagocyte NADPH oxidase assembly itself, the homologs are now referred to as the NOX family of NADPH oxidases. NOX are complex multidomain proteins with varying requirements for assembly with combinations of other proteins for activity. The recent structural insights acquired on both prokaryotic and eukaryotic NOX open new perspectives for the understanding of the molecular mechanisms inherent to NOX regulation and ROS production (superoxide or hydrogen peroxide). This new structural information will certainly inform new investigations of human disease. As specialized ROS producers, NOX enzymes participate in numerous crucial physiological processes, including host defense, the post-translational processing of proteins, cellular signaling, regulation of gene expression, and cell differentiation. These diversities of physiological context will be discussed in this review. We also discuss NOX misregulation, which can contribute to a wide range of severe pathologies, such as atherosclerosis, hypertension, diabetic nephropathy, lung fibrosis, cancer, or neurodegenerative diseases, giving this family of membrane proteins a strong therapeutic interest.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/antiox10060890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228183PMC
June 2021

SARS-CoV-2 spike protein removes lipids from model membranes and interferes with the capacity of high density lipoprotein to exchange lipids.

J Colloid Interface Sci 2021 Nov 12;602:732-739. Epub 2021 Jun 12.

Biofilms - Research Center for Biointerfaces and Department of Biomedical Science, Faculty of Health and Society, Malmö University, 20506 Malmö, Sweden. Electronic address:

Cholesterol has been shown to affect the extent of coronavirus binding and fusion to cellular membranes. The severity of Covid-19 infection is also known to be correlated with lipid disorders. Furthermore, the levels of both serum cholesterol and high-density lipoprotein (HDL) decrease with Covid-19 severity, with normal levels resuming once the infection has passed. Here we demonstrate that the SARS-CoV-2 spike (S) protein interferes with the function of lipoproteins, and that this is dependent on cholesterol. In particular, the ability of HDL to exchange lipids from model cellular membranes is altered when co-incubated with the spike protein. Additionally, the S protein removes lipids and cholesterol from model membranes. We propose that the S protein affects HDL function by removing lipids from it and remodelling its composition/structure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jcis.2021.06.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8195693PMC
November 2021

DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist.

PLoS Pathog 2021 05 20;17(5):e1009576. Epub 2021 May 20.

Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, Grenoble, France.

The efficient spread of SARS-CoV-2 resulted in a unique pandemic in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLRS) of antigen-presenting cells, widely present in respiratory mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2+ Vero E6 cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. These data have been then confirmed using authentic SARS-CoV-2 virus and human respiratory cell lines. Thus, we described a mechanism potentiating viral spreading of infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1009576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8136665PMC
May 2021

Protein Mannosylation as a Diagnostic and Prognostic Biomarker of Lupus Nephritis: An Unusual Glycan Neoepitope in Systemic Lupus Erythematosus.

Arthritis Rheumatol 2021 Apr 21. Epub 2021 Apr 21.

Institute for Research and Innovation in Health (i3S), University of Porto, Porto, Portugal, Faculty of Medicine, University of Porto, Porto, Portugal, and Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal.

Objective: Changes in protein glycosylation are a hallmark of immune-mediated diseases. Glycans are master regulators of the inflammatory response and are important molecules in self-nonself discrimination. This study was undertaken to investigate whether lupus nephritis (LN) exhibits altered cellular glycosylation to identify a unique glycosignature that characterizes LN pathogenesis.

Methods: A comprehensive tissue glycomics characterization was performed in kidney specimens from patients with systemic lupus erythematosus and biopsy-proven LN. A combination of advanced tissue mass spectrometry, in situ glyco-characterization, and ex vivo glycophenotyping was performed to structurally map the repertoire of N-glycans in LN tissue samples.

Results: LN exhibited a unique glycan signature characterized by increased abundance and spatial distribution of unusual mannose-enriched glycans that are typically found in lower microorganisms. This glycosignature was specific for LN, as it was not observed in other kidney diseases. Exposure of mannosylated glycans in LN was shown to occur at the cell surface of kidney cells, promoting increased recognition by specific glycan-recognizing receptors expressed by immune cells. This abnormal glycosignature of LN was shown to be due to a deficient complex N-glycosylation pathway and a proficient O-mannosylation pathway. Moreover, mannosylation levels detected in kidney biopsy samples from patients with LN at the time of diagnosis were demonstrated to predict the development of chronic kidney disease (CKD) with 93% specificity.

Conclusion: Cellular mannosylation is a marker of LN, predicting the development of CKD, and thus representing a potential glycobiomarker to be included in the diagnostic and prognostic algorithm of LN.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/art.41768DOI Listing
April 2021

New lipophilic glycomimetic DC-SIGN ligands: Stereoselective synthesis and SPR-based binding inhibition assays.

Bioorg Chem 2021 02 19;107:104566. Epub 2020 Dec 19.

Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via Moruzzi 13, 56125 Pisa, Italy. Electronic address:

The design and synthesis of efficient ligands for DC-SIGN is a topic of high interest, because this C-type lectin has been implicated in the early stages of many infection processes. DC-SIGN membrane-protein presents four carbohydrate-binding domains (CRD) that specifically recognize mannose and fucose. Therefore, antagonists of minimal disaccharide epitope Manα(1,2)Man, represent potentially interesting antibacterial and antiviral agents. In the recent past, we were able to develop efficient antagonists, mimics of the natural moiety, characterized by the presence of a real d-carbamannose unit which confers greater stability to enzymatic breakdown than the corresponding natural disaccharide ligand. Herein, we present the challenging stereoselective synthesis of four new amino or azide glycomimetic DC-SIGN antagonists with attractive orthogonal lipophilic substituents in C(3), C(4) or C(6) positions of the real carba unit, which were expected to establish crucial interactions with lipophilic areas of DC-SIGN CRD. The activity of the new ligands was evaluated by SPR binding inhibition assays. The interesting results obtained, allow to acquire important information about the influence of the lipophilic substituents present in specific positions of the carba scaffold. Furthermore, C(6) benzyl C(4) tosylamide pseudodisaccharide displayed a good affinity for DC-SIGN with a more favorable IC value than those of the previously described real carba-analogues. This study provides valuable knowledge for the implementation of further structural modifications towards improved inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bioorg.2020.104566DOI Listing
February 2021

Chemo-Enzymatic Synthesis of S. mansoni O-Glycans and Their Evaluation as Ligands for C-Type Lectin Receptors MGL, DC-SIGN, and DC-SIGNR.

Chemistry 2020 Oct 17;26(56):12818-12830. Epub 2020 Sep 17.

CIC biomaGUNE, Glycotechnology Group, Paseo Miramón 182, 20014, San Sebastian, Spain.

Due to their interactions with C-type lectin receptors (CLRs), glycans from the helminth Schistosoma mansoni represent promising leads for treatment of autoimmune diseases, allergies or cancer. We chemo-enzymatically synthesized nine O-glycans based on the two predominant O-glycan cores observed in the infectious stages of schistosomiasis, the mucin core 2 and the S. mansoni core. The O-glycans were fucosylated next to a selection of N-glycans directly on a microarray slide using a recombinant fucosyltransferase and GDP-fucose or GDP-6-azidofucose as donor. Binding assays with fluorescently labelled human CLRs DC-SIGN, DC-SIGNR and MGL revealed the novel O-glycan O8 as the best ligand for MGL from our panel. Significant binding to DC-SIGN was also found for azido-fucosylated glycans. Contrasting binding specificities were observed between the monovalent carbohydrate recognition domain (CRD) and the tetravalent extracellular domain (ECD) of DC-SIGNR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.202000291DOI Listing
October 2020

Lectin recognition and hepatocyte endocytosis of GalNAc-decorated nanostructured lipid carriers.

J Drug Target 2021 01 2;29(1):99-107. Epub 2020 Oct 2.

Univ. Grenoble Alpes, CEA, CNRS, IRIG, SyMMES, Grenoble, France, IRIG-SyMMES, University Grenoble Alpes, Grenoble, France.

Liver is the main organ for metabolism but is also subject to various pathologies, from viral, genetic, cancer or metabolic origin. There is thus a crucial need to develop efficient liver-targeted drug delivery strategies. Asialoglycoprotein receptor (ASGPR) is a C-type lectin expressed in the hepatocyte plasma membrane that efficiently endocytoses glycoproteins exposing galactose (Gal) or N-acetylgalactosamine (GalNAc). Its targeting has been successfully used to drive the uptake of small molecules decorated with three or four GalNAc, thanks to an optimisation of their spatial arrangement. Herein, we assessed the biological properties of highly stable nanostructured lipid carriers (NLC) made of FDA-approved ingredients and formulated with increasing amounts of GalNAc. Cellular studies showed that a high density of GalNAc was required to favour hepatocyte internalisation via the ASGPR pathway. Interaction studies using surface plasmon resonance and the macrophage galactose-lectin as GalNAc-recognising lectin confirmed the need of high GalNAc density for specific recognition of these NLC. This work is the first step for the development of efficient nanocarriers for prolonged liver delivery of active compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/1061186X.2020.1806286DOI Listing
January 2021

Targeting of the C-Type Lectin Receptor Langerin Using Bifunctional Mannosylated Antigens.

Front Cell Dev Biol 2020 14;8:556. Epub 2020 Jul 14.

Department of Molecular Cell Biology and Immunology, Cancer Center Amsterdam, Amsterdam Infection and Immunity Institute, Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, Amsterdam, Netherlands.

Langerhans cells (LCs) are antigen-presenting cells that reside in the skin. They uniquely express high levels of the C-type lectin receptor Langerin (CD207), which is an attractive target for antigen delivery in immunotherapeutic vaccination strategies against cancer. We here assess a library of 20 synthetic, well-defined mannoside clusters, built up from one, two, and three of six monomannosides, dimannosides, or trimannosides, appended to an oligopeptide backbone, for binding with Langerin using surface plasmon resonance and flow cytometric quantification. It is found that Langerin binding affinity increases with increasing number of mannosides. Hexavalent presentation of the mannosides resulted in binding affinities ranging from 3 to 12 μM. Trivalent presentation of the dimannosides and trimannosides led to Langerin affinity in the same range. The model melanoma gp100 antigenic peptide was subsequently equipped with a hexavalent cluster of the dimannosides and trimannosides as targeting moieties. Surprisingly, although the bifunctional conjugates were taken up in LCs in a Langerin-dependent manner, limited antigen presentation to cytotoxic T cells was observed. These results indicate that targeting glycan moieties on immunotherapeutic vaccines should not only be validated for target binding, but also on the continued effects on biology, such as antigen presentation to both CD8 and CD4 T cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcell.2020.00556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371993PMC
July 2020

Influence of the reducing-end anomeric configuration of the Man epitope on DC-SIGN recognition.

Org Biomol Chem 2020 Aug 30;18(31):6086-6094. Epub 2020 Jul 30.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), CSIC - Universidad de Sevilla, Av. Américo Vespucio 49, Seville 41092, Spain.

High-mannose (ManGlcNAc) is the main carbohydrate unit present in viral envelope glycoproteins such as gp120 of HIV and the GP1 of Ebola virus. This oligosaccharide comprises the Man epitope conjugated to two terminal N-acetylglucosamines by otherwise rarely-encountered β-mannose glycosidic bond. Formation of this challenging linkage is the bottleneck of the few synthetic approaches described to prepare high mannose. Herein, we report the synthesis of the Man epitope with both alpha and beta configurations at the reducing end, and subsequent evaluation of the impact of this configuration on binding to natural receptor of high-mannose, DC-SIGN. Using fluorescence polarization assays, we demonstrate that both anomers bind to DC-SIGN with comparable affinity. These relevant results therefore indicate that the more synthetically-accesible Man alpha epitope may be deployed as ligand for DC-SIGN in both in vitro and in vivo biological assays.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0ob01380cDOI Listing
August 2020

TETRALEC, Artificial Tetrameric Lectins: A Tool to Screen Ligand and Pathogen Interactions.

Int J Mol Sci 2020 Jul 25;21(15). Epub 2020 Jul 25.

Institut de Biologie Structurale, CEA, CNRS, University of Grenoble Alpes, F-38000 Grenoble, France.

C-type lectin receptor (CLR)/carbohydrate recognition occurs through low affinity interactions. Nature compensates that weakness by multivalent display of the lectin carbohydrate recognition domain (CRD) at the cell surface. Mimicking these low affinity interactions in vitro is essential to better understand CLR/glycan interactions. Here, we present a strategy to create a generic construct with a tetrameric presentation of the CRD for any CLR, termed TETRALEC. We applied our strategy to a naturally occurring tetrameric CRD, DC-SIGNR, and compared the TETRALEC ligand binding capacity by synthetic N- and O-glycans microarray using three different DC-SIGNR constructs i) its natural tetrameric counterpart, ii) the monomeric CRD and iii) a dimeric Fc-CRD fusion. DC-SIGNR TETRALEC construct showed a similar binding profile to that of its natural tetrameric counterpart. However, differences observed in recognition of low affinity ligands underlined the importance of the CRD spatial arrangement. Moreover, we further extended the applications of DC-SIGNR TETRALEC to evaluate CLR/pathogens interactions. This construct was able to recognize heat-killed by flow cytometry and confocal microscopy, a so far unreported specificity of DC-SIGNR. In summary, the newly developed DC-SIGNR TETRALEC tool proved to be useful to unravel novel CLR/glycan interactions, an approach which could be applied to other CLRs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21155290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432041PMC
July 2020

Interdomain Flexibility within NADPH Oxidase Suggested by SANS Using LMNG Stealth Carrier.

Biophys J 2020 08 3;119(3):605-618. Epub 2020 Jul 3.

University Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, Grenoble, France. Electronic address:

Small angle neutron scattering (SANS) provides a method to obtain important low-resolution information for integral membrane proteins (IMPs), challenging targets for structural determination. Specific deuteration furnishes a "stealth" carrier for the solubilized IMP. We used SANS to determine a structural envelope of SpNOX, the Streptococcus pneumoniae NADPH oxidase (NOX), a prokaryotic model system for exploring structure and function of eukaryotic NOXes. SpNOX was solubilized in the detergent lauryl maltose neopentyl glycol, which provides optimal SpNOX stability and activity. Using deuterated solvent and protein, the lauryl maltose neopentyl glycol was experimentally undetected in SANS. This affords a cost-effective SANS approach for obtaining novel structural information on IMPs. Combining SANS data with molecular modeling provided a first, to our knowledge, structural characterization of an entire NOX enzyme. It revealed a distinctly less compact structure than that predicted from the docking of homologous crystal structures of the separate transmembrane and dehydrogenase domains, consistent with a flexible linker connecting the two domains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2020.06.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7399496PMC
August 2020

Development of C-type lectin-oriented surfaces for high avidity glycoconjugates: towards mimicking multivalent interactions on the cell surface.

Org Biomol Chem 2020 07;18(25):4763-4772

Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, 38000 Grenoble, France.

Multivalent interactions between complex carbohydrates and oligomeric C-type lectins govern a wide range of immune responses. Up to date, standard SPR (surface plasmon resonance) competitive assays have largely been to evaluate binding properties from monosaccharide units (low affinity, mM) to multivalent elemental antagonists (moderate affinity, μM). Herein, we report typical case-studies of SPR competitive assays showing that they underestimate the potency of glycoclusters to inhibit the interaction between DC-SIGN and immobilized glycoconjugates. This paper describes the design and implementation of a SPR direct interaction over DC-SIGN oriented surfaces, extendable to other C-type lectin surfaces as such Langerin. This setup provides an overview of intrinsic avidity generation emanating simultaneously from multivalent glycoclusters and from DC-SIGN tetramers organized in nanoclusters at the cell membrane. To do so, covalent biospecific capture of DC-SIGN via StreptagII/StrepTactin interaction preserves tetrameric DC-SIGN, accessibility and topology of its active sites, that would have been dissociated using standard EDC-NHS procedure under acidic conditions. From the tested glycoclusters libraries, we demonstrated that the scaffold architecture, the valency and the glycomimetic-based ligand are crucial to reach nanomolar affinities for DC-SIGN. The glycocluster 3·D illustrates the tightest binding partner in this set for a DC-SIGN surface (KD = 18 nM). Moreover, the selectivity at monovalent scale of glycomimetic D can be easily analyzed at multivalent scale comparing its binding over different C-type lectin immobilized surfaces. This approach may give rise to novel insights into the multivalent binding mechanisms responsible for avidity and make a major contribution to the full characterization of the binding potency of promising specific and multivalent immodulators.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0ob00781aDOI Listing
July 2020

Second-Generation Dendrimers with Chondroitin Sulfate Type-E Disaccharides as Multivalent Ligands for Langerin.

Biomacromolecules 2020 07 24;21(7):2726-2734. Epub 2020 Jun 24.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.

Chondroitin sulfate type-E (CS-E) is a sulfated polysaccharide that shows several interesting biological activities, such as modulation of the neuronal growth factor signaling and its interaction with langerin, a C-type lectin with a crucial role in the immunological system. However, applications of CS-E are hampered by the typical heterogeneous structure of the natural polysaccharide. Well-defined, homogeneous CS-E analogues are highly demanded. Here, we report the synthesis of monodispersed, structurally well-defined second-generation glycodendrimers displaying up to 18 CS-E disaccharide units. These complex multivalent systems have a molecular weight and a number of disaccharide repeating units comparable with those of the natural polysaccharides. In addition, surface plasmon resonance experiments revealed a calcium-independent interaction between these glycodendrimers and langerin, in the micromolar range, highlighting the utility of these compounds as CS-E mimetics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biomac.0c00476DOI Listing
July 2020

Rapid On-Chip Synthesis of Complex Glycomimetics from N-Glycan Scaffolds for Improved Lectin Targeting.

Chemistry 2020 Oct 11;26(56):12809-12817. Epub 2020 Sep 11.

CIC biomaGUNE, Paseo Miramón 182, 20009, San Sebastian, Spain.

C-type lectin receptor (CLR) carbohydrate binding proteins found on immune cells with important functions in pathogen recognition as well as self and non-self-differentiation are increasingly moving into the focus of drug developers as targets for the immune therapy of cancer autoimmune diseases and inflammation and to improve the efficacy of vaccines. The development of molecules with increased affinity and selectivity over the natural glycan binders has largely focused on the synthesis of mono and disaccharide mimetics but glycan array binding experiments have shown increased binding selectivity and affinity for selected larger oligosaccharides that are able to engage in additional favorable interactions beyond the primary binding site. Here, a platform for the rapid preparation and screening of N-glycan mimetics on microarrays is presented that turns a panel of complex glycan core structures into structurally diverse glycomimetics by a combination of enzymatic glycosylation with a nonnatural donor and subsequent cycloaddition with a collection of alkynes. All surface-based reactions were monitored by MALDI-TOF MS to assess conversion and purity of spot compositions. Screening the collection of 374 N-glycomimetics against the plant lectin WFA and the 2 human immune lectins MGL ECD and Langerin ECD produced a number of high affinity binders as lead structures for more selective lectin targeting probes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.202000026DOI Listing
October 2020

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.

Nat Commun 2020 02 6;11(1):741. Epub 2020 Feb 6.

Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38000, Grenoble, France.

Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the μs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-14537-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005145PMC
February 2020

Systematic Dual Targeting of Dendritic Cell C-Type Lectin Receptor DC-SIGN and TLR7 Using a Trifunctional Mannosylated Antigen.

Front Chem 2019 4;7:650. Epub 2019 Oct 4.

Department of Molecular Cell Biology and Immunology, Cancer Center Amsterdam, Amsterdam Infection and Immunity Institute, Amsterdam Universitair Medische Centra, Vrije Universiteit Amsterdam, Amsterdam, Netherlands.

Dendritic cells (DCs) are important initiators of adaptive immunity, and they possess a multitude of Pattern Recognition Receptors (PRR) to generate an adequate T cell mediated immunity against invading pathogens. PRR ligands are frequently conjugated to tumor-associated antigens in a vaccination strategy to enhance the immune response toward such antigens. One of these PPRs, DC-SIGN, a member of the C-type lectin receptor (CLR) family, has been extensively targeted with Lewis structures and mannose glycans, often presented in multivalent fashion. We synthesized a library of well-defined mannosides (mono-, di-, and tri-mannosides), based on known "high mannose" structures, that we presented in a systematically increasing number of copies ( = 1, 2, 3, or 6), allowing us to simultaneously study the effect of mannoside configuration and multivalency on DC-SIGN binding via Surface Plasmon Resonance (SPR) and flow cytometry. Hexavalent presentation of the clusters showed the highest binding affinity, with the hexa-α1,2-di-mannoside being the most potent ligand. The four highest binding hexavalent mannoside structures were conjugated to a model melanoma gp100-peptide antigen and further equipped with a Toll-like receptor 7 (TLR7)-agonist as adjuvant for DC maturation, creating a trifunctional vaccine conjugate. Interestingly, DC-SIGN affinity of the mannoside clusters did not directly correlate with antigen presentation enhancing properties and the α1,2-di-mannoside cluster with the highest binding affinity in our library even hampered T cell activation. Overall, this systematic study has demonstrated that multivalent glycan presentation can improve DC-SIGN binding but enhanced binding cannot be directly translated into enhanced antigen presentation and the sole assessment of binding affinity is thus insufficient to determine further functional biological activity. Furthermore, we show that well-defined antigen conjugates combining two different PRR ligands can be generated in a modular fashion to increase the effectiveness of vaccine constructs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fchem.2019.00650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6787163PMC
October 2019

Enhancing Potency and Selectivity of a DC-SIGN Glycomimetic Ligand by Fragment-Based Design: Structural Basis.

Chemistry 2019 Nov 18;25(64):14659-14668. Epub 2019 Oct 18.

Université Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, 38044, Grenoble, France.

Chemical modification of pseudo-dimannoside ligands guided by fragment-based design allowed for the exploitation of an ammonium-binding region in the vicinity of the mannose-binding site of DC-SIGN, leading to the synthesis of a glycomimetic antagonist (compound 16) of unprecedented affinity and selectivity against the related lectin langerin. Here, the computational design of pseudo-dimannoside derivatives as DC-SIGN ligands, their synthesis, their evaluation as DC-SIGN selective antagonists, the biophysical characterization of the DC-SIGN/16 complex, and the structural basis for the ligand activity are presented. On the way to the characterization of this ligand, an unusual bridging interaction within the crystals shed light on the plasticity and potential secondary binding sites within the DC-SIGN carbohydrate recognition domain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.201903391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899773PMC
November 2019

Human Macrophage Galactose-Type Lectin (MGL) Recognizes the Outer Core of Escherichia coli Lipooligosaccharide.

Chembiochem 2019 07 24;20(14):1778-1782. Epub 2019 Jun 24.

Department of Chemical Sciences, University of Naples Federico II, via Cintia 4, 80126, Napoli, Italy.

Carbohydrate-lectin interactions intervene in and mediate most biological processes, including a crucial modulation of immune responses to pathogens. Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. Herein, a novel molecular interaction between the human macrophage galactose-type lectin (MGL) and the lipooligosaccharide (LOS) of Escherichia coli strain R1 is described. Saturation transfer difference NMR spectroscopy analysis, supported by computational studies, demonstrated that MGL bound to the purified deacylated LOS mainly through recognition of its outer core and established crucial interactions with the terminal Galα(1,2)Gal epitope. These results assess the ability of MGL to recognise glycan moieties exposed on Gram-negative bacterial surfaces.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201900087DOI Listing
July 2019

Unprecedented Thiacalixarene Fucoclusters as Strong Inhibitors of Ebola cis-Cell Infection and HCMV-gB Glycoprotein/DC-SIGN C-type Lectin Interaction.

Bioconjug Chem 2019 04 4;30(4):1114-1126. Epub 2019 Apr 4.

Laboratoire de Glycochimie des Antimicrobiens et des Agroressources (LG2A-UMR7378-CNRS) , Université de Picardie Jules Verne , 10 Rue Baudelocque , 80039 , Amiens , France.

Glycan-protein interactions control numerous biological events from cell-cell recognition and signaling to pathogen host cell attachment for infections. To infect cells, some viruses bind to immune cells with the help of DC-SIGN (dendritic cell [DC]-specific ICAM3-grabbing nonintegrin) C-type lectin expressed on dendritic and macrophage cell membranes, via their envelope protein. Prevention of this infectious interaction is a serious therapeutic option. Here, we describe the synthesis of the first water-soluble tetravalent fucocluster pseudopeptide-based 1,3-alternate thiacalixarenes as viral antigen mimics designed for the inhibition of DC-SIGN, to prevent viral particle uptake. Their preparation exploits straightforward convergent strategies involving one-pot Ugi four-component (Ugi-4CR) and azido-alkyne click chemistry reactions as key steps. Surface plasmon resonance showed strong inhibition of DC-SIGN interaction properties by tetravalent ligands designed with high relative potencies and β avidity factors. All ligands block DC-SIGN active sites at nanomolar IC preventing cis-cell infection by Ebola viral particles pseudotyped with EBOV glycoprotein (Zaire species of Ebola virus) on Jurkat cells that express DC-SIGN. In addition, we observed strong inhibition of DC-SIGN/human cytomegalovirus (HCMV)-gB recombinant glycoprotein interaction. This finding opens the way to the simple development of new models of water-soluble glycocluster-based thia-calixarenes with wide-ranging antimicrobial activities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.bioconjchem.9b00066DOI Listing
April 2019

Assemblies of lauryl maltose neopentyl glycol (LMNG) and LMNG-solubilized membrane proteins.

Biochim Biophys Acta Biomembr 2019 05 15;1861(5):939-957. Epub 2019 Feb 15.

Univ. Grenoble Alpes, CNRS, CEA, Institute for Structural Biology (IBS), 38000 Grenoble, France. Electronic address:

Laurylmaltose neopentylglycol (LMNG) bears two linked hydrophobic chains of equal length and two hydrophilic maltoside groups. It arouses a strong interest in the field of membrane protein biochemistry, since it was shown to efficiently solubilize and stabilize membrane proteins often better than the commonly used dodecylmaltopyranoside (DDM), and to allow structure determination of some challenging membrane proteins. However, LMNG was described to form large micelles, which could be unfavorable for structural purposes. We thus investigated its auto-assemblies and the association state of different membrane proteins solubilized in LMNG by analytical ultracentrifugation, size exclusion chromatography coupled to light scattering, centrifugation on sucrose gradient and/or small angle scattering. At high concentrations (in the mM range), LMNG forms long rods, and it stabilized the membrane proteins investigated herein, i.e. a bacterial multidrug transporter, BmrA; a prokaryotic analogous of the eukaryotic NADPH oxidases, SpNOX; an E. coli outer membrane transporter, FhuA; and the halobacterial bacteriorhodopsin, bR. BmrA, in the Apo and the vanadate-inhibited forms showed reduced kinetics of limited proteolysis in LMNG compared to DDM. Both SpNOX and BmrA display an increased specific activity in LMNG compared to DDM. The four proteins form LMNG complexes with their usual quaternary structure and with usual amount of bound detergent. No heterogeneous complexes related to the large micelle size of LMNG alone were observed. In conditions where LMNG forms assemblies of large size, FhuA crystals diffracting to 4.0 Å were obtained by vapor diffusion. LMNG large micelle size thus does not preclude membrane protein homogeneity and crystallization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamem.2019.02.003DOI Listing
May 2019

Microbe-focused glycan array screening platform.

Proc Natl Acad Sci U S A 2019 02 22;116(6):1958-1967. Epub 2019 Jan 22.

Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany;

Interactions between glycans and glycan binding proteins are essential for numerous processes in all kingdoms of life. Glycan microarrays are an excellent tool to examine protein-glycan interactions. Here, we present a microbe-focused glycan microarray platform based on oligosaccharides obtained by chemical synthesis. Glycans were generated by combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis, and chemoenzymatic methods. The current library of more than 300 glycans is as diverse as the mammalian glycan array from the Consortium for Functional Glycomics and, due to its microbial focus, highly complementary. This glycan platform is essential for the characterization of various classes of glycan binding proteins. Applications of this glycan array platform are highlighted by the characterization of innate immune receptors and bacterial virulence factors as well as the analysis of human humoral immunity to pathogenic glycans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1800853116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6369816PMC
February 2019

Quantitative live-cell imaging and 3D modeling reveal critical functional features in the cytosolic complex of phagocyte NADPH oxidase.

J Biol Chem 2019 03 10;294(11):3824-3836. Epub 2019 Jan 10.

From the Laboratoire de Chimie Physique, CNRS, Université Paris-Sud, Université Paris-Saclay, 91405, Orsay France,

Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections. However, uncontrolled ROS production contributes to inflammation, making NADPH oxidase a major drug target. It consists of two membranous (Nox2 and p22) and three cytosolic subunits (p40, p47, and p67) that undergo structural changes during enzyme activation. Unraveling the interactions between these subunits and the resulting conformation of the complex could shed light on NADPH oxidase regulation and help identify inhibition sites. However, the structures and the interactions of flexible proteins comprising several well-structured domains connected by intrinsically disordered protein segments are difficult to investigate by conventional techniques such as X-ray crystallography, NMR, or cryo-EM. Here, we developed an analytical strategy based on FRET-fluorescence lifetime imaging (FLIM) and fluorescence cross-correlation spectroscopy (FCCS) to structurally and quantitatively characterize NADPH oxidase in live cells. We characterized the inter- and intramolecular interactions of its cytosolic subunits by elucidating their conformation, stoichiometry, interacting fraction, and affinities in live cells. Our results revealed that the three subunits have a 1:1:1 stoichiometry and that nearly 100% of them are present in complexes in living cells. Furthermore, combining FRET data with small-angle X-ray scattering (SAXS) models and published crystal structures of isolated domains and subunits, we built a 3D model of the entire cytosolic complex. The model disclosed an elongated complex containing a flexible hinge separating two domains ideally positioned at one end of the complex and critical for oxidase activation and interactions with membrane components.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.006864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422089PMC
March 2019

On-Chip Screening of a Glycomimetic Library with C-Type Lectins Reveals Structural Features Responsible for Preferential Binding of Dectin-2 over DC-SIGN/R and Langerin.

Chemistry 2018 Sep 3;24(54):14448-14460. Epub 2018 Sep 3.

Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133, Milano, Italy.

A library of mannose- and fucose-based glycomimetics was synthesized and screened in a microarray format against a set of C-type lectin receptors (CLRs) that included DC-SIGN, DC-SIGNR, langerin, and dectin-2. Glycomimetic ligands able to interact with dectin-2 were identified for the first time. Comparative analysis of binding profiles allowed their selectivity against other CLRs to be probed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.201802577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220942PMC
September 2018

Chemoenzymatic Synthesis of N-glycan Positional Isomers and Evidence for Branch Selective Binding by Monoclonal Antibodies and Human C-type Lectin Receptors.

ACS Chem Biol 2018 08 21;13(8):2269-2279. Epub 2018 Jun 21.

CIC biomaGUNE, Glycotechnology Laboratory , Paseo Miramón 182 , 20014 San Sebastian , Spain.

Here, we describe a strategy for the rapid preparation of pure positional isomers of complex N-glycans to complement an existing array comprising a larger number of N-glycans and smaller glycan structures. The expanded array was then employed to study context-dependent binding of structural glycan fragments by monoclonal antibodies and C-type lectins. A partial enzymatic elongation of semiprotected core structures was combined with the protecting-group-aided separation of positional isomers by preparative HPLC. This methodology, which avoids the laborious chemical differentiation of antennae, was employed for the preparation of eight biantennary N-glycans with Galβ1,4GlcNAc (LN), GalNAcβ1,4GlcNAc (LDN), and GalNAcβ1,4[Fucα1,3]GlcNAc (LDNF) motifs presented on either one or both antennae. Screening of the binding specificities of three anti-Le monoclonal IgM antibodies raised against S. mansoni glycans and three C-type lectin receptors of the innate immune system, namely DC-SIGN, DC-SIGNR, and LSECtin, revealed a surprising context-dependent fine specificity for the recognition of the glycan motifs. Moreover, we observed a striking selection of one individual positional isomer over the other by the C-type lectins tested, underscoring the biological relevance of the structural context of glycan elements in molecular recognition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.8b00431DOI Listing
August 2018
-->