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1.
Sci Rep ; 13(1): 20488, 2023 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-37993516

RESUMO

The development of effective recombinant vaccines against parasitic nematodes has been challenging and so far mostly unsuccessful. This has also been the case for Ostertagia ostertagi, an economically important abomasal nematode in cattle, applying recombinant versions of the protective native activation-associated secreted proteins (ASP). To gain insight in key elements required to trigger a protective immune response, the protein structure and N-glycosylation of the native ASP and a non-protective Pichia pastoris recombinant ASP were compared. Both antigens had a highly comparable protein structure, but different N-glycan composition. After mimicking the native ASP N-glycosylation via the expression in Nicotiana benthamiana plants, immunisation of calves with these plant-produced recombinants resulted in a significant reduction of 39% in parasite egg output, comparable to the protective efficacy of the native antigen. This study provides a valuable workflow for the development of recombinant vaccines against other parasitic nematodes.


Assuntos
Doenças dos Bovinos , Ostertagíase , Bovinos , Animais , Ostertagia/genética , Ostertagíase/prevenção & controle , Ostertagíase/veterinária , Vacinação/veterinária , Vacinas Sintéticas/genética , Proteínas Recombinantes/genética , Contagem de Ovos de Parasitas
2.
Int J Mol Sci ; 24(21)2023 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-37958949

RESUMO

Cells use glycans to encode information that modulates processes ranging from cell-cell recognition to programmed cell death. This information is encoded within a glycocode, and its decoding is performed by carbohydrate-binding proteins. Among these, lectins stand out due to their specific and reversible interaction with carbohydrates. Changes in glycosylation patterns are observed in several pathologies, including cancer, where abnormal glycans are found on the surfaces of affected tissues. Given the importance of the bioprospection of promising biomolecules, the current work aimed to determine the structural properties and anticancer potential of the mannose-specific lectin from seeds of Canavalia villosa (Cvill). Experimental elucidation of the primary and 3D structures of the lectin, along with glycan array and molecular docking, facilitated the determination of its fine carbohydrate-binding specificity. These structural insights, coupled with the lectin's specificity, have been combined to explain the antiproliferative effect of Cvill against cancer cell lines. This effect is dependent on the carbohydrate-binding activity of Cvill and its uptake in the cells, with concomitant activation of autophagic and apoptotic pathways.


Assuntos
Canavalia , Lectinas , Lectinas/farmacologia , Lectinas/análise , Canavalia/metabolismo , Simulação de Acoplamento Molecular , Lectinas de Plantas/metabolismo , Sementes/metabolismo , Carboidratos/análise , Polissacarídeos/análise
3.
Chemistry ; 29(52): e202301494, 2023 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-37347819

RESUMO

The isolation from organisms and readily available glycoproteins has become an increasingly convenient source of N-glycans for multiple applications including glycan microarrays, as reference standards in glycan analysis or as reagents that improve bioavailability of protein and peptide therapeutics through conjugation. A problematic step in the isolation process on a preparative scale can be the attachment of a linker for the improved purification, separation, immobilization and quantification of the glycan structures. Addressing this issue, we firstly aimed for the development of an UV active linker for a fast and reliable attachment to anomeric glycosylamines via urea bond formation. Secondly, we validated the new linker on glycan arrays in a comparative study with a collection of N-glycans which were screened against various lectins. In total, we coupled four structurally varied N-glycans to four different linkers, immobilized all constructs on a microarray and compared their binding affinities to four plant and fungal lectins of widely described specificity. Our study shows that the urea type linker showed an overall superior performance for lectin binding and once more, highlights the often neglected influence of the choice of linker on lectin recognition.


Assuntos
Glicoproteínas , Lectinas , Análise em Microsséries , Glicoproteínas/metabolismo , Lectinas/química , Ligação Proteica , Polissacarídeos/química
4.
J Biol Chem ; 299(5): 104627, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36944399

RESUMO

The FimH type-1 fimbrial adhesin allows pathogenic Escherichia coli to adhere to glycoproteins in the epithelial linings of human bladder and intestinal tract, by using multiple fimbriae simultaneously. Pauci- and high-mannose type N-glycans are natural FimH receptors on those glycoproteins. Oligomannose-3 and oligomannose-5 bind with the highest affinity to FimH by using the same Manα1,3Man branch. Oligomannose-6 is generated from oligomannose-5 in the next step of the biogenesis of high-mannose N-glycans, by the transfer of a mannose in α1,2-linkage onto this branch. Using serial crystallography and by measuring the kinetics of binding, we demonstrate that shielding the high-affinity epitope drives the binding of multiple FimH molecules. First, we profiled FimH glycan binding on a microarray containing paucimannosidic N-glycans and in a FimH LEctPROFILE assay. To make the transition to oligomannose-6, we measured the kinetics of FimH binding using paucimannosidic N-glycans, glycoproteins and all four α-dimannosides conjugated to bovine serum albumin. Equimolar mixed interfaces of the dimannosides present in oligomannose-6 and molecular dynamics simulations suggest a positive cooperativity in the bivalent binding of Manα1,3Manα1 and Manα1,6Manα1 dimannosides. The binding of core α1,6-fucosylated oligomannose-3 in cocrystals of FimH is monovalent but interestingly the GlcNAc1-Fuc moiety retains highly flexibility. In cocrystals with oligomannose-6, two FimH bacterial adhesins bind the Manα1,3Manα1 and Manα1,6Manα1 endings of the second trimannose core (A-4'-B). This cooperative switch towards bivalent binding appears sustainable beyond a molar excess of oligomannose-6. Our findings provide important novel structural insights for the design of multivalent FimH antagonists that bind with positive cooperativity.


Assuntos
Adesinas de Escherichia coli , Receptor de Manose , Modelos Moleculares , Humanos , Adesinas de Escherichia coli/química , Adesinas de Escherichia coli/metabolismo , Aderência Bacteriana , Escherichia coli/metabolismo , Glicoproteínas/metabolismo , Manose/metabolismo , Receptor de Manose/química , Receptor de Manose/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Simulação de Acoplamento Molecular
5.
Glycoconj J ; 40(1): 85-95, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36287345

RESUMO

The Dalbergieae lectin group encompasses several lectins with significant differences in their carbohydrate specificities and biological properties. The current work reports on the purification and characterization of a GalNAc/Gal-specific lectin from Vataireopsis araroba (Aguiar) Ducke, designated as VaL. The lectin was purified from the seeds in a single step using guar gum affinity chromatography. The lectin migrated as a single band of about 35 kDa on SDS-PAGE and, in native conditions, occurs as a homodimer. The purified lectin is stable at temperatures up to 60 °C and in a pH range from 7 to 8 and requires divalent cations for its activity. Sugar-inhibition assays demonstrate the lectin specificity towards N-acetyl-D-galactosamine, D-galactose and related sugars. Furthermore, glycan array analyses show that VaL interacts preferentially with glycans containing terminal GalNAc/Galß1-4GlcNAc. Biological activity assays were performed using three insect cell lines: CF1 midgut cells from the spruce budworm Choristoneura fumiferana, S2 embryo cells from the fruit fly Drosophila melanogaster, and GutAW midgut cells from the corn earworm Helicoverpa zea. In vitro assays indicated a biostatic effect for VaL on CF1 cells, but not on S2 and GutAW cells. The lectin presented a biostatic effect by reducing the cell growth and inducing cell agglutination, suggesting an interaction with glycans on the cell surface. VaL has been characterized as a galactoside-specific lectin of the Dalbergieae tribe, with sequence similarity to lectins from Vatairea and Arachis.


Assuntos
Fabaceae , Lectinas , Animais , Lectinas/metabolismo , Fabaceae/química , Fabaceae/metabolismo , Drosophila melanogaster , Carboidratos/análise , Sementes/química , Polissacarídeos/metabolismo , Galactosídeos/análise , Galactosídeos/metabolismo , Lectinas de Plantas/química
6.
ACS Cent Sci ; 8(10): 1415-1423, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36313162

RESUMO

The molecular recognition features of LSECtin toward asymmetric N-glycans have been scrutinized by NMR and compared to those occurring in glycan microarrays. A pair of positional glycan isomers (LDN3 and LDN6), a nonelongated GlcNAc4Man3 N-glycan (G0), and the minimum binding epitope (the GlcNAcß1-2Man disaccharide) have been used to shed light on the preferred binding modes under both experimental conditions. Strikingly, both asymmetric LDN3 and LDN6 N-glycans are recognized by LSECtin with similar affinities in solution, in sharp contrast to the results obtained when those glycans are presented on microarrays, where only LDN6 was efficiently recognized by the lectin. Thus, different results can be obtained using different experimental approaches, pointing out the tremendous difficulty of translating in vitro results to the in vivo environment.

7.
Stud Health Technol Inform ; 295: 183-186, 2022 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-35773838

RESUMO

During the COVID-19 pandemic, there was a growing need to characterise the disease. A very important aspect is the ability to measure the immunisation extent, which can be achieved using antigen microarrays that quantitively measure the presence of COVID-related antibodies. A significant limitation for these tests was the complexity of manually analysing the results, and the limited availability of software for its analysis. In this paper, we describe the development of COVID-BIOCHIP, an ad-hoc web-based solution for the automatic analysis and visualisation of COVID-19 antigen microarray data results.


Assuntos
COVID-19 , Humanos , Análise em Microsséries , Pandemias , Software
8.
Cell Rep ; 38(13): 110611, 2022 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-35354052

RESUMO

The HIV-1 Envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs). Env is heavily glycosylated with host-derived N-glycans, and many bnAbs bind to, or are dependent upon, Env glycans for neutralization. Although glycan-binding bnAbs are frequently detected in HIV-infected individuals, attempts to elicit them have been unsuccessful because of the poor immunogenicity of Env N-glycans. Here, we report cross-reactivity of glycan-binding bnAbs with self- and non-self N-glycans and glycoprotein antigens from different life-stages of Schistosoma mansoni. Using the IAVI Protocol C HIV infection cohort, we examine the relationship between S. mansoni seropositivity and development of bnAbs targeting glycan-dependent epitopes. We show that the unmutated common ancestor of the N332/V3-specific bnAb lineage PCDN76, isolated from an HIV-infected donor with S. mansoni seropositivity, binds to S. mansoni cercariae while lacking reactivity to gp120. Overall, these results present a strategy for elicitation of glycan-reactive bnAbs which could be exploited in HIV-1 vaccine development.


Assuntos
Infecções por HIV , HIV-1 , Parasitos , Animais , Anticorpos Neutralizantes , Anticorpos Amplamente Neutralizantes , Anticorpos Anti-HIV , Humanos , Parasitos/metabolismo , Polissacarídeos/metabolismo
9.
Int J Biol Macromol ; 193(Pt B): 1562-1571, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34740693

RESUMO

Lectins from plants of the Diocleinae subtribe often exhibit specificity towards mannose/glucose and derived sugars, with some plants also displaying a second lectin specific to lactose/GalNAc. Here, we present a novel lectin from Collaea speciosa, named CsL, that displays specificity for GlcNAc/glucose. The lectin was extracted from Collaea speciosa seeds and purified by a single chromatographic step on a Sephadex G-50 matrix. In solution, the lectin appears as a dimeric protein composed of 25 kDa monomers. The protein is stable at pH 7-8 and dependent on divalent cations. CsL maintained its agglutination activity after heating to 90 °C for 1 h. Glycan array studies revealed that CsL binds to N-glycans with terminal GlcNAc residues, chitobiose and chitotriose moieties. The partial amino acid sequence of the lectin is similar to that of some lactose-specific lectins from the same subtribe. In contrast to other ConA-like lectins, CsL is not toxic to Artemia. Because of its remarkably different properties and specificity, this lectin could be the first member of a new group inside the Diocleinae lectins.


Assuntos
Fabaceae/química , Lectinas de Plantas/química , Polissacarídeos/química , Polissacarídeos/metabolismo , Sementes/química , Sequência de Aminoácidos , Animais , Artemia/metabolismo , Glucose/metabolismo , Hemaglutinação , Manose/metabolismo
10.
Sci Rep ; 11(1): 17958, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504130

RESUMO

Several plant lectins, or carbohydrate-binding proteins, interact with glycan moieties on the surface of immune cells, thereby influencing the immune response of these cells. Orysata, a mannose-binding lectin from rice, has been reported to exert immunomodulatory activities on insect cells. While the natural lectin is non-glycosylated, recombinant Orysata produced in the yeast Pichia pastoris (YOry) is modified with a hyper-mannosylated N-glycan. Since it is unclear whether this glycosylation can affect the YOry activity, non-glycosylated rOrysata was produced in Escherichia coli (BOry). In a comparative analysis, both recombinant Orysata proteins were tested for their carbohydrate specificity on a glycan array, followed by the investigation of the carbohydrate-dependent agglutination of red blood cells (RBCs) and the carbohydrate-independent immune responses in Drosophila melanogaster S2 cells. Although YOry and BOry showed a similar carbohydrate-binding profiles, lower concentration of BOry were sufficient for the agglutination of RBCs and BOry induced stronger immune responses in S2 cells. The data are discussed in relation to different hypotheses explaining the weaker responses of glycosylated YOry. In conclusion, these observations contribute to the understanding how post-translational modification can affect protein function, and provide guidance in the selection of the proper expression system for the recombinant production of lectins.


Assuntos
Drosophila melanogaster/citologia , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Lectinas de Ligação a Manose/metabolismo , Lectinas de Ligação a Manose/farmacologia , Oryza/química , Fagócitos/efeitos dos fármacos , Fagócitos/imunologia , Lectinas de Plantas/metabolismo , Lectinas de Plantas/farmacologia , Polissacarídeos/metabolismo , Animais , Linhagem Celular , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosilação , Hemaglutinação/efeitos dos fármacos , Lectinas de Ligação a Manose/genética , Fagócitos/metabolismo , Lectinas de Plantas/genética , Ligação Proteica , Coelhos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Saccharomycetales/genética , Saccharomycetales/metabolismo
11.
Glycobiology ; 31(8): 1005-1017, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-33909073

RESUMO

Paucimannosidic glycans are restricted to the core structure [Man1-3GlcNAc2Fuc0-1] of N-glycans and are rarely found in mammalian tissues. Yet, especially [Man2-3GlcNAc2Fuc1] have been found significantly upregulated in tumors, including in colorectal and liver cancer. Mannitou IgM is a murine monoclonal antibody that was previously shown to recognize Man3GlcNAc2 with an almost exclusive selectivity. Here, we have sought the definition of the minimal glycan epitope of Mannitou IgM, initiated by screening on a newly designed paucimannosidic glycan microarray; among the best binders were Man3GlcNAc2 and its α1,6 core-fucosylated variant, Man3GlcNAc2Fuc1. Unexpectedly and in contrast to earlier findings, Man5GlcNAc2-type structures bind equally well and a large tolerance was observed for substitutions on the α1,6 arm. It was confirmed that any substitution on the single α1,3-linked mannose completely abolishes binding. Surface plasmon resonance for kinetic measurements of Mannitou IgM binding, either directly on the glycans or as presented on omega-1 and kappa-5 soluble egg antigens from the helminth parasite Schistosoma mansoni, showed submicromolar affinities. To characterize the epitope in greater and atomic detail, saturation transfer difference nuclear magnetic resonance spectroscopy was performed with the Mannitou antigen-binding fragment. The STD-NMR data demonstrated the strongest interactions with the aliphatic protons H1 and H2 of the α1-3-linked mannose and weaker imprints on its H3, H4 and H5 protons. In conclusion, Mannitou IgM binding requires a nonsubstituted α1,3-linked mannose branch of paucimannose also on proteins, making it a highly specific tool for the distinction of concurrent human tumor-associated carbohydrate antigens.


Assuntos
Glicoproteínas , Schistosoma mansoni , Animais , Proteínas de Ligação a DNA , Epitopos/química , Fucose/metabolismo , Glicoproteínas/metabolismo , Humanos , Imunoglobulina M , Mamíferos/metabolismo , Proteínas de Membrana , Camundongos , Polissacarídeos/química , Schistosoma mansoni/química , Schistosoma mansoni/metabolismo
12.
Pathogens ; 10(4)2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33917609

RESUMO

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). A better understanding of its immunogenicity can be important for the development of improved diagnostics, therapeutics, and vaccines. Here, we report the longitudinal analysis of three COVID-19 patients with moderate (#1) and mild disease (#2 and #3). Antibody serum responses were analyzed using spike glycoprotein enzyme linked immunosorbent assay (ELISA), full-proteome peptide, and glycan microarrays. ELISA immunoglobulin A, G, and M (IgA, IgG, and IgM) signals increased over time for individuals #1 and #2, whereas #3 only showed no clear positive IgG and IgM result. In contrast, peptide microarrays showed increasing IgA/G signal intensity and epitope spread only in the moderate patient #1 over time, whereas early but transient IgA and stable IgG responses were observed in the two mild cases #2 and #3. Glycan arrays showed an interaction of antibodies to fragments of high-mannose and core N-glycans, present on the viral shield. In contrast to protein ELISA, microarrays allow for a deeper understanding of IgA, IgG, and IgM antibody responses to specific epitopes of the whole proteome and glycans of SARS-CoV-2 in parallel. In the future, this may help to better understand and to monitor vaccination programs and monoclonal antibodies as therapeutics.

13.
ACS Catal ; 11(15): 9052-9065, 2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-35662980

RESUMO

FUT8 is an essential α-1,6-fucosyltransferase that fucosylates the innermost GlcNAc of N-glycans, a process called core fucosylation. In vitro, FUT8 exhibits substrate preference for the biantennary complex N-glycan oligosaccharide (G0), but the role of the underlying protein/peptide to which N-glycans are attached remains unclear. Here, we explored the FUT8 enzyme with a series of N-glycan oligosaccharides, N-glycopeptides, and an Asn-linked oligosaccharide. We found that the underlying peptide plays a role in fucosylation of paucimannose (low mannose) and high-mannose N-glycans but not for complex-type N-glycans. Using saturation transfer difference (STD) NMR spectroscopy, we demonstrate that FUT8 recognizes all sugar units of the G0 N-glycan and most of the amino acid residues (Asn-X-Thr) that serve as a recognition sequon for the oligosaccharyltransferase (OST). The largest STD signals were observed in the presence of GDP, suggesting that prior FUT8 binding to GDP-ß-l-fucose (GDP-Fuc) is required for an optimal recognition of N-glycans. We applied genetic engineering of glycosylation capacities in CHO cells to evaluate FUT8 core fucosylation of high-mannose and complex-type N-glycans in cells with a panel of well-characterized therapeutic N-glycoproteins. This confirmed that core fucosylation mainly occurs on complex-type N-glycans, although clearly only at selected glycosites. Eliminating the capacity for complex-type glycosylation in cells (KO mgat1) revealed that glycosites with complex-type N-glycans when converted to high mannose lost the core Fuc. Interestingly, however, for erythropoietin that is uncommon among the tested glycoproteins in efficiently acquiring tetra-antennary N-glycans, two out of three N-glycosites obtained Fuc on the high-mannose N-glycans. An examination of the N-glycosylation sites of several protein crystal structures indicates that core fucosylation is mostly affected by the accessibility and nature of the N-glycan and not by the nature of the underlying peptide sequence. These data have further elucidated the different FUT8 acceptor substrate specificities both in vitro and in vivo in cells, revealing different mechanisms for promoting core fucosylation.

14.
Allergy ; 76(1): 233-246, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32568414

RESUMO

BACKGROUND: In high-income, temperate countries, IgE to allergen extracts is a risk factor for, and mediator of, allergy-related diseases (ARDs). In the tropics, positive IgE tests are also prevalent, but rarely associated with ARD. Instead, IgE responses to ubiquitous cross-reactive carbohydrate determinants (CCDs) on plant, insect and parasite glycoproteins, rather than to established major allergens, are dominant. Because anti-CCD IgE has limited clinical relevance, it may impact ARD phenotyping and assessment of contribution of atopy to ARD. METHODS: Using an allergen extract-based test, a glycan and an allergen (glyco)protein microarray, we mapped IgE fine specificity among Ugandan rural Schistosoma mansoni (Sm)-endemic communities, proximate urban communities, and importantly in asthmatic and nonasthmatic schoolchildren. RESULTS: Overall, IgE sensitization to extracts was highly prevalent (43%-73%) but allergen arrays indicated that this was not attributable to established major allergenic components of the extracts (0%-36%); instead, over 40% of all participants recognized CCD-bearing components. Using glycan arrays, we dissected IgE responses to specific glycan moieties and found that reactivity to classical CCD epitopes (core ß-1,2-xylose, α-1,3-fucose) was positively associated with sensitization to extracts, rural environment and Sm infection, but not with skin reactivity to extracts or sensitization to their major allergenic components. Interestingly, we discovered that reactivity to only a subset of core α-1,3-fucose-carrying N-glycans was inversely associated with asthma. CONCLUSIONS: CCD reactivity is not just an epiphenomenon of parasite exposure hampering specificity of allergy diagnostics; mechanistic studies should investigate whether specific CCD moieties identified here are implicated in the protective effect of certain environmental exposures against asthma.


Assuntos
Asma , Fucose , Alérgenos , Asma/diagnóstico , Asma/epidemiologia , Asma/etiologia , Carboidratos , Criança , Reações Cruzadas , Epitopos , Humanos , Imunoglobulina E
15.
Int J Mol Sci ; 21(15)2020 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-32722514

RESUMO

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 Candida albicans 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.


Assuntos
Candida albicans/metabolismo , Citometria de Fluxo , Fragmentos Fc das Imunoglobulinas/química , Lectinas Tipo C/química , Proteínas Recombinantes de Fusão/química , Candida albicans/citologia , Fragmentos Fc das Imunoglobulinas/genética , Lectinas Tipo C/genética , Ligantes , Proteínas Recombinantes de Fusão/genética
16.
Nat Commun ; 11(1): 973, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080177

RESUMO

Core-fucosylation is an essential biological modification by which a fucose is transferred from GDP-ß-L-fucose to the innermost N-acetylglucosamine residue of N-linked glycans. A single human enzyme α1,6-fucosyltransferase (FUT8) is the only enzyme responsible for this modification via the addition of an α-1,6-linked fucose to N-glycans. To date, the details of substrate recognition and catalysis by FUT8 remain unknown. Here, we report the crystal structure of FUT8 complexed with GDP and a biantennary complex N-glycan (G0), which provides insight into both substrate recognition and catalysis. FUT8 follows an SN2 mechanism and deploys a series of loops and an α-helix which all contribute in forming the binding site. An exosite, formed by one of these loops and an SH3 domain, is responsible for the recognition of branched sugars, making contacts specifically to the α1,3 arm GlcNAc, a feature required for catalysis. This information serves as a framework for inhibitor design, and helps to assess its potential as a therapeutic target.


Assuntos
Fucosiltransferases/química , Fucosiltransferases/metabolismo , Biocatálise , Sequência de Carboidratos , Domínio Catalítico , Cristalografia por Raios X , Glicosilação , Guanosina Difosfato/metabolismo , Humanos , Análise em Microsséries , Modelos Moleculares , Polissacarídeos/química , Polissacarídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Domínios de Homologia de src
17.
Sci Rep ; 9(1): 3522, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30837526

RESUMO

Core ß-1,2-xylose and α-1,3-fucose are antigenic motifs on schistosome N-glycans, as well as prominent IgE targets on some plant and insect glycoproteins. To map the association of schistosome infection with responses to these motifs, we assessed plasma IgE and IgG reactivity using microarray technology among Ugandans from rural Schistosoma mansoni (Sm)-endemic islands (n = 209), and from proximate urban communities with lower Sm exposure (n = 62). IgE and IgG responses to core ß-1,2-xylose and α-1,3-fucose modified N-glycans were higher in rural versus urban participants. Among rural participants, IgE and IgG to core ß-1,2-xylose were positively associated with Sm infection and concentration peaks coincided with the infection intensity peak in early adolescence. Responses to core α-1,3-fucose were elevated regardless of Sm infection status and peaked before the infection peak. Among urban participants, Sm infection intensity was predominantly light and positively associated with responses to both motifs. Principal component and hierarchical cluster analysis reduced the data to a set of variables that captured core ß-1,2-xylose- and α-1,3-fucose-specific responses, and confirmed associations with Sm and the rural environment. Responses to core ß-1,2-xylose and α-1,3-fucose have distinctive relationships with Sm infection and intensity that should further be explored for associations with protective immunity, and cross-reactivity with other exposures.


Assuntos
Imunoglobulina E/sangue , Imunoglobulina G/sangue , Polissacarídeos/imunologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/patologia , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Feminino , Glicosilação , Humanos , Masculino , Análise em Microsséries , Análise de Componente Principal , População Rural , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/epidemiologia , Esquistossomose mansoni/parasitologia , Uganda , População Urbana , Adulto Jovem
18.
Nanoscale Res Lett ; 13(1): 360, 2018 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-30421263

RESUMO

Carbohydrate-protein interactions mediate fundamental biological processes, such as fertilization, cell signaling, or host-pathogen communication. However, because of the enormous complexity of glycan recognition events, new tools enabling their analysis or applications emerge in recent years. Here, we describe the first preparation of neoglycoprotein functionalized fluorescent gold nanoclusters, containing a biantennary N-glycan G0 as targeting molecule, ovalbumin as carrier/model antigen, and a fluorescent gold core as imaging probe (G0-OVA-AuNCs). Subsequently, we demonstrate the utility of generated G0-OVA-AuNCs for specific sensing of plant lectins and in vitro imaging of dendritic cells.

19.
Anal Chem ; 90(21): 12536-12543, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30350619

RESUMO

A solution-phase enzymatic assay has been developed to track bacterial glycosyl hydrolase activity by surface-assisted MALDI-TOF mass spectrometry. Lactose was equipped with an azide-functionalized linker and was supplemented to bacterial cultures as an artificial substrate for bacterial ß-galactosidase enzyme. The azide linked glycoside probe was then covalently captured on an alkyne-functionalized indium tin oxide sample plate via a bio-orthogonal copper-catalyzed azide alkyne cycloaddition (CuAAC). The noncovalent immobilization of the alkyne capture tag via hydrophobic interactions on the ITO-sample plate allowed the analysis of the probe conjugate by surface-based mass spectrometry. The ratio of digested to nondigested lactose probe was then employed as a measure for bacterial hydrolase activity, which correlated well with bacterial growth measured by optical density. In addition, we established in a proof of concept experiment that the setup was well suited to identify antibiotic susceptibility of bacterial strains with a performance comparable to current state-of-the-art methods. While the proof of concept version is limited to the identification of a single enzyme activity, we envisage that the use of multiple substrate probes in a multiplexed version will allow the quantification of various glycosyl hydrolase activities with clinical relevance in a single experiment.


Assuntos
Alcinos/química , Azidas/química , Lactose/análogos & derivados , Sondas Moleculares/química , beta-Galactosidase/análise , Ampicilina/farmacologia , Antibacterianos/farmacologia , Aspergillus oryzae/enzimologia , Aspergillus oryzae/crescimento & desenvolvimento , Química Click , Cobre/química , Reação de Cicloadição , Ensaios Enzimáticos/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Técnicas de Sonda Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , beta-Galactosidase/química
20.
Chemistry ; 24(54): 14448-14460, 2018 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-29975429

RESUMO

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.

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