RESUMO
Herein, we describe the efficient gram-scale synthesis of α2,3- and α2,6-sialyllactose oligosaccharides as well as mimetics from N-acyl mannosamines and lactose in metabolically engineered bacterial cells grown at high cell density. We designed new Escherichia coli strains co-expressing sialic acid synthase and N-acylneuraminate cytidylyltransferase from Campylobacter jejuni together with the α2,3-sialyltransferase from Neisseria meningitidis or the α2,6-sialyltransferase from Photobacterium sp. JT-ISH-224. Using their mannose transporter, these new strains actively internalized N-acetylmannosamine (ManNAc) and its N-propanoyl (N-Prop), N-butanoyl (N-But) and N-phenylacetyl (N-PhAc) analogs and converted them into the corresponding sialylated oligosaccharides, with overall yields between 10 % and 39 % (200-700â mg.L-1 of culture). The three α2,6-sialyllactose analogs showed similar binding affinity for Sambucus nigra SNA-I lectin as for the natural oligosaccharide. They also proved to be stable competitive inhibitors of Vibrio cholerae neuraminidase. These N-acyl sialosides therefore hold promise for the development of anti-adhesion therapy against influenza viral infections.
Assuntos
Lactose , Neuraminidase , Neuraminidase/metabolismo , Escherichia coli/metabolismo , Sialiltransferases/metabolismo , Oligossacarídeos/químicaRESUMO
Influenza viruses bind to their target through a multivalent interaction of their hemagglutinins (HAs) with sialosides at the host cell surface. To fight the virus, one therapeutic approach consists in developing sialylated multivalent structures that can saturate the virus HAs and prevent the binding to host cells. We describe herein the biotechnological production of sialylated solid lipid microparticles (SSLMs) in 3 steps: (i) a microbiological step leading to the large-scale production of sialylated maltodextrins by metabolic engineering of an Escherichia coli strain, (ii) a new in vitro glycosylation process using the amylomaltase MalQ, based on the transglycosylation of the terminal sialoside ligand of the sialylated maltodextrin onto a long-chain alkyl glucoside, and (iii) the formulation of the final SSLMs presenting a multivalent sialic acid. We also describe the morphology and structure of the SSLMs and demonstrate their very promising properties as influenza virus inhibitors using hemagglutination inhibition and microneutralization assays on the human A/H1N1 pdm09 virus.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Humana , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A/metabolismo , Influenza Humana/tratamento farmacológico , Hemaglutininas Virais , Lipídeos , Glicoproteínas de Hemaglutininação de Vírus da InfluenzaRESUMO
l-Fucose and l-fucose-containing polysaccharides, glycoproteins or glycolipids play an important role in a variety of biological processes. l-Fucose-containing glycoconjugates have been implicated in many diseases including cancer and rheumatoid arthritis. Interest in fucose and its derivatives is growing in cancer research, glyco-immunology, and the study of host-pathogen interactions. l-Fucose can be extracted from bacterial and algal polysaccharides or produced (bio)synthetically. While deuterated glucose and galactose are available, and are of high interest for metabolic studies and biophysical studies, deuterated fucose is not easily available. Here, we describe the production of perdeuterated l-fucose, using glyco-engineered Escherichia coli in a bioreactor with the use of a deuterium oxide-based growth medium and a deuterated carbon source. The final yield was 0.2 g L-1 of deuterated sugar, which was fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We anticipate that the perdeuterated fucose produced in this way will have numerous applications in structural biology where techniques such as NMR, solution neutron scattering and neutron crystallography are widely used. In the case of neutron macromolecular crystallography, the availability of perdeuterated fucose can be exploited in identifying the details of its interaction with protein receptors and notably the hydrogen bonding network around the carbohydrate binding site.
Assuntos
Escherichia coli/metabolismo , Polissacarídeos/biossíntese , Polissacarídeos/químicaRESUMO
Strigolactones (SLs) influence the ability of legumes to associate with nitrogen-fixing bacteria. In this study, we determine the precise stage at which SLs influence nodulation. We show that SLs promote infection thread formation, as a null SL-deficient pea (Pisum sativum) mutant forms significantly fewer infection threads than wild-type plants, and this reduction can be overcome by the application of the synthetic SL GR24. We found no evidence that SLs influence physical events in the plant before or after infection thread formation, since SL-deficient plants displayed a similar ability to induce root hair curling in response to rhizobia or Nod lipochitooligosaccharides (LCOs) and SL-deficient nodules appear to fix nitrogen at a similar rate to those of wild-type plants. In contrast, an SL receptor mutant displayed no decrease in infection thread formation or nodule number, suggesting that SL deficiency may influence the bacterial partner. We found that this influence of SL deficiency was not due to altered flavonoid exudation or the ability of root exudates to stimulate bacterial growth. The influence of SL deficiency on infection thread formation was accompanied by reduced expression of some early nodulation genes. Importantly, SL synthesis is down-regulated by mutations in genes of the Nod LCO signaling pathway, and this requires the downstream transcription factor NSP2 but not NIN This, together with the fact that the expression of certain SL biosynthesis genes can be elevated in response to rhizobia/Nod LCOs, suggests that Nod LCOs may induce SL biosynthesis. SLs appear to influence nodulation independently of ethylene action, as SL-deficient and ethylene-insensitive double mutant plants display essentially additive phenotypes, and we found no evidence that SLs influence ethylene synthesis or vice versa.
Assuntos
Lactonas/farmacologia , Pisum sativum/fisiologia , Rhizobium/fisiologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Regulação para Baixo , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Lactonas/metabolismo , Lipopolissacarídeos/farmacologia , Mutação , Pisum sativum/efeitos dos fármacos , Pisum sativum/genética , Pisum sativum/microbiologia , Fenótipo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nodulação/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Simbiose/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
A conjugatable form of the tumour-associated carbohydrate antigen sialyl-Tn (Neu5Ac-α-2,6-GalNAc) was efficiently produced in Escherichia coli. Metabolically engineered E.â coli strains overexpressing the 6-sialyltransferase gene of Photobacterium sp. and CMP-Neu5Ac synthetase genes of Neisseria meningitidis were cultivated at high density in the presence of GalNAc-α-propargyl as the exogenous acceptor. The target disaccharides, which were produced on the scale of several hundreds of milligrams, were then conjugated by using copper(I)-catalysed azide-alkyne cycloaddition click chemistry to a fully synthetic and immunogenic scaffold with the aim to create a candidate anticancer vaccine. Four sialyl-Tn epitopes were introduced on the upper face of an azido-functionalised multivalent cyclopeptide scaffold, the lower face of which was previously modified by an immunogenic polypeptide, PADRE. The ability of the resulting glycoconjugate to interact with oncofoetal sialyl-Tn monoclonal antibodies was confirmed in ELISA assays.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Escherichia coli/metabolismo , Vacinas Sintéticas/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Antígenos Glicosídicos Associados a Tumores/química , Antígenos Glicosídicos Associados a Tumores/genética , Antígenos Glicosídicos Associados a Tumores/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/metabolismo , Cromatografia em Camada Fina , Química Click , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Epitopos/metabolismo , Engenharia Metabólica , Neisseria/enzimologia , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/imunologia , Peptídeos Cíclicos/metabolismo , Photobacterium/enzimologia , Sialiltransferases/genética , Sialiltransferases/metabolismo , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Glycan-protein interactions play a crucial role in physiological and pathological events. Hence, improving the isolation of carbohydrate-binding proteins (i.e., lectins and anti-glycan antibodies) from complex media might not only lead to a better understanding of their function, but also provide solutions for public health issues, such as water contamination or the need for universal blood plasma. Here we report a rapid and efficient method for producing carbohydrate-based affinity adsorbents combining enzymatic synthesis and metal-free click chemistry. Both simple and complex glycans (maltose, blood group antigensâ A, B, and H) were readily modified by the addition of a furyl group at the reducing end without the need for protecting groups and were then efficiently conjugated to maleimide-activated Sepharose particles through Diels-Alder cycloaddition. These neoglycoconjugates showed high efficiency for the purification of lectins (concanavalinâ A and Ulex europaeus agglutinin), as well as for the capture of anti-A and anti-B blood group antibodies, opening new prospects for glycoproteomics and for the development of universal blood plasma.
Assuntos
Furanos/química , Maleimidas/química , Oligossacarídeos/química , Polímeros/química , Receptores de Superfície Celular/química , Química Click , Concanavalina A/química , Concanavalina A/metabolismo , Reação de Cicloadição , Fluoresceína-5-Isotiocianato/química , Lectinas/química , Lectinas/metabolismo , Microscopia de Fluorescência , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Sefarose/química , Espectrofotometria InfravermelhoRESUMO
Root hairs are involved in water and nutrient uptake, and thereby in plant autotrophy. In legumes, they also play a crucial role in establishment of rhizobial symbiosis. To obtain a holistic view of Medicago truncatula genes expressed in root hairs and of their regulation during the first hours of the engagement in rhizobial symbiotic interaction, a high throughput RNA sequencing on isolated root hairs from roots challenged or not with lipochitooligosaccharides Nod factors (NF) for 4 or 20 h was carried out. This provided a repertoire of genes displaying expression in root hairs, responding or not to NF, and specific or not to legumes. In analyzing the transcriptome dataset, special attention was paid to pumps, transporters, or channels active at the plasma membrane, to other proteins likely to play a role in nutrient ion uptake, NF electrical and calcium signaling, control of the redox status or the dynamic reprogramming of root hair transcriptome induced by NF treatment, and to the identification of papilionoid legume-specific genes expressed in root hairs. About 10% of the root hair expressed genes were significantly up- or down-regulated by NF treatment, suggesting their involvement in remodeling plant functions to allow establishment of the symbiotic relationship. For instance, NF-induced changes in expression of genes encoding plasma membrane transport systems or disease response proteins indicate that root hairs reduce their involvement in nutrient ion absorption and adapt their immune system in order to engage in the symbiotic interaction. It also appears that the redox status of root hair cells is tuned in response to NF perception. In addition, 1176 genes that could be considered as "papilionoid legume-specific" were identified in the M. truncatula root hair transcriptome, from which 141 were found to possess an ortholog in every of the six legume genomes that we considered, suggesting their involvement in essential functions specific to legumes. This transcriptome provides a valuable resource to investigate root hair biology in legumes and the roles that these cells play in rhizobial symbiosis establishment. These results could also contribute to the long-term objective of transferring this symbiotic capacity to non-legume plants.
RESUMO
A fast chemoenzymatic synthesis of sialylated oligosaccharides containing C5-modified neuraminic acids is reported. Analogues of GM3 and GM2 ganglioside saccharidic portions where the acetyl group of NeuNAc has been replaced by a phenylacetyl (PhAc) or a propanoyl (Prop) moiety have been efficiently prepared with metabolically engineered E. coli bacteria. GM3 analogues were either obtained by chemoselective modification of biosynthetic N-acetyl-sialyllactoside (GM3 NAc) or by direct bacterial synthesis using C5-modified neuraminic acid precursors. The latter strategy proved to be very versatile as it led to an efficient synthesis of GM2 analogues. These glycomimetics were assessed against hemagglutinins and sialidases. In particular, the GM3 NPhAc displayed a binding affinity for Maackia amurensis agglutinin (MAA) similar to that of GM3 NAc, while being resistant to hydrolysis by Vibrio cholerae (VC) neuraminidase. A preliminary study with influenza viruses also confirmed a selective inhibition of N1 neuraminidase by GM3 NPhAc, suggesting potential developments for the detection of flu viruses and for fighting them.
Assuntos
Hemaglutininas/metabolismo , Engenharia Metabólica , Ácidos Neuramínicos/síntese química , Neuraminidase/antagonistas & inibidores , Oligossacarídeos/síntese química , Ácidos Siálicos/síntese química , Vibrio cholerae/enzimologia , Aglutininas/metabolismo , Animais , Bovinos , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrólise , Maackia/metabolismo , Ácidos Neuramínicos/química , Ácidos Neuramínicos/metabolismo , Ácidos Neuramínicos/farmacologia , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Oligossacarídeos/farmacologia , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Ácidos Siálicos/farmacologiaRESUMO
Blood transfusions are critically important in many medical procedures, but the presence of antigens on red blood cells (RBCs, erythrocytes) means that careful blood-typing must be carried out prior to transfusion to avoid adverse and sometimes fatal reactions following transfusion. Enzymatic removal of the terminal N-acetylgalactosamine or galactose of A- or B-antigens, respectively, yields universal O-type blood, but is inefficient. Starting with the family 98 glycoside hydrolase from Streptococcus pneumoniae SP3-BS71 (Sp3GH98), which cleaves the entire terminal trisaccharide antigenic determinants of both A- and B-antigens from some of the linkages on RBC surface glycans, through several rounds of evolution, we developed variants with vastly improved activity toward some of the linkages that are resistant to cleavage by the wild-type enzyme. The resulting enzyme effects more complete removal of blood group antigens from cell surfaces, demonstrating the potential for engineering enzymes to generate antigen-null blood from donors of various types.
Assuntos
Antígenos de Grupos Sanguíneos/metabolismo , Glicosídeo Hidrolases/metabolismo , Polissacarídeos/metabolismo , Antígenos de Grupos Sanguíneos/química , Configuração de Carboidratos , Sequência de Carboidratos , Eritrócitos/química , Eritrócitos/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Polissacarídeos/química , Streptococcus pneumoniae/enzimologiaRESUMO
Establishment of arbuscular mycorrhizal interactions involves plant recognition of diffusible signals from the fungus, including lipochitooligosaccharides (LCOs) and chitooligosaccharides (COs). Nitrogen-fixing rhizobial bacteria that associate with leguminous plants also signal to their hosts via LCOs, the so-called Nod factors. Here, we have assessed the induction of symbiotic signaling by the arbuscular mycorrhizal (Myc) fungal-produced LCOs and COs in legumes and rice (Oryza sativa). We show that Myc-LCOs and tetra-acetyl chitotetraose (CO4) activate the common symbiosis signaling pathway, with resultant calcium oscillations in root epidermal cells of Medicago truncatula and Lotus japonicus. The nature of the calcium oscillations is similar for LCOs produced by rhizobial bacteria and by mycorrhizal fungi; however, Myc-LCOs activate distinct gene expression. Calcium oscillations were activated in rice atrichoblasts by CO4, but not the Myc-LCOs, whereas a mix of CO4 and Myc-LCOs activated calcium oscillations in rice trichoblasts. In contrast, stimulation of lateral root emergence occurred following treatment with Myc-LCOs, but not CO4, in M. truncatula, whereas both Myc-LCOs and CO4 were active in rice. Our work indicates that legumes and non-legumes differ in their perception of Myc-LCO and CO signals, suggesting that different plant species respond to different components in the mix of signals produced by arbuscular mycorrhizal fungi.
Assuntos
Lotus/microbiologia , Medicago truncatula/microbiologia , Micorrizas/fisiologia , Oryza/microbiologia , Transdução de Sinais , Simbiose , Sinalização do Cálcio/efeitos dos fármacos , Quitina/análogos & derivados , Quitina/farmacologia , Quitosana , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucuronidase/metabolismo , Lipopolissacarídeos/farmacologia , Medicago truncatula/efeitos dos fármacos , Medicago truncatula/genética , Dados de Sequência Molecular , Micorrizas/efeitos dos fármacos , Oligossacarídeos/farmacologia , Oryza/efeitos dos fármacos , Oryza/genética , Plântula/efeitos dos fármacos , Plântula/microbiologia , Transdução de Sinais/efeitos dos fármacos , Simbiose/efeitos dos fármacosRESUMO
Lipochitin oligosaccharides (LCOs) are signaling molecules required by ecologically and agronomically important bacteria and fungi to establish symbioses with diverse land plants. In plants, oligo-chitins and LCOs can differentially interact with different lysin motif (LysM) receptors and affect innate immunity responses or symbiosis-related pathways. In animals, oligo-chitins also induce innate immunity and other physiological responses but LCO recognition has not been demonstrated. Here LCO and LCO-like compounds are shown to be biologically active in mammals in a structure dependent way through the modulation of angiogenesis, a tightly-regulated process involving the induction and growth of new blood vessels from existing vessels. The testing of 24 LCO, LCO-like or oligo-chitin compounds resulted in structure-dependent effects on angiogenesis in vitro leading to promotion, or inhibition or nil effects. Like plants, the mammalian LCO biological activity depended upon the presence and type of terminal substitutions. Un-substituted oligo-chitins of similar chain lengths were unable to modulate angiogenesis indicating that mammalian cells, like plant cells, can distinguish between LCOs and un-substituted oligo-chitins. The cellular mode-of-action of the biologically active LCOs in mammals was determined. The stimulation or inhibition of endothelial cell adhesion to vitronectin or fibronectin correlated with their pro- or anti-angiogenic activity. Importantly, novel and more easily synthesised LCO-like disaccharide molecules were also biologically active and de-acetylated chitobiose was shown to be the primary structural basis of recognition. Given this, simpler chitin disaccharides derivatives based on the structure of biologically active LCOs were synthesised and purified and these showed biological activity in mammalian cells. Since important chronic disease states are linked to either insufficient or excessive angiogenesis, LCO and LCO-like molecules may have the potential to be a new, carbohydrate-based class of therapeutics for modulating angiogenesis.
Assuntos
Glycine max/química , Lipopolissacarídeos/farmacologia , Mamíferos/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Simbiose/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Acilação/efeitos dos fármacos , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Dissacarídeos/química , Dissacarídeos/farmacologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Técnicas In Vitro , Integrinas/metabolismo , Lipopolissacarídeos/química , Ratos Endogâmicos F344RESUMO
While chitooligosaccharides (COs) derived from fungal chitin are potent elicitors of defense reactions, structurally related signals produced by certain bacteria and fungi, called lipo-chitooligosaccharides (LCOs), play important roles in the establishment of symbioses with plants. Understanding how plants distinguish between friend and foe through the perception of these signals is a major challenge. We report the synthesis of a range of COs and LCOs, including photoactivatable probes, to characterize a membrane protein from the legume Medicago truncatula. By coupling photoaffinity labeling experiments with proteomics and transcriptomics, we identified the likely LCO-binding protein as LYR3, a lysin motif receptor-like kinase (LysM-RLK). LYR3, expressed heterologously, exhibits high-affinity binding to LCOs but not COs. Homology modeling, based on the Arabidopsis CO-binding LysM-RLK AtCERK1, suggests that LYR3 could accommodate the LCO in a conserved binding site. The identification of LYR3 opens up ways for the molecular characterization of LCO/CO discrimination.
Assuntos
Quitina/análogos & derivados , Medicago truncatula/fisiologia , Oligossacarídeos/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Lipídeos/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oligossacarídeos/química , Proteínas de Plantas/química , Alinhamento de Sequência , SimbioseRESUMO
Functionalized oligosaccharides are useful intermediates to prepare products for biological research or for the development of advanced functional materials. Here, we report the unprecedented use of aniline as an efficient organocatalyst reaction with "clickable" (azide or alkyne) amine for the transimination-mediated reductive amination of a chitooligosaccharide. Moreover, we demonstrate that alkyne-bearing aniline constitutes an excellent tool for the easy derivatization of chitosan oligosaccharides. Evidence for such improvement has been illustrated by the straightforward design of a FRET substrate to probe chitinase activity and of amphiphilic polycaprolactone-grafted-chitosan. This efficient methodology paves the way to the preparation of novel chitosan oligosaccharide-based advanced materials.
Assuntos
Compostos de Anilina/química , Química Click , Oligossacarídeos/síntese química , Aminação , Configuração de Carboidratos , Catálise , Dados de Sequência Molecular , Oligossacarídeos/química , OxirreduçãoRESUMO
A chemo-biotechnological approach is reported for the synthesis of TMG-chitooligomycins, CO-n (NMe(3)). Their abilities to inhibit ß-N-acetylhexosaminidases (HexNAcases), from Aspergillus oryzae (AoHex, fungi), Canavalia ensiformis (CeHex, plant) HexNAcases and a chitobiase from Serratia marcescens (SmCHB, bacteria) were studied and compared with their precursors CO-n (N). CO-n (NMe(3)) were revealed as potent inhibitors for AoHex and SmCHB with a proved chain length effect while CO-n (N) was a highly selective inhibitor of SmCHB. This route can be considered as the privileged way to produce easily and in large scale a wide range of size-defined chitooligosaccharide-based inhibitors to fine-tune the structure-activity relationships for inhibition of HexNAcases from various origins.
Assuntos
Inibidores Enzimáticos/química , Glicosídeo Hidrolases/metabolismo , Álcoois Açúcares/química , Álcoois Açúcares/farmacologia , beta-N-Acetil-Hexosaminidases/metabolismo , Acetilglucosaminidase/metabolismo , Aspergillus oryzae/enzimologia , Canavalia/enzimologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Relação Estrutura-Atividade , Álcoois Açúcares/síntese químicaRESUMO
The α-Gal epitope is a carbohydrate structure, Galα-3Galß-4GlcNAc-R, expressed on glycoconjuguates in many mammals, but not in humans. Species that do not express this epitope have present in their serum large amounts of natural anti-Gal antibodies, which contribute to organ hyperacute rejection during xenotransplantation. We first describe the efficient conversion of lactose into isoglobotriaose (Galα-3Galß-4Glc) using high cell density cultures of a genetically engineered Escherichia coli strain expressing the bovine gene for α-1,3-galactosyltransferase. Attempts to produce the Galili pentasaccharide (Galα-3Galß-4GlcNAcß-3Galß-4Glc) by additionally expressing the Neisseria meningitis lgtA gene for ß-1,3-N-acetylglucosaminyltransferase and the Helicobacter pylori gene for ß-1,4-galactosyltransferase were unsuccessful and led to the formation of a series of long chain oligosaccharides formed by the repeated addition of the trisaccharide motif [Galß-4GlcNAcß-3Galα-3] onto a lacto-N-neotetraose primer. The replacement of LgtA by a more specific ß-1,3-N-acetylglucosaminyltransferase from H. pylori, which was unable to glycosylate α-galactosides, prevented the formation of these unwanted compounds and allowed the successful formation of the Galili pentasaccharide and longer α-Gal epitopes.
Assuntos
Escherichia coli/metabolismo , Engenharia Metabólica , Trissacarídeos/biossíntese , Escherichia coli/química , Escherichia coli/genética , Trissacarídeos/química , Trissacarídeos/metabolismoRESUMO
The cluster of genes of capsular K5 heparosan is composed of three regions, involved in the synthesis and the exportation of the polysaccharide. The region 2 possesses all the necessary genes involved in the synthesis of heparosan, namely kfiA, encoding alpha-4-N-acetylglucosaminyltransferase, kfiD, encoding ß-3-glucuronyl transferase, kfiC, encoding UDP-glucose dehydrogenase (UDP-glucuronic acid synthesis), and kfiB encoding a protein of unknown function. The cloning and expression of kfiADCB into Escherichia coli K-12 were found to be sufficient for the production of heparosan, which accumulates in the cells due to a lack of the exporting system. The concentration of recombinant heparosan reached one gram per liter under fed-batch cultivation. The cytoplasmic localization of heparosan inside the bacteria allowed subsequent enzymatic modifications such as a partial degradation with K5 lyase when expressed intracellularly. Under these conditions, the production of DP 2-10 oligosaccharides occurred intracellularly, at a concentration similar to that of recombinant intracellular heparosan.
Assuntos
Dissacarídeos/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Liases/genética , Liases/metabolismo , Engenharia Metabólica , Oligossacarídeos/biossíntese , Dissacarídeos/química , Dissacarídeos/genética , Escherichia coli/citologia , Oligossacarídeos/química , Oligossacarídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The formation of root nodules and arbuscular mycorrhizal (AM) roots is controlled by a common signaling pathway including the calcium/calmodulin-dependent kinase Doesn't Make Infection3 (DMI3). While nodule initiation by lipochitooligosaccharide (LCO) Nod factors is well characterized, diffusible AM fungal signals were only recently identified as sulfated and nonsulfated LCOs. Irrespective of different outcomes, the perception of symbiotic LCOs in Medicago truncatula is mediated by the LysM receptor kinase M. truncatula Nod factor perception (MtNFP). To shed light on transcriptional responses toward symbiotic LCOs and their dependence on MtNFP and Ca(2+) signaling, we performed genome-wide expression studies of wild-type, Nod-factor-perception mutant1, and dmi3 mutant roots challenged with Myc- and Nod-LCOs. We show that Myc-LCOs lead to transient, quick responses in the wild type, whereas Nod-LCOs require prolonged incubation for maximal expression activation. While Nod-LCOs are most efficient for an induction of persistent transcriptional changes, sulfated Myc-LCOs are less active, and nonsulfated Myc-LCOs display the lowest capacity to activate and sustain expression. Although all symbiotic LCOs up-regulated a common set of genes, discrete subsets were induced by individual LCOs, suggesting common and specific functions for these in presymbiotic signaling. Surprisingly, even sulfated fungal Myc-LCOs and Sinorhizobium meliloti Nod-LCOs, having very similar structures, each elicited discrete subsets of genes, while a mixture of both Myc-LCOs activated responses deviating from those induced by single treatments. Focusing on the precontact phase, we identified signaling-related and transcription factor genes specifically up-regulated by Myc-LCOs. Comparative gene expression studies in symbiotic mutants demonstrated that transcriptional reprogramming by AM fungal LCOs strictly depends on MtNFP and largely requires MtDMI3.
Assuntos
Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Medicago truncatula/genética , Micorrizas/fisiologia , Proteínas de Plantas/metabolismo , Simbiose/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Bioensaio , Difusão/efeitos dos fármacos , Genes de Plantas/genética , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Cinética , Medicago truncatula/efeitos dos fármacos , Medicago truncatula/microbiologia , Modelos Biológicos , Mutação/genética , Micorrizas/efeitos dos fármacos , Proteínas de Plantas/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Simbiose/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
The prion protein (PrP) resides in lipid rafts in vivo, and lipids modulate misfolding of the protein to infectious isoforms. Here we demonstrate that binding of recombinant PrP to model raft membranes requires the presence of ganglioside GM1. A combination of liquid- and solid-state NMR revealed the binding sites of PrP to the saccharide head group of GM1. The binding epitope for GM1 was mapped to the folded C-terminal domain of PrP, and docking simulations identified key residues in the C-terminal region of helix C and the loop between strand S2 and helix B. Crucially, this region of PrP is linked to prion resistance in vivo, and structural changes caused by lipid binding in this region may explain the requirement for lipids in the generation of infectious prions in vitro.
Assuntos
Gangliosídeo G(M1)/metabolismo , Microdomínios da Membrana/metabolismo , Príons/metabolismo , Sítios de Ligação , Simulação por Computador , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Príons/química , Príons/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
We have previously reported the efficient conversion of lactose into 3'-sialyllactose by high cell density cultures of a genetically engineered Escherichia coli strain expressing the Neisseria meningitidis gene for alpha-(2-->3)-sialyltransferase [Fierfort, N.; Samain, E. J. Biotechnol. 2008, 134, 261-265.]. First attempts to use a similar strategy to produce 6'-sialyllactose with a strain expressing alpha-(2-->6)-sialyltransferase from the Photobacterium sp. JT-ISH-224 led to the production of a trisaccharide that was identified as KDO-lactose (2-keto-3-deoxy-manno-octonyllactose). This result showed that alpha-(2-->6)-sialyltransferase was able to use CMP-KDO as sugar donor and preferentially used CMP-KDO over CMP-Neu5Ac. By reducing the expression level of the sialyltransferase gene and increasing that of the neuABC genes, we have been able to favour the formation of 6'-sialyllactose and to prevent the formation of KDO-lactose. However, in this case, a third lactose derivative, which was identified as 6,6'-disialyllactose, was also produced. Formation of 6,6'-disialyllactose was mainly observed under conditions of lactose shortage. On the other hand, when the culture was continuously fed with an excess of lactose, 6'-sialyllactose was almost the only product detected and its final concentration was higher than 30g/L of culture medium.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética/métodos , Lactose/biossíntese , Photobacterium/enzimologia , Sialiltransferases/genética , Expressão Gênica , Lactose/análogos & derivados , Espectroscopia de Ressonância Magnética , Photobacterium/genética , Trissacarídeos/biossínteseRESUMO
The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-beta-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-beta-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.