RESUMEN
Free polymannose-type oligosaccharides (fOS) are processed by cytosolic enzymes to generate Man5GlcNAc which is transferred to lysosomes and degraded. Lysosomal fOS import was demonstrated in vitro but is poorly characterized in part due to lack of convenient substrates. As chitooligosaccharides (COS, oligomers ß1,4-linked GlcNAc) block [3H]Man5GlcNAc transport into lysosomes, we asked if COS are themselves transported and if so, can they be chemically modified to generate fluorescent substrates. We show that COS are degraded by lysosomal hydrolases to generate GlcNAc, and robust ATP-dependent transport of [3H]COS2/4 di and tetrasaccharides into intact rat liver lysosomes was observed only after blocking lysosomal [3H]GlcNAc efflux with cytochalasin B. As oligosaccharides with unmodified reducing termini are the most efficient inhibitors of [3H]COS2/4 and [3H]Man5GlcNAc transport, the non-reducing GlcNAc residue of COS2-4 was de-N-acetylated using Sinorhizobium meliloti NodB, and the resulting amine substituted with rhodamine B (RB) to yield RB-COS2-4. The fluorescent compounds inhibit [3H]Man5GlcNAc transport and display temperature-sensitive, ATP-dependent transport into a sedimentable compartment that is ruptured with the lysosomotropic agent L-methyl methionine ester. Once in this compartment, RB-COS3 is converted to RB-COS2 further identifying it as the lysosomal compartment. RB-COS2/3 and [3H]Man5GlcNAc transports are blocked similarly by competing sugars, and are partially inhibited by the vacuolar ATPase inhibitor bafilomycin and high concentrations of the P-type ATPase inhibitor orthovanadate. These data show that Man5GlcNAc, COS2/4 and RB-COS2/3 are transported into lysosomes by the same or closely related mechanism and demonstrate the utility of COS modified at their non-reducing terminus to study lysosomal oligosaccharide transport.
Asunto(s)
Hígado , Lisosomas , Ratas , Animales , Hígado/metabolismo , Lisosomas/metabolismo , Oligosacáridos/metabolismo , Transporte Biológico , Adenosina Trifosfato/metabolismoRESUMEN
Lysin motif receptor-like kinases (LysM-RLKs) are involved in the perception of chitooligosaccharides (COs) and related lipochitooligosaccharides (LCOs) in plants. Expansion and divergence of the gene family during evolution have led to various roles in symbiosis and defense. By studying proteins of the LYR-IA subclass of LysM-RLKs of the Poaceae, we show here that they are high-affinity LCO-binding proteins with a lower affinity for COs, consistent with a role in LCO perception to establish arbuscular mycorrhiza (AM). In Papilionoid legumes, whole-genome duplication has resulted in two LYR-IA paralogs, MtLYR1 and MtNFP in Medicago truncatula, with MtNFP playing an essential role in root nodule symbiosis with nitrogen-fixing rhizobia. We show that MtLYR1 has retained the ancestral LCO-binding characteristic and is dispensable for AM. Domain swapping between the three LysMs of MtNFP and MtLYR1 and mutagenesis in MtLYR1 suggest that the MtLYR1 LCO-binding site is on the second LysM and that divergence in MtNFP led to better nodulation, but surprisingly with decreased LCO binding. These results suggest that divergence of the LCO-binding site has been important for the evolution of a role of MtNFP in nodulation with rhizobia.
Asunto(s)
Medicago truncatula , Micorrizas , Medicago truncatula/genética , Medicago truncatula/metabolismo , Proteínas de Plantas/metabolismo , Micorrizas/metabolismo , Simbiosis/genética , Quitina/metabolismoRESUMEN
Soluble fragments of peptidoglycan called muropeptides are released from the cell wall of bacteria as part of their metabolism or as a result of biological stresses. These compounds trigger immune responses in mammals and plants. In bacteria, they play a major role in the induction of antibiotic resistance. The development of efficient methods to produce muropeptides is, therefore, desirable both to address their mechanism of action and to design new antibacterial and immunostimulant agents. Herein, we engineered the peptidoglycan recycling pathway of Escherichia coli to produce N-acetyl-ß-D-glucosaminyl-(1â4)-1,6-anhydro-N-acetyl-ß-D-muramic acid (GlcNAc-anhMurNAc), a common precursor of Gram-negative and Gram-positive muropeptides. Inactivation of the hexosaminidase nagZ gene allowed the efficient production of this key disaccharide, providing access to Gram-positive muropeptides through subsequent chemical peptide conjugation. E. coli strains deficient in both NagZ hexosaminidase and amidase activities further enabled the inâ vivo production of Gram-negative muropeptides containing meso-diaminopimelic acid, a rarely available amino acid.
Asunto(s)
Escherichia coli , Peptidoglicano , Escherichia coli/metabolismo , Peptidoglicano/metabolismo , Bacterias/metabolismo , Pared Celular/metabolismo , HexosaminidasasRESUMEN
In the barley ß-D-glucan glucohydrolase, a glycoside hydrolase family 3 (GH3) enzyme, the Trp286/Trp434 clamp ensures ß-D-glucosides binding, which is fundamental for substrate hydrolysis during plant growth and development. We employ mutagenesis, high-resolution X-ray crystallography, and multi-scale molecular modelling methods to examine the binding and conformational behaviour of isomeric ß-D-glucosides during substrate-product assisted processive catalysis that operates in GH3 hydrolases. Enzyme kinetics reveals that the W434H mutant retains broad specificity, while W434A behaves as a strict (1,3)-ß-D-glucosidase. Investigations of reactant movements on the nanoscale reveal that processivity is sensitive to mutation-specific alterations of the tryptophan clamp. While wild-type and W434H utilise a lateral cavity for glucose displacement and sliding of (1,3)-linked hydrolytic products through the catalytic site without dissociation, consistent with their high hydrolytic rates, W434A does not adopt processive catalysis. Phylogenomic analyses of GH3 hydrolases disclose the evolutionary advantage of the tryptophan clamp that confers broad specificity, high catalytic efficiency, and processivity.
Asunto(s)
Glicósido Hidrolasas , Triptófano , Cristalografía por Rayos X , Glucosa , Glucosidasas/química , Glucósidos , Glicósido Hidrolasas/metabolismo , Glicósidos , Cinética , Plantas/metabolismo , Especificidad por SustratoRESUMEN
Chitin oligosaccharides (COs) hold high promise as organic fertilizers in the ongoing agro-ecological transition. Short- and long-chain COs can contribute to the establishment of symbiotic associations between plants and microorganisms, facilitating the uptake of soil nutrients by host plants. Long-chain COs trigger plant innate immunity. A fine investigation of these different signaling pathways requires improving the access to high-purity COs. Here, we used the response surface methodology to optimize the production of COs by enzymatic hydrolysis of water-soluble chitin (WSC) with hen egg-white lysozyme. The influence of WSC concentration, its acetylation degree, and the reaction time course were modelled using a Box-Behnken design. Under optimized conditions, water-soluble COs up to the nonasaccharide were formed in 51% yield and purified to homogeneity. This straightforward approach opens new avenues to determine the complex roles of COs in plants.
Asunto(s)
Quitina/química , Muramidasa/química , Oligosacáridos/química , Acetilación , HidrólisisRESUMEN
Lipo-chitinoligosaccharides (LCOs) are key molecules for the establishment of plant-microorganisms symbiosis. Interactions of leguminous crops with nitrogen-fixing rhizobial bacteria involve Nod factors, while Myc-LCOs improve the association of most plants with arbuscular mycorrhizal fungi. Both Nod factors and Myc-LCOs are composed of a chitinoligosaccharide fatty acylated at the non-reducing end accompanied with various substituting groups. One straightforward way to access LCOs is starting from chitin hydrolysate, an abundant polysaccharide found in crustacean shells, followed by regioselective enzymatic cleavage of an acetyl group from the non-reducing end of chitin tetra- or pentaose, and subsequent chemical introduction of N-acyl group. In the present work, we describe the in vitro synthesis of LCO precursors on preparative scale. To this end, Sinorhizobium meliloti chitin deacetylase NodB was produced in high yield in E. coli as a thioredoxin fusion protein. The recombinant enzyme was expressed in soluble and catalytically active form and used as an efficient biocatalyst for N-deacetylation of chitin tetra- and pentaose.
Asunto(s)
Amidohidrolasas/biosíntesis , Amidohidrolasas/metabolismo , Lipopolisacáridos/biosíntesis , Rhizobium/metabolismo , Amidohidrolasas/aislamiento & purificación , Lipopolisacáridos/química , Estructura Molecular , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Rhizobium/enzimologíaRESUMEN
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.
Asunto(s)
Furanos/química , Maleimidas/química , Oligosacáridos/química , Polímeros/química , Receptores de Superficie Celular/química , Química Clic , Concanavalina A/química , Concanavalina A/metabolismo , Reacción de Cicloadición , Fluoresceína-5-Isotiocianato/química , Lectinas/química , Lectinas/metabolismo , Microscopía Fluorescente , Unión Proteica , Receptores de Superficie Celular/metabolismo , Sefarosa/química , Espectrofotometría InfrarrojaRESUMEN
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.