Oyster mushrooms are an interesting source of biologically active glucans and other polysaccharides. This work is devoted to the isolation and structural characterization of polysaccharides from basidiocarps of the cultivated oyster mushroom, Pleurotus ostreatus. Five polysaccharidic fractions were obtained by subsequent extraction with cold water, hot water and two subsequent extractions with 1 m sodium hydroxide. Branched partially methoxylated mannogalactan and slightly branched (1→6)-β-d-glucan predominated in cold- and hot-water-soluble fractions, respectively. Alternatively, these polysaccharides were obtained by only hot water extraction and subsequent two-stage chromatographic separation. The alkali-soluble parts originating from the first alkali extraction were then fractionated by dissolution in dimethyl sulfoxide (DMSO). The polysaccharide insoluble in DMSO was identified as linear (1→3)-α-d-glucan, while branched (1→3)(1→6)-β-d-glucans were found to be soluble in DMSO. The second alkaline extract contained the mentioned branched β-d-glucan together with some proteins. Finally, the alkali insoluble part was a cell wall complex of chitin and β-d-glucans.
- MeSH
- chemická frakcionace MeSH
- chromatografie MeSH
- fungální polysacharidy chemie izolace a purifikace MeSH
- fytonutrienty chemie izolace a purifikace MeSH
- glukany chemie MeSH
- molekulární struktura MeSH
- monosacharidy chemie MeSH
- Pleurotus chemie MeSH
- plodnice hub chemie MeSH
- spektrální analýza MeSH
- Publikační typ
- časopisecké články MeSH
The linear interaction energy (LIE) method to compute binding free energies is applied to lectin-monosaccharide complexes. Here, we calculate the binding free energies of monosaccharides to the Ralstonia solanacearum lectin (RSL) and the Pseudomonas aeruginosa lectin-II (PA-IIL). The standard LIE model performs very well for RSL, whereas the PA-IIL system, where ligand binding involves two calcium ions, presents a major challenge. To overcome this, we explore a new variant of the LIE model, where ligand-metal ion interactions are scaled separately. This model also predicts the saccharide binding preference of PA-IIL on mutation of the receptor, which may be useful for protein engineering of lectins.
In this study, interactions of selected monosaccharides with the Pseudomonas aeruginosa Lectin II (PA-IIL) are analyzed in detail. An interesting feature of the PA-IIL binding is that the monosaccharide is interacting via two calcium ions and the binding is unusually strong for protein-saccharide interaction. We have used Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) and normal mode analysis to calculate the free energy of binding. The impact of intramolecular hydrogen bond network for the lectin/monosaccharide interaction is also analyzed.
- MeSH
- bakteriální adheziny chemie metabolismus MeSH
- entropie MeSH
- konformace proteinů MeSH
- konformace sacharidů MeSH
- lektiny chemie metabolismus MeSH
- monosacharidy chemie metabolismus MeSH
- Pseudomonas aeruginosa MeSH
- simulace molekulární dynamiky MeSH
- statická elektřina MeSH
- vápník metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Three new nervogenic acid glycosides, 1-O-alpha-L-rhamnopyranosyl 3,5-bis(3-methyl-but-2-enyl)-4-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl]-benzoate, 3,5-bis(3-methyl-but-2-enyl)-4-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl]-benzoic acid, and bis{3,5-bis(3-methyl-but-2-enyl)-4-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl]-benzoyl} 1,2-O-beta-d-glucopyranose, which we named condobulbosides A-C, were isolated from a methanol extract of the leaves of Liparis condylobulbon together with an apigenin C-glycoside, schaftoside. Their structures were established on the basis of spectral techniques, namely, UV, IR, HR-MS spectroscopy, both 1D and 2D NMR experiments, and chemical reactions.
- MeSH
- elektrolyty chemie terapeutické užití MeSH
- financování organizované MeSH
- lidé MeSH
- monosacharidy chemie terapeutické užití MeSH
- osmolární koncentrace MeSH
- osmotický tlak fyziologie účinky léků MeSH
- parenterální infuze metody využití MeSH
- salinita MeSH
- statistika jako téma MeSH
- tekutinová terapie metody využití MeSH
- způsoby aplikace léků MeSH
- Check Tag
- lidé MeSH
This article is focused on the application of two types of docking software, AutoDock and DOCK. It is aimed at studying the interactions of a calcium-dependent bacterial lectin PA-IIL (from Pseudomonas aeruginosa) and its in silico mutants with saccharide ligands. The effect of different partial charges assigned to the calcium ions was tested and evaluated in terms of the best agreement with the crystal structure. The results of DOCK were further optimized by molecular dynamics and rescored using AMBER. For both software, the agreement of the docked structures and the provided binding energies were evaluated in terms of prediction accuracy. This was carried out by comparing the computed results to the crystal structures and experimentally determined binding energies, respectively. The performance of both docking software applied on a studied problem was evaluated as well. The molecular docking methods proved efficient in identifying the correct binding modes in terms of geometry and partially also in predicting the preference changes caused by mutation. Obtaining a reasonable in silico method for the prediction of lectin-saccharide interactions may be possible in the future.
- MeSH
- bakteriální adheziny genetika chemie MeSH
- financování organizované MeSH
- informatika MeSH
- kvarterní struktura proteinů MeSH
- lektiny genetika chemie MeSH
- ligandy MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- monosacharidy chemie MeSH
- mutageneze cílená statistika a číselné údaje MeSH
- počítačová simulace MeSH
- Pseudomonas aeruginosa genetika chemie MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- software MeSH
- termodynamika MeSH
- vápník chemie MeSH
- vazebná místa genetika MeSH
- MeSH
- arabinosa chemie izolace a purifikace MeSH
- Chlorella cytologie chemie MeSH
- chromatografie plynová metody využití MeSH
- finanční podpora výzkumu jako téma MeSH
- galaktosa chemie izolace a purifikace MeSH
- kyselina glukarová chemie izolace a purifikace MeSH
- monosacharidy analýza chemie MeSH
- polysacharidy analýza chemie MeSH
- spektrální analýza metody využití MeSH
BACKGROUND: Lectins are proteins of non-immune origin capable of binding saccharide structures with high specificity and affinity. Considering the high encoding capacity of oligosaccharides, this makes lectins important for adhesion and recognition. The present study is devoted to the PA-IIL lectin from Pseudomonas aeruginosa, an opportunistic human pathogen capable of causing lethal complications in cystic fibrosis patients. The lectin may play an important role in the process of virulence, recognizing specific saccharide structures and subsequently allowing the bacteria to adhere to the host cells. It displays high values of affinity towards monosaccharides, especially fucose--a feature caused by unusual binding mode, where two calcium ions participate in the interaction with saccharide. Investigating and understanding the nature of lectin-saccharide interactions holds a great potential of use in the field of drug design, namely the targeting and delivery of active compounds to the proper site of action. RESULTS: In vitro site-directed mutagenesis of the PA-IIL lectin yielded three single point mutants that were investigated both structurally (by X-ray crystallography) and functionally (by isothermal titration calorimetry). The mutated amino acids (22-23-24 triad) belong to the so-called specificity binding loop responsible for the monosaccharide specificity of the lectin. The mutation of the amino acids resulted in changes to the thermodynamic behaviour of the mutants and subsequently in their relative preference towards monosaccharides. Correlation of the measured data with X-ray structures provided the molecular basis for rationalizing the affinity changes. The mutations either prevent certain interactions to be formed or allow formation of new interactions--both of afore mentioned have strong effects on the saccharide preferences. CONCLUSION: Mutagenesis of amino acids forming the specificity binding loop allowed identification of one amino acid that is crucial for definition of the lectin sugar preference. Altering specificity loop amino acids causes changes in saccharide-binding preferences of lectins derived from PA-IIL, via creation or blocking possible binding interactions. This finding opens a gate towards protein engineering and subsequent protein design to refine the desired binding properties and preferences, an approach that could have strong potential for drug design.
- MeSH
- bakteriální adheziny genetika chemie MeSH
- chromatografie afinitní MeSH
- financování organizované MeSH
- jednonukleotidový polymorfismus MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- lektiny genetika chemie MeSH
- molekulární modely MeSH
- monosacharidy chemie MeSH
- mutageneze cílená MeSH
- proteinové inženýrství MeSH
- Pseudomonas aeruginosa genetika MeSH
- Ralstonia solanacearum chemie MeSH
- rekombinantní proteiny genetika izolace a purifikace MeSH
- rostlinné lektiny chemie MeSH
- substituce aminokyselin MeSH
- vazebná místa MeSH
- MeSH
- genetické inženýrství MeSH
- glykosylace MeSH
- monosacharidy biosyntéza chemie MeSH
- nemoc z ukládání kyseliny sialové enzymologie metabolismus MeSH
- neurony metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- virové nemoci genetika metabolismus patologie MeSH
- Publikační typ
- přehledy MeSH
- techniky in vitro MeSH
