Project: LECARB – Protein-Carbohydrate Interactions: Pathogen Recognition Phenomena
Person in Charge: Assoc. prof. RNDr. Michaela Wimmerová, Ph.D.
Host institution: National Centre for Biomolecular Research, Faculty of Science, Masaryk University
Country of Origin: Slovak republic
Country of scientific activity: USA, Ireland
Project duration: 35 months
Scientific panel: Life sciences
Carbohydrates play an important role in many biological processes like cell recognition, signalling, differentiation, cancerogenesis and others. Carbohydrate interactions with proteins - lectins are also involved in the first step of pathogenic bacteria adhesion, invasion and infectivity. This project proposal is focussed on the detailed studies of lectin-carbohydrate systems predominantly by number of new NMR techniques which are complementary to surface plasmon resonance and microcalorimetry methods. NMR is the most powerful method for detailed structural study of molecules involved in interactions. The advanced NMR techniques should give details on the binding site, the identities of lectin amino acids involved and functional groups of carbohydrate ligand involved in the interaction and other details. This information is extremely important for effective new drug design. The proposed project will benefit from experience with lectin-carbohydrate interactions already studied at the National Centre for Biomolecular Research by other physico-chemical methods and from current NMR instrumentation available at the Centre.
The ongoing project summary
This project is focussed on a study of lectins that might play a key role in primary recognition of host organism by pathogens. The main project objectives for this 1st year period were to study lectin-carbohydrate interactions by nuclear magnetic resonance (NMR) techniques and by measurement and interpretation of appropriate NMR spectra. This objective was achieved. The lectin that was studied originates from bacterium Ralstonia solanacearum (RS lectin, RSL). This lectin was chosen due to its properties as a good model system. The interactions of this lectin with carbohydrate ligands were studied by saturation transfer difference (STD) NMR method. Large number of STD experiments was performed (for example STD experiments with the lectin and L-fucose – as its strongest binding ligand, with mixture of monosaccharides as the potential ligands, experiments distinguishing between anomeric configuration with α-OMe-fucoside and β-OMe-fucoside, a pair of experiments with L-galactose and D-galactose, a series of experiments with the lectin mutants and STD experiments with blood group B trisaccharide that involved 2D STD HSQC experiment (heteronuclear single quantum coherence)). A transferred nuclear Overhauser effect (NOE) experiment was also performed. The lectin interactions with fucose were further studied by titration of the lectin by fucose while acquiring relatively fast 2D 15N-1H HSQC spectra that correlate protons with nitrogens. So it was necessary to isotopically label the lectin at least by 15N. Change of positions and intensities of some cross-peaks in the spectrum during the titrations were indicative of involvement in the interaction. Next, the cross-peaks in question needed to be assigned to appropriate N-H pair. Instead of time consuming complete assignment of all amino acids in the sequence we opted for assignment of only key amino acids involved in the interaction as determined from the crystal structure of the lectin-fucoside complex. The preparation of selectively labelled lectin, its single point mutants and measurement of their NMR spectra in solution were required for the partial assignment of lectin’s hydrogen-nitrogen pairs. So we prepared selectively labelled RS lectin for tryptophans and two of its single point mutants in order to facilitate the assignment of tryptophans in both active binding sites of the lectin.
It was proven by the results from aforementioned experiments that RS lectin has the highest affinity towards fucose among common monosaccharides. NMR experiments suggest that this lectin binds both α and β anomers of free-reducing L-fucose equally well. Also it was shown that the tryptophans in both binding sites are involved in the interaction where four of them can be considered the key residues in the interaction.
Obtained results so far were presented to the scientific community on two conferences. It is planned to present these results at pan-european carbohydrate conference, The 16th European Carbohydrate Symposium (Eurocarb 16) and the preparation of their publication as a full length article in a scientific foreign impacted journal is under way.