Lectins are a group of ubiquitous proteins which specifically recognize and reversibly bind sugar moieties of glycoprotein and glycolipid constituents on cell surfaces. The mutagenesis approach is often employed to characterize lectin binding properties. As lectins are not enzymes, it is not easy to perform a rapid specificity screening of mutants using chromogenic substrates. It is necessary to use different binding assays such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), microscale thermophoresis (MST), enzyme-linked lectin assays (ELLA), or glycan arrays for their characterization. These methods often require fluorescently labeled proteins (MST), highly purified proteins (SPR) or high protein concentrations (ITC). Mutant proteins may often exhibit problematic behaviour, such as poor solubility or low stability. Lectin-based cell agglutination is a simple and low-cost technique which can overcome most of these problems. In this work, a modified method of the agglutination of human erythrocytes and yeast cells with microscopy detection was successfully used for a specificity study of the newly prepared mutant lectin RS-IIL_A22S, which experimentally completed studies on sugar preferences of lectins in the PA-IIL family. Results showed that the sensitivity of this method is comparable with ITC, is able to determine subtle differences in lectin specificity, and works directly in cell lysates. The agglutination method with microscopy detection was validated by comparison of the results with results obtained by agglutination assay in standard 96-well microtiter plate format. In contrast to this assay, the microscopic method can clearly distinguish between hemagglutination and hemolysis. Therefore, this method is suitable for examination of lectins with known hemolytic activity as well as mutant or uncharacterized lectins, which could damage red blood cells. This is due to the experimental arrangement, which includes very short sample incubation time in combination with microscopic detection of agglutinates, that are easily observed by a small portable microscope.

• MeSH
• Agglutination drug effects   MeSH
• Agglutination Tests * methods   MeSH
• Bacterial Proteins pharmacology   MeSH
• Erythrocytes cytology   drug effects   MeSH
• Hemagglutination drug effects   MeSH
• Hemolysis drug effects   MeSH
• Yeasts cytology   drug effects   MeSH
• Lectins pharmacology   MeSH
• Humans MeSH
• Microscopy MeSH
• Surface Plasmon Resonance MeSH
• Escherichia coli Proteins pharmacology   MeSH
• Saccharomyces cerevisiae cytology   drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Lectins MeSH
• Escherichia coli Proteins MeSH

This article focuses on designing mutations of the PA-IIL lectin from Pseudomonas aeruginosa that lead to change in specificity. Following the previous results revealing the importance of the amino acid triad 22-23-24 (so-called specificity-binding loop), saturation in silico mutagenesis was performed, with the intent of finding mutations that increase the lectin's affinity and modify its specificity. For that purpose, a combination of docking, molecular dynamics and binding free energy calculation was used. The combination of methods revealed mutations that changed the performance of the wild-type lectin and its mutants to their preferred partners. The mutation at position 22 resulted in 85% in inactivation of the binding site, and the mutation at 23 did not have strong effects thanks to the side chain being pointed away from the binding site. Molecular dynamics simulations followed by binding free energy calculation were performed on mutants with promising results from docking, and also at those where the amino acid at position 24 was replaced for bulkier or longer polar chain. The key mutants were also prepared in vitro and their binding properties determined by isothermal titration calorimetry. Combination of the used methods proved to be able to predict changes in the lectin performance and helped in explaining the data observed experimentally.

• MeSH
• Adhesins, Bacterial chemistry   genetics   metabolism   MeSH
• Computer-Aided Design MeSH
• Lectins chemistry   genetics   metabolism   MeSH
• Carbohydrate Metabolism MeSH
• Mutation MeSH
• Mutagenesis * MeSH
• Computer Simulation MeSH
• Pseudomonas aeruginosa chemistry   genetics   metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Molecular Docking Simulation MeSH
• Thermodynamics MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• adhesin, Pseudomonas MeSH Browser
• Adhesins, Bacterial MeSH
• Lectins 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
• Adhesins, Bacterial chemistry   genetics   MeSH
• Chromatography, Affinity MeSH
• Polymorphism, Single Nucleotide MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Lectins chemistry   genetics   MeSH
• Models, Molecular MeSH
• Monosaccharides chemistry   MeSH
• Mutagenesis, Site-Directed MeSH
• Protein Engineering MeSH
• Pseudomonas aeruginosa genetics   MeSH
• Ralstonia solanacearum chemistry   MeSH
• Recombinant Proteins chemistry   isolation & purification   MeSH
• Plant Lectins chemistry   MeSH
• Amino Acid Substitution MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• adhesin, Pseudomonas MeSH Browser
• Adhesins, Bacterial MeSH
• Lectins MeSH
• Monosaccharides MeSH
• Recombinant Proteins MeSH
• Plant Lectins MeSH