Tri- and tetravalent mannoclusters cross-link and aggregate BC2L-A lectin from Burkholderia cenocepacia

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Investigation of the Binding Affinity of a Broad Array of l-Fucosides with Six Fucose-Specific Lectins of Bacterial and Fungal Origin

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Synthesis of β-d-galactopyranoside-Presenting Glycoclusters, Investigation of Their Interactions with Pseudomonas aeruginosa Lectin A (PA-IL) and Evaluation of Their Anti-Adhesion Potential

Pseudomonas aeruginosa is an opportunistic human pathogen associated with cystic fibrosis. This bacterium produces, among other virulence factors, a soluble d-galactose-specific lectin PA-IL (LecA). PA-IL plays an important role in the adhesion to the host cells and is also cytotoxic. Therefore, this protein is an interesting therapeutic target, suitable for inhibition by carbohydrate-based compounds. In the current study, ß-d-galactopyranoside-containing tri- and tetravalent glycoclusters were synthesized. Methyl gallate and pentaerythritol equipped with propargyl groups were chosen as multivalent scffolds and the galactoclusters were built from the above-mentioned cores by coupling ethylene or tetraethylene glycol-bridges and peracetylated propargyl ß-d-galactosides using 1,3-dipolar azide-alkyne cycloaddition. The interaction between galactoside derivatives and PA-IL was investigated by several biophysical methods, including hemagglutination inhibition assay, isothermal titration calorimetry, analytical ultracentrifugation, and surface plasmon resonance. Their ability to inhibit the adhesion of P. aeruginosa to bronchial cells was determined by ex vivo assay. The newly synthesized multivalent galactoclusters proved to be significantly better ligands than simple d-galactose for lectin PA-IL and as a result, two representatives of the dendrimers were able to decrease adhesion of P. aeruginosa to bronchial cells to approximately 32% and 42%, respectively. The results may provide an opportunity to develop anti-adhesion therapy for the treatment of P. aeruginosa infection

Terminology of bioanalytical methods (IUPAC Recommendations 2018)

Recommendations are given concerning the terminology of methods of bioanalytical chemistry. With respect to dynamic development particularly in the analysis and investigation of biomacromolecules, terms related to bioanalytical samples, enzymatic methods, immunoanalytical methods, methods used in genomics and nucleic acid analysis, proteomics, metabolomics, glycomics, lipidomics, and biomolecules interaction studies are introduced

Terminology of bioanalytical methods (IUPAC Recommendations 2018)

free accessRecommendations are given concerning the terminology of methods of bioanalytical chemistry. With respect to dynamic development particularly in the analysis and investigation of biomacromolecules, terms related to bioanalytical samples, enzymatic methods, immunoanalytical methods, methods used in genomics and nucleic acid analysis, proteomics, metabolomics, glycomics, lipidomics, and biomolecules interaction studies are introduced.Peer reviewe

Burkholderia cenocepacia BC2L-C Is a Super Lectin with Dual Specificity and Proinflammatory Activity

Lectins and adhesins are involved in bacterial adhesion to host tissues and mucus during early steps of infection. We report the characterization of BC2L-C, a soluble lectin from the opportunistic pathogen Burkholderia cenocepacia, which has two distinct domains with unique specificities and biological activities. The N-terminal domain is a novel TNF-α-like fucose-binding lectin, while the C-terminal part is similar to a superfamily of calcium-dependent bacterial lectins. The C-terminal domain displays specificity for mannose and l-glycero-d-manno-heptose. BC2L-C is therefore a superlectin that binds independently to mannose/heptose glycoconjugates and fucosylated human histo-blood group epitopes. The apo form of the C-terminal domain crystallized as a dimer, and calcium and mannose could be docked in the binding site. The whole lectin is hexameric and the overall structure, determined by electron microscopy and small angle X-ray scattering, reveals a flexible arrangement of three mannose/heptose-specific dimers flanked by two fucose-specific TNF-α-like trimers. We propose that BC2L-C binds to the bacterial surface in a mannose/heptose-dependent manner via the C-terminal domain. The TNF-α-like domain triggers IL-8 production in cultured airway epithelial cells in a carbohydrate-independent manner, and is therefore proposed to play a role in the dysregulated proinflammatory response observed in B. cenocepacia lung infections. The unique architecture of this newly recognized superlectin correlates with multiple functions including bacterial cell cross-linking, adhesion to human epithelia, and stimulation of inflammation

Microscopy examination of red blood and yeast cell agglutination induced by bacterial lectins.

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

Synthesis of Tetravalent Thio- and Selenogalactoside-Presenting Galactoclusters and Their Interactions with Bacterial Lectin PA-IL from Pseudomonas aeruginosa

Synthesis of tetravalent thio- and selenogalactopyranoside-containing glycoclusters using azide-alkyne click strategy is presented. Prepared compounds are potential ligands of Pseudomonas aeruginosa lectin PA-IL. P. aeruginosa is an opportunistic human pathogen associated with cystic fibrosis, and PA-IL is one of its virulence factors. The interactions of PA-IL and tetravalent glycoconjugates were investigated using hemagglutination inhibition assay and compared with mono- and divalent galactosides (propargyl 1-thio- and 1-seleno-β-d-galactopyranoside, digalactosyl diselenide and digalactosyl disulfide). The lectin-carbohydrate interactions were also studied by saturation transfer difference NMR technique. Both thio- and seleno-tetravalent glycoconjugates were able to inhibit PA-IL significantly better than simple d-galactose or their intermediate compounds from the synthesis

Bioanalytical Chemistry

This chapter provides a terminology of bioanalytical chemistry in general and analysis of biomacromolecules in particular. The vocabulary given in this chapter is largely taken from Labuda et al. “Terminology of bioanalytical methods (IUPAC Recommendations 2018)”,1 which becomes the immediate source reference for definitions of terms in this chapter that are not otherwise attributed. Reference to secondary sources follow the entry as “see also:” Terms are taken from the IUPAC Recommendations published in 1994 covering mostly the analytical terminology related to body fluids, enzymology, and immunology.2 Selected terms related to bioanalysis are included within recommendations and reports devoted to the unit “katal”,3 biotechnology,4 clinical chemistry,5 toxicology,6,7 medicinal chemistry,8,9 proteomics,10 electrochemical biosensors,11,12 and physical organic chemistry.13 Definitions of some terms have been updated here with respect to new reports and considerations, and a number of new terms has been introduced particularly on the topics of “–omics”, DNA analysis and studies of the interactions between biomolecules. Terms from earlier IUPAC Recommendations that are replaced by ref. 1 are not otherwise referenced but can be found as references in ref. 1