Abstract
Pseudomonas aeruginosa is a widespread opportunistic pathogen that is capable of colonizing various human tissues and is resistant to many antibiotics. LecA is a galactose binding tetrameric lectin involved in adhesion, infection and biofilm formation. This study reports on the binding characteristics of mono- and divalent (chelating) ligands to LecA using different techniques. These techniques include affinity capillary electrophoresis, bio-layer interferometry, native mass spectrometry and a thermal shift assay. Aspects of focus include: affinity, selectivity, binding kinetics and residence time. The affinity of a divalent ligand was determined to be in the low-nanomolar range for all of the used techniques and with a ligand residence time of approximately 7 h, while no strong binding was seen to related lectin tetramers. Each of the used techniques provides a unique and complementary insight into the chelation based binding mode of the divalent ligand to the LecA tetramer.
Highlights
Lectins are carbohydrate-binding proteins with diverse functions that are found in all domains of life (Chemani et al 2009); (Huang et al 2018)
The first was the carboxylated galactoside (1). This was used as the reference monovalent ligand and its negatively charged carboxylate increases the chances of a protein shift in Capillary electrophoresis (CE) where charges are the important mobility determinant
We used the bivalent carbohydrate (2), previously optimized for bivalent binding (Yu et al 2019b). These glycoligands were added to the optimized CE background electrolyte (BGE) and the protein LecA was injected
Summary
Lectins are carbohydrate-binding proteins with diverse functions that are found in all domains of life (Chemani et al 2009); (Huang et al 2018) These proteins play crucial roles in various processes such as cell–cell recognition, infection processes and immune defense (Lee and Lee 1995); (Brown et al 2018); (Bernardi et al 2013). They are generally characterized by an intermediate to low affinity toward their carbohydrate ligands, a limitation that is frequently overcome in nature through multivalency of both the lectin receptors and their carbohydrate ligands, resulting in enhanced binding or inhibition (Cecioni et al 2015); (Fasting et al 2012); (Wittmann and Pieters 2013). Inhibition of LecA has been examined by numerous studies as a novel approach to control P. aeruginosa infection (Chemani et al 2009); (Goyard et al 2019)
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