Generating novel recombinant prokaryotic lectins with altered carbohydrate binding properties through mutagenesis of the PA-IL protein from Pseudomonas aeruginosa

Keogh, Damien; Thompson, Roisin; Larragy, Ruth; McMahon, Kenneth; O'Connell, Michael; O’Connor, Brendan; Clarke, Paul

doi:10.1016/j.bbagen.2014.01.020

Cited by 15 publications

(10 citation statements)

References 48 publications

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“…Mutagenesis is frequently employed for detailed investigation of lectin properties and/or the preparation of new lectins with novel binding preferences. This is often done using directed-evolution methods that generate thousands of different mutants with unknown binding properties [38–40]. This makes using commonly used approaches, which include the purification of proteins for their detailed characterization, nearly impossible.…”

Section: Resultsmentioning

confidence: 99%

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

2019

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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.

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Section: Resultsmentioning

confidence: 99%

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

2019

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“…(described in detail below) [22], who tailored a novel sialic acid-binding lectin from an R-type galactose-binding protein using random mutagenesis and a ribosome-display method under the concept of 'natural evolution-mimicry' [23]. Since their work, a significant number of reports of protein engineering base lectin engineering have come out of different laboratories [24][25][26][27][28][29][30][31][32][33][34][35][36]. Aptamer and chemistry-based lectin engineering are also promising and challenging.…”

Section: Current State Of the Art Of Lectin Engineeringmentioning

confidence: 99%

Lectin engineering: the possible and the actual

Hirabayashi

Arai

2019

Interface Focus.

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Lectins are a widespread group of sugar-binding proteins occurring in all types of organisms including animals, plants, bacteria, fungi and even viruses. According to a recent report, there are more than 50 lectin scaffolds (∼Pfam), for which three-dimensional structures are known and sugar-binding functions have been confirmed in the literature, which far exceeds our view in the twentieth century (Fujimoto et al. 2014 Methods Mol. Biol. 1200 , 579–606 ( doi:10.1007/978-1-4939-1292-6_46 )). This fact suggests that new lectins will be discovered either by a conventional screening approach or just by chance. It is also expected that new lectin domains including those found in enzymes as carbohydrate-binding modules will be generated in the future through evolution, although this has never been attempted on an experimental level. Based on the current state of the art, various methods of lectin engineering are available, by which lectin specificity and/or stability of a known lectin scaffold can be improved. However, the above observation implies that any protein scaffold, including those that have never been described as lectins, may be modified to acquire a sugar-binding function. In this review, possible approaches to confer sugar-binding properties on synthetic proteins and peptides are described.

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“…Glycan recognition is a ubiquitous theme in nature and intimately related to targeting, transport, adhesion and signalling processes both on an intracellular and extracellular level . Most cellular envelopes are covered with glycan matrices and addressing these surfaces with tailor‐made protein‐binders has impact both in diagnostics and therapy . This cell surface localization and involvement in reversible signalling events results in protein–carbohydrate complexes with relatively low affinities at single sites that gain functionality within a multivalent glycan–glycan binding environment .…”

Section: Introductionmentioning

confidence: 99%

Increasing the Affinity of an O‐Antigen Polysaccharide Binding Site in Shigella flexneri Bacteriophage Sf6 Tailspike Protein

Kunstmann

Engström

Wehle

et al. 2020

Chemistry A European J

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Broad and unspecific use of antibiotics accelerates spread of resistances. Sensitive and robust pathogen detection is thus important for a more targeted application. Bacteriophages contain a large repertoire of pathogen‐binding proteins. These tailspike proteins (TSP) often bind surface glycans and represent a promising design platform for specific pathogen sensors. We analysed bacteriophage Sf6 TSP that recognizes the O‐polysaccharide of dysentery‐causing Shigella flexneri to develop variants with increased sensitivity for sensor applications. Ligand polyrhamnose backbone conformations were obtained from 2D 1H,1H‐trNOESY NMR utilizing methine–methine and methine–methyl correlations. They agreed well with conformations obtained from molecular dynamics (MD), validating the method for further predictions. In a set of mutants, MD predicted ligand flexibilities that were in good correlation with binding strength as confirmed on immobilized S. flexneri O‐polysaccharide (PS) with surface plasmon resonance. In silico approaches combined with rapid screening on PS surfaces hence provide valuable strategies for TSP‐based pathogen sensor design.

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Generating novel recombinant prokaryotic lectins with altered carbohydrate binding properties through mutagenesis of the PA-IL protein from Pseudomonas aeruginosa

Cited by 15 publications

References 48 publications

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

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

Lectin engineering: the possible and the actual

Increasing the Affinity of an O‐Antigen Polysaccharide Binding Site in Shigella flexneri Bacteriophage Sf6 Tailspike Protein

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