Abstract
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.
Highlights
Sugars in various forms occur in all organisms, where they have a wide range of functions
The inhibition ratio obtained by red blood cell (RBC) and yeast agglutination inhibition methods were close to the ratio of binding affinities determined by enzyme-linked lectin assays (ELLA) and isothermal titration calorimetry (ITC) measurements [36], enabling a semi-quantification of lectin affinity
The results showed that both agglutination inhibition assays performed directly with the bacterial extracts gave the same results as experiments with purified lectins, i.e., the inhibition ratio for mannose/fucose was 8 with CV-IIL (Fig 7) and 16 with the mutant RS-IIL_A22S (Fig 8)
Summary
Sugars in various forms occur in all organisms, where they have a wide range of functions. The glycoproteins and glycolipids present on cell surfaces can bear terminal oligosaccharides which are readily accessible These sugar moieties are recognized and bound by a special class of proteins called lectins [1]. Lectins are neither antibodies nor enzymes, and they differ from proteins that are specialized for the transport of free saccharides They bind a wide range of carbohydrates reversibly and with a very high specificity, they participate in recognition and interactions at the cell level. As they are ubiquitous, their precise function depends on the organism in which they occur or their localization in a particular tissue. Lectins produced by pathogens are involved in the recognition of host cells and subsequent adhesion to the cell surface during the initial step of infection, and lectins are often counted as virulence factors [3]
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