Dataset Information

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

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.

SUBMITTER: Mrazkova J

PROVIDER: S-EPMC6657890 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Mrázková Jana J Malinovská Lenka L Wimmerová Michaela M

PloS one 20190725 7

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 resonanc ...[more]

PMID: 31344098

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Project description:With the use of engineered nano-materials (ENM) becoming more prevalent, it is essential to determine potential human health impacts. Specifically, the effects on biological lipid membranes will be important for determining molecular events that may contribute to both toxicity and suitable biomedical applications. To better understand the mechanisms of ENM-induced hemolysis and membrane permeability, fluorescence lifetime imaging microscopy (FLIM) was performed on human red blood cells (RBC) exposed to titanium dioxide ENM, zinc oxide ENM, or micron-sized crystalline silica. In the FLIM images, changes in the intensity-weighted fluorescence lifetime of the lipophilic fluorescence probe Di-4-ANEPPDHQ were used to identify localized changes to membrane. Time-resolved fluorescence anisotropy and FLIM of RBC treated with methyl-ß-cyclodextrin was performed to aid in interpreting how changes to membrane order influence changes in the fluorescence lifetime of the probe. Treatment of RBC with methyl-ß-cyclodextrin caused an increase in the wobble-in-a-cone angle and shorter fluorescence lifetimes of di-4-ANEPPDHQ. Treatment of RBC with titanium dioxide caused a significant increase in fluorescence lifetime compared to non-treated samples, indicating increased membrane order. Crystalline silica also increased the fluorescence lifetime compared to control levels. In contrast, zinc oxide decreased the fluorescence lifetime, representing decreased membrane order. However, treatment with soluble zinc sulfate resulted in no significant change in fluorescence lifetime, indicating that the decrease in order of the RBC membranes caused by zinc oxide ENM was not due to zinc ions formed during potential dissolution of the nanoparticles. These results give insight into mechanisms for how these three materials might disrupt RBC membranes and membranes of other cells. The results also provide evidence for a direct correlation between the size, interaction-available surface area of the nano-material and cell membrane disruption.

Dataset Information

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

Publications

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

Similar Datasets

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