Molecular Interactions of Agaricus Bisporus Lectin with Dietary Carbohydrates and Phenolic Compounds: Binding Specificity and Structural Insights

Agaricus bisporus lectin (ABL) has garnered increasing interest as a potential therapeutic agent for treating cancer and immune system disorders due to its ability to recognize carbohydrates in epithelial cells. A deep understanding of ABL-carbohydrate interactions is also essential to uncovering further applications, such as the potential role in managing diabetes through diet. Previous studies using enzyme-linked lectin sorbent assay (ELLSA) have suggested that intermediate to high concentrations of glucose and galactose can alter the physiological activity of ABL. However, other studies on ABL interactions with dietary carbohydrates have failed to detect binding in crystalline form using techniques like X-ray crystallography and frontal affinity chromatography (FAC).

In addition to ABL-carbohydrate interactions, phenolic compounds are a diverse group of plant-derived secondary metabolites, including flavonoids, phenolic acids, tannins and lignans, with significant implications for food technology, nutrition and health, but their interactions with ABL are underresearched. Phenolic compound interactions with proteins can influence key sensory attributes such as flavor and texture, as well as impact the shelf life of food products. Despite Agaricus bisporus being a rich source of both proteins and phenolic compounds, no prior research has investigated potential interactions between ABL and phenolic compounds. Therefore, exploring these interactions could provide new insights into the functional properties of ABL and its role in food systems.

Given the above, the primary objective of this PhD research was to comprehensively investigate the molecular interactions and binding affinities between ABL and dietary carbohydrates, as well as phenolic compounds, in solutions. Using intrinsic fluorescence quenching, a highly sensitive analytical technique, this study delved into the thermodynamic aspects of these interactions.

The first experimental chapter examined the binding between ABL and dietary carbohydrates, including glucose, galactose and their N-acetylated derivatives (Nacetyl-D-glucosamine and N-acetyl-D-galactosamine) at pH 7.2 and ambient temperature. Intrinsic fluorescence quenching confirmed binding interactions between ABL and all investigated ligands, with glucose exhibiting the strongest interaction. Molecular docking identified the likely binding sites, revealing stable interactions between ABL and these sugars. Notably, the epimeric hydroxyl group at carbon four of the sugar ring influenced binding location, with galactose-containing molecules favoring the T-antigen binding site, while glucose-containing molecules favored the opposite site. Spectroscopic techniques, including Fourier transform infrared (FTIR) and circular dichroism (CD), indicated structural changes in ABL upon ligand binding, suggesting alterations in its secondary structure.

Building on this, the second experimental phase explored the effect of carbohydrate chain length on ABL binding affinity. This study investigated a series of glucose-based oligosaccharides—glucose, maltose, maltotetraose, maltohexaose, maltoheptaose and maltodecaose. Fluorescence quenching analysis revealed that binding strength increased with carbohydrate chain length, peaking at six glucose units (maltohexaose) before declining for longer carbohydrates. This trend was consistent with the secondary structure modifications observed via FTIR and CD, which showed the most significant changes for maltohexaose. Molecular docking further confirmed stable interactions between ABL and all six ligands, with multiple hydrogen bonds and van der Waals forces stabilizing the complexes.

Following these carbohydrate studies, the research shifted to investigating ABL’s interactions with phenolic compounds. The third experimental phase focused on 4-hydroxybenzoic acid (4HBA) and p-coumaric acid (p-CA) at pH 7.2 and ambient temperature. UV-vis spectroscopy confirmed that the interactions were non-covalent in nature. Secondary structure analysis revealed a reduction in ABL’s α-helical content by approximately 3% and 4% upon complexation with 4HBA and p-CA, respectively. Fluorescence quenching analysis indicated stronger binding between ABL and 4HBA compared to p-CA, a trend further supported by molecular docking, which showed a more favorable binding energy (ΔG) for 4HBA. Interestingly, docking results suggested that these phenolic acids bind in the glucose-binding region of ABL, with Arg103 emerging as a key residue in stabilizing the interactions—mirroring its suspected role in glucose binding.

The final experimental phase investigated ABL’s interactions with genistein, an isoflavone, and its glycosylated form, genistin, at pH 7.2 and ambient temperature. FTIR and CD analyses indicated significant increases in the β-sheet content of ABL (approximately 8.0% and 6.5%, respectively), accompanied by a corresponding reduction in α-helical structures. Molecular docking suggested that these structural modifications were induced by ligand interactions within the glucose-binding domain of ABL. Fluorescence quenching analysis revealed that genistein exhibited stronger binding affinity than genistin, demonstrating that glycosylation did not enhance binding strength. Instead, genistein’s ability to form π-alkyl hydrophobic forces with ABL contributed to its stronger interaction.

Overall, the findings from these four experimental chapters provide valuable insights into the molecular interactions between ABL and both dietary carbohydrates and naturally occurring phenolic compounds by elucidating their binding mechanisms, affinities and structural implications. This research enhances our understanding of ABL’s potential bioactive properties in functional foods and nutraceuticals while advancing knowledge of its molecular binding mechanisms. These discoveries may further support the application of ABL as a functional ingredient in highly selective therapeutic formulations.