Abstract
Multivalent ligand–protein interactions are a commonly employed approach by nature in many biological processes. Single glycan–protein interactions are often weak, but their affinity and specificity can be drastically enhanced by engaging multiple binding sites. Microarray technology allows for quick, parallel screening of such interactions. Yet, current glycan microarray methodologies usually neglect defined multivalent presentation. Our laser-based array technology allows for a flexible, cost-efficient, and rapid in situ chemical synthesis of peptide scaffolds directly on functionalized glass slides. Using copper(I)-catalyzed azide–alkyne cycloaddition, different monomer sugar azides were attached to the scaffolds, resulting in spatially defined multivalent glycopeptides on the solid support. Studying their interaction with several different lectins showed that not only the spatially defined sugar presentation, but also the surface functionalization and wettability, as well as accessibility and flexibility, play an essential role in such interactions. Therefore, different commercially available functionalized glass slides were equipped with a polyethylene glycol (PEG) linker to demonstrate its effect on glycan–lectin interactions. Moreover, different monomer sugar azides with and without an additional PEG-spacer were attached to the peptide scaffold to increase flexibility and thereby improve binding affinity. A variety of fluorescently labeled lectins were probed, indicating that different lectin–glycan pairs require different surface functionalization and spacers for enhanced binding. This approach allows for rapid screening and evaluation of spacing-, density-, ligand and surface-dependent parameters, to find optimal lectin binders.
Highlights
Glycan-protein interactions exist in many biological processes, such as protein folding, cell-cell interaction, cell-adhesion, and signaling
We demonstrate the importance of surface accessibility and wettability on glycan-glycan binding proteins (GBPs) interactions, enabling us to study a wide range of plant lectins in a high-throughput manner
The amino acid pattern was coupled in an oven to the acceptor slide, the surface was washed, capped and Fmoc deprotected. Repeating these in-situ solid phase synthesis steps, peptides were generated in the array format on the acceptor
Summary
Glycan-protein interactions exist in many biological processes, such as protein folding, cell-cell interaction, cell-adhesion, and signaling Their understanding is of fundamental importance (Varki, 2009). Glycans play a key role in diseases and virulence (e.g., diabetes, inflammation, cancer, infections), rendering scientists to investigate their structural and functional characteristics (Zhou and Cobb, 2021) Their interaction with other cells, and their recognition by glycan binding proteins (GBPs), so called lectins, triggered the investigation of their binding ability, and molecular mechanism (Raman et al, 2016; Valverde et al, 2020). The recognition process that nature has evolved to enhance the binding strength and specificity is called multivalency This effect enables high binding affinities via simultaneous recognition of one or several glycans by GBPs, which have multiple and spatially well-defined glycan binding sites (Fasting et al, 2012; Haag, 2015). For a strong multivalent interaction, the type(s) of sugar(s), and their spatial orientation, their accessibility, and the carrier scaffold are important, to achieve optimum distance with the binding pockets of the multivalent receptor
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