Editor's Choice Protein engineering strategies to develop lectins by design.

Glycans regulate a wide array of biological processes, making them central to studies of cell biology. Thus, it is essential to characterize the spatiotemporal dynamics of glycans on cells and tissues, and to elucidate how glycan structures affect protein and cell function. Among the available molecular tools, glycan-binding proteins (GBPs), including naturally occurring lectins, are uniquely suited to provide this information at single-cell resolution. However, the diversity of cell-surface glycans far exceeds the number of readily available GBPs. Moreover, conventional lectins often possess shallow binding pockets that limit their recognition to terminal glycan epitopes, and such recognition often proceeds with low binding affinity. Protein engineering offers a promising strategy to expand GBP specificity, enhance affinity, and introduce novel binding capabilities. Currently, large gaps remain between the available protein design principles and their application to GBP engineering. This has somewhat slowed progress in the development of glycan-targeted tools. In this review, we outline recent efforts that use rational design to inform GBP engineering for specific tasks. We also present methods to select suitable protein scaffolds and the application of directed evolution for optimizing lectin design. This includes our recent efforts to modify glycosyltransferases into GBPs, which potentially offers a predictive strategy to design lectins based on desired properties. Together, the presentation offers a roadmap for developing next-generation glycan binding proteins capable of decoding the complex glycan landscape of cells.