2025-08-14 バッファロー大学(UB)
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An illustration of the pig enzyme ST3Gal1 (green) bound to its sugar ligand (yellow sticks). Binding interfaces contain green (carbon), red (oxygen), and blue (nitrogen) surfaces. Credit: University at Buffalo.
<関連情報>
- https://www.buffalo.edu/news/releases/2025/08/glycan-sugar-pattern-study.html
- https://www.nature.com/articles/s41467-025-62018-z#author-information
- https://academic.oup.com/glycob/advance-article/doi/10.1093/glycob/cwaf041/8210292
- https://onlinelibrary.wiley.com/doi/abs/10.1002/smll.202502318
哺乳類表面表示プラットフォームを用いて、糖転移酵素を糖鎖結合タンパク質に組み込む Engineering glycosyltransferases into glycan binding proteins using a mammalian surface display platform
Ryoma Hombu,Lauren E. Beatty,John Tomaszewski,Sheldon Park & Sriram Neelamegham
Nature Communications Published:18 July 2025
DOI:https://doi.org/10.1038/s41467-025-62018-z
Abstract
Traditional lectins exhibit broad binding specificity for cell-surface carbohydrates, and generating anti-glycan antibodies is challenging due to low immunogenicity. Nevertheless, it is necessary to develop glycan binding proteins for single-cell glycosylation pathway analysis. Here, we test the hypothesis that protein engineering of mammalian glycosyltransferases can yield glycan-binding proteins with defined specificity. Introducing an H302A mutation, based on rational design, into porcine ST3Gal1 abolishes its enzymatic activity, but results in a lectin that specifically binds sialylated core-2 O-linked glycans (Neu5Acα2-3Galβ1-3[GlcNAc(β1-6)]GalNAcα). To improve binding, we develop a mammalian cell-surface display platform to screen variants. One ST3Gal1 mutant (sCore2) with three mutations, H302A/A312I/F313S exhibits enhanced binding specificity. Spectral flow cytometry and tissue microarray analysis using sCore2 reveal distinct cell- and tissue-specific sialyl core-2 staining patterns in human blood cells and paraffin-embedded tissue sections. Overall, glycosyltransferases can be engineered to generate specific glycan binding proteins, suggesting that a similar approach may be extended to other glycoenzymes.
設計に基づくレクチン開発のためのタンパク質工学戦略 Protein engineering strategies to develop lectins by design
Ryoma Hombu , Lauren E Beatty , Sriram Neelamegham
Glycobiology Published:22 July 2025
DOI:https://doi.org/10.1093/glycob/cwaf041
Abstract
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.
糖転移酵素の高スループットタンパク質工学のための哺乳類細胞表面表示 Mammalian Cell Surface Display for High-Throughput Protein Engineering of Glycosyltransferases
Ryoma Hombu, Sriram Neelamegham
Small Published: 26 May 2025
DOI:https://doi.org/10.1002/smll.202502318
Abstract
High-throughput methods are needed to study structure-function relationships of mammalian glycosyltransferases due to their essential role in assembling the glycocalyx. Mutations in these enzymes result in Congenital Disorders of Glycosylation, and improvements of enzyme properties can yield new biocatalysts. In this manuscript, a cell-based glycosyltransferase activity assay is developed using mammalian surface display. It is demonstrated that coupling this approach with click chemistry enables rapid quantification of glycosyltransferase activity. Screening of 1680 different pig ST3Gal1 mutants yielded α(2,3)sialyltransferases with improved enzymatic properties. Using endogenous cell-surface substrates, the method is extended to other human sialyltransferases, including ST6Gal1, ST3Gal4, and ST6GalNAc2. Additionally, the approach is used to screen putative sialyltransferases from diverse organisms not characterized in the CAZy (Carbohydrate-Active EnZymes) database. Overall, a facile, robust, high-throughput, low-cost method is presented to study glycosyltransferase structure-function relationships.
