Engineering glycosyltransferases into glycan binding proteins using a mammalian surface display platform

Ryoma Hombu, Lauren E. Beatty, John Tomaszewski, Sheldon Park and Sriram Neelamegham ()
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Ryoma Hombu: State University of New York
Lauren E. Beatty: State University of New York
John Tomaszewski: State University of New York
Sheldon Park: State University of New York
Sriram Neelamegham: State University of New York

Nature Communications, 2025, vol. 16, issue 1, 1-18

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-025-62018-z

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