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

Author

Listed:
  • 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
    State University of New York
    State University of New York)

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.

Suggested Citation

  • Ryoma Hombu & Lauren E. Beatty & John Tomaszewski & Sheldon Park & Sriram Neelamegham, 2025. "Engineering glycosyltransferases into glycan binding proteins using a mammalian surface display platform," Nature Communications, Nature, vol. 16(1), pages 1-18, December.
  • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-62018-z
    DOI: 10.1038/s41467-025-62018-z
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