An insect β1–3-galactosyltransferase enables efficient synthesis of multi-sites T antigen glycoconjugates

Tumor-associated carbohydrate antigens (TACAs), particularly the T antigen characterized by the Galβ1–3GalNAc structure, are commonly expressed in various human cancers. However, the in vitro enzymatic synthesis of the Galβ1–3GalNAc structure has remained a significant challenge. In this study, we report the characterization of a β1–3-galactosyltransferase, DmC1GalT1, derived from D. melanogaster, which exhibits remarkable tolerance to a broad temperature (16–45 °C) and pH tolerance (pH 5.0–8.5). Additionally, DmC1GalT1 exhibits approximately 16-fold higher catalytic efficiency compared to the CjCgtBΔ30 mutant and effectively prevents further Gal-modification of the product. The enzyme shows strict donor specificity, utilizing only the native sugar nucleotide UDP-Gal (yields ∼100 %) and its derivative UDP-GalNH₂ (yields ∼15 %). We successfully applied DmC1GalT1 to synthesize a variety of glycoconjugates, including polysaccharides, glycopeptides, and glycoproteins. These findings not only expand the synthetic routes for the Galβ1–3GalNAc structure but also highlight the potential of DmC1GalT1 for enzymatic synthesis of important glycoconjugates, offering a more efficient and sustainable approach for future applications in glycoscience and biomedicine.