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.