Rational design of N-glycosyltransferases from Aggregatibacter aphrophilus to synthesize Gal-modified glycoconjugates targeting hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) significantly impacts human health due to its tendency to develop multi-drug resistance. Regarding the treatment of HCC, galactosylated conjugates can target drug delivery. Bacterial N-glycosyltransferases (NGTs) catalyze glucosylation or galactosylation of the Asn residue motif Asn⁰-X⁺¹-Ser/Thr⁺² (X ≠ P). However, the limited utilization of the sugar donor UDP-Gal by NGTs severely restricts the potential applications in the development of glycol-drugs and vaccines. In this study, we rationally designed an NGT derived from Aggregatibacter aphrophilus and generated a mutant called AaFQ, which has a catalytic activity 18.8 times greater than that of wild-type towards UDP-Gal. AaFQ could rapidly synthesize Gal-modified peptides, which have been demonstrated to possess high targeting activity for HCC cells HepG2. Moreover, AaFQ exhibited a marked affinity for sugar donors with an equatorial bond-linked hydroxyl group at the C-2 position in monosaccharides. The mutant led to a significantly enhanced binding of K440 to the hydroxyl group at the C-2 position, which might be a major factor contributing to the significantly increased ability to utilize UDP-Gal. This work provides a crucial enzymatic synthetic tool for the targeted therapy of HCC and also offers a new perspective for the engineering modification and application of NGTs.