Carbohydr Res. 2026 Jun 26;567:110019. doi: 10.1016/j.carres.2026.110019. Online ahead of print.

ABSTRACT

Antibody Fc glycosylation critically influences effector functions, motivating the development of chemoenzymatic strategies to generate homogeneous glycoforms. However, limited availability and high cost of mammalian glycosyltransferases remain to be significant constraints. Here, we report the high-yield expression of human and bovine β-1,4-galactosyltransferases (B4GalT1) and their single-point mutants (Y285L and Y289L, respectively) as maltose-binding protein fusions in Escherichia coli. A comparative analysis revealed that human B4GalT1 and its Y285L mutant exhibited substantially higher activity than bovine enzymes in transferring galactose (Gal) and N-acetylgalactosamine (GalNAc) from the corresponding UDP-sugar to terminal N-acetylglucosamine residues on Fc N-glycans of intact antibodies. These enzymes enabled the synthesis of antibody glycoforms bearing GalNAcβ1-4GlcNAc (LacdiNAc) in place of LacNAc. An unexpected finding was that the α-2,6-sialyltransferase from Photobacterium damselae (Pd2,6ST) and the α-2,3-sialyltransferase mutant PmST1 M144D from Pasteurella multocida recognized terminal GalNAc, but not Gal, in the intact antibody for sialylation. Interestingly, sialylation of terminal Gal was restored Pd2,6ST for the trastuzumab variant carrying a point F241A mutation. These results suggest that the intramolecular interaction between the terminal galactose and aromatic residues such as F241 in the Fc domain may hinder the enzymatic sialylation, while the F241A mutation partially releases such an interaction thus enhancing the sialylation efficiency. Receptor binding analysis showed that the antibodies carrying a LacdiNAc moiety exhibited comparable affinity to the FcγIIIA receptor and C1q protein as the LacNAc terminated glycoform, while the sialylated LacdiNAc glycoform could still bind to Siglec-2, albeit at lower affinity than the common sialylated LacNAc glycoform. These results expand the enzymatic toolkit for antibody glycoengineering and highlight the functional impact of LacdiNAc incorporation.

PMID:42378775 | DOI:10.1016/j.carres.2026.110019