Enhanced antibody-defucosylation capability of α-L-fucosidase by proximity-based protein fusion.

Up to date, the reported fucosidases generally show poor activities toward the IgG core-fucose, which limits the efficiency of ENGase-catalyzed glycoengineering process. However, EndoS or EndoS2 owns excellent activity and great selectivity towards the N-glycosylation of IgGs, and their non-catalytic domains are deduced to have specific interactions to IgG Fc domain that result in the great activity and selectivity. Herein, we constructed a series fusion protein of AlfC (an α-l-fucosidase from Lactobacillus casei BL23) with EndoS/S2 non-catalytic domain by replacing the catalytic GH (glycan hydrolase) domain of EndoS/S2 with the AlfC.

One-step synthesis of site-specific antibody-drug conjugates by reprograming IgG glycoengineering with LacNAc-based substrates.

Glycosite-specific antibody‒drug conjugatess (gsADCs), harnessing Asn297 -glycan of IgG Fc as the conjugation site for drug payloads, usually require multi-step glycoengineering with two or more enzymes, which limits the substrate diversification and complicates the preparation process. Herein, we report a series of novel disaccharide-based substrates, which reprogram the IgG glycoengineering to one-step synthesis of gsADCs, catalyzed by an --acetylglucosaminidase (ENGase) of Endo-S2. IgG glycoengineering ENGases usually has two steps: deglycosylation by wild-type (WT) ENGases and transglycosylation by mutated ENGases.

Cloning, characterization, and production of three α-L-fucosidases from Clostridium perfringens ATCC 13124.

α-L-Fucosidases are key enzymes for the degradation of intestinal glycans by gut microbes. In this work, three putative α-L-fucosidases (Afc1, Afc2, and Afc3) genes from Clostridium perfringens ATCC 13124 were cloned and expressed in Escherichia coli. Afc1 had the α-L-fucosidase domain of glycoside hydrolase (GH) 29 family but showed no enzyme activity toward all the substrates examined.

Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions.

The fine structures of Fc N-glycans can modulate the effector functions of IgG antibodies. It has been demonstrated that lack of the core fucose on the Fc N-glycans leads to drastic enhancement of antibody-dependent cellular cytotoxicity (ADCC), while terminal α2,6-sialylation of Fc glycan plays a critical role for the anti-inflammatory activity of human intravenous immunoglobulin (IVIG). We describe in this paper a highly efficient chemoenzymatic method for site-selective Fc glycoengineering of intact monoclonal antibody and IVIG.

Remarkable transglycosylation activity of glycosynthase mutants of endo-D, an endo-β-N-acetylglucosaminidase from Streptococcus pneumoniae.

Endo-β-N-acetylglucosaminidase from Streptococcus pneumoniae (Endo-D) is an endoglycosidase capable of hydrolyzing the Fc N-glycan of intact IgG antibodies after sequential removal of the sialic acid, galactose, and internal GlcNAc residues in the N-glycan. Endo-D also possesses transglycosylation activity with sugar oxazoline as the donor substrate, but the transglycosylation yield is low due to enzymatic hydrolysis of the donor substrate and the product. We report here our study on the hydrolytic and transglycosylation activity of recombinant Endo-D and its selected mutants.