Defucosylation of Glycoproteins with FucosEXO

Efficient Removal of α1-2,3,4 Linked Fucose

Analysis of glycoproteins modified with complex glycan structures can be challenging and requires efficient and specific enzymatic tools. FucosEXO is a mix of α-fucosidases for efficient hydrolysis of α1-2, α1-3 and α1-4 linked fucose residues on N- and O-glycoproteins or oligosaccharides, without the need for co-factors or additives.

Schematic overview of the FucosEXO activity.

galactosidase Array GalactEXO

Substrate specificity of FucosEXO

Fucose are present on both N- and O-glycans in different linkages. α1-2, α1-3, and α1-4 linked fucose most commonly occurs in O-glycans and as antenna fucosylation of N-glycans whereas α1-6 linked fucose is found as a modification of the N-glycan core. We measured release of fucose by FucosEXO from a panel of different oligosaccharide substrates representing the different linkages. FucosEXO efficiently releases α1-2, α1-3, and α1-4 linked fucose, without activity on α1-6 linked fucose residues (Fig. 1).

Figure 1. The substrate specificity of FucosEXO was analyzed on equal molar amounts of synthetic oligosaccharides; α1-2 (2’- fucosyllactose,) α1-3 (3-fucosyllactose), α1-4 (Lewis a) and α1-6 (α1-6 fucosylated chitobiose). Substrates were incubated with FucosEXO for 30 min at 37°C and the amount of released fucose measured spectrometrically using an L-Fucose Assay Kit (Megazymes).

Performance Testing on Glycoproteins

Fucosylation of O-glycans is involved in the synthesis of functionally important glycan epitopes such as blood group antigens and the Lewis structures. Analysis of glycoproteins modified with such complex glycan structures can be challenging and requires efficient and specific enzymatic tools. We tested FucosEXO on a number of glycoengineered TNFR proteins carrying up to 11 O-glycans decorated with fucose in different linkages. We compared the activity to other commercially available fucosidases (Fig. 2). FucosEXO is able to defucosylate all three TNFR proteins within 1 hour, while treatment with the other fucosidases only led to partial removal of fucose or no removal at all.

Figure 2. Glycoengineered TNFR proteins were incubated with FucosEXO for 1h at 37°C or with other fucosidases according to the manufacturers recommendations. The resulting proteins were analyzed by reverse phase LC-MS on a Waters BioAccord system equipped with a Waters BioResolve RP mAb column (2.1 x 50 mm).

No Co-factors or Additives Required

FucosEXO is active in physiological buffers and neutral pH, making it compatible with most samples. Moreover, the FucosEXO activity is not dependent on any cofactors, such as BSA, that may otherwise impair LC-MS analysis. Depending on the nature of the substrate, FucosEXO hydrolyzes terminal α1-2,3,4 linked fucose residues on glycoproteins within 1 to 2 hours, while longer incubation may be required for very complex samples. FucoseEXO is also available immobilized in spin columns for fast and convenient processing up to 0.5 mg of glycoprotein. Using the spin column format, the glycoprotein sample is incubated with the Immobilized FucosEXO resin and the digested glycoproteins are then easily collected in a centrifugation step.