α 1 3 4 fucosidase

Removal of N-linked glycans was performed using PNGaseF (New England
Biolabs, Ipswich, USA) at a concentration of 125 U/μg protein.
Removal of O-linked glycans was performed using O-glycosidase (New
England Biolabs, 20,000 U/μg protein), α2-3,6,8 neuraminidase
(New England Biolabs, 25 U/μg protein), and α-N-acetyl-galactosaminidase (New England Biolabs, 10 U/μg
protein). Removal of both N-linked and O-linked glycans was performed
using PNGaseF (New England Biolabs, 125 U/μg protein), O-glycosidase
(New England Biolabs, 20,000 U/μg protein), α2-3,6,8 neuraminidase
(New England Biolabs, 25 U/μg protein), and α-N-acetyl-galactosaminidase (New England Biolabs, 10 U/μg
protein). Removal of sialic acids was performed using the α2-3,6,8
neuraminidase (New England Biolabs, 50 U/μg protein). Removal
of fucose was performed using α1-2,4,5,6 fucosidase O (New England
Biolabs, 2 U/μg protein) and α1-3,4 fucosidase (New England
Biolabs, 4 U/μg protein). All enzymatic reactions were performed
as a 1-step reaction with 1× Glycobuffer 2 (New England Biolabs)
and 10 μg of RBD produced in CHO-S-, HEK293F-, or Lec3.2.8.1
cells and incubation at 37 °C for 24 h. As heat-treated controls,
peptides were incubated at 37 °C for 24 h but without additional
enzymes.

Enzymatic removal of fucose residues was conducted according to the supplier’s protocol (α1-3,4 Fucosidase, α1-2 Fucosidase, New England Biolabs) with minor modifications. Briefly, 20 μg of protein sample was used for the enzymatic treatment by adding 2 μl of the exoglycosidase. The resulting mixture was incubated at 37°C overnight for the removal of the fucose residues, followed by analyzing with SDS–PAGE and immunoblotting analysis.

To remove fucose from glycan samples, the purified sample solution of 2AB-GSLs from cells (20 μL→S.V.) and 10 pmol/μL × 2 μL (20 pmol) Control:2AB-labeled LNFP II/III (Seikagaku Corporation) were treated with 20 mU of α1-2 fucosidase (Takara Bio) in 20 mM sodium phosphate buffer (pH 8.6) (total: 10 μL) or with 8 U of α1-3/4 fucosidase (P0769, New England Biolabs) in 20 mM sodium phosphate buffer (pH 6.0) (total: 10 μL) at 37 °C for at least 18 h. If the fucose cleavage of the sample was incomplete, digestion was repeated until complete cleavage was confirmed.