Aminoquinoline Fluorescent Labels Obstruct Efficient Removal of N-Glycan Core α(1–6) Fucose by Bovine Kidney α-l-Fucosidase (BKF)

• DOI: 10.1021/acs.jproteome.7b00580
• Publication Date: 2017-09-27T18:17:51Z
• Authors: Róisín O’Flaherty; Aoife M. Harbison; Philip J. Hanley; Christopher H. Taron; Elisa Fadda; Pauline M. Rudd
• Keywords: alpha-L-Fucosidase; 0301 basic medicine; 570; Binding Sites; Staining and Labeling; 540; 03 medical and health sciences; Polysaccharides; Aminoquinolines; Animals; Cattle; Fluorescent Dyes; Fucose

Here we report evidence that new aminoquinoline N-glycan fluorescent labels interfere with the release of core α(1-6) fucose from N-glycans by bovine kidney α-l-fucosidase (BKF). BKF is a commonly employed exoglycosidase for core α(1-6) fucose determination. Molecular simulations of the bound and unbound Fuc-α(1-6)-GlcNAc, where GlcNAc is situated at the reducing end for all N-glycans, suggest that the reduced BKF activity may be due to a nonoptimal fit of the highest populated conformers in the BKF active binding site at room temperature. Population analysis and free energy estimates suggest that an enhanced flexibility of the labeled sugar, which facilitates recognition and binding, can be achievable with extended reaction conditions. We provide these experimental conditions using a sequential exoglycosidase digestion array using high concentrations of BKF.