Detecting human milk oligosaccharides through enzymatic replacement with azido fucose

Human milk oligosaccharides (HMOs) are complex sugars. These sugars possess prebiotic, antibiotic and immunomodulatory properties and therefore are important for the health and well-being of newborn babies. The backbones of HMOs are terminated either with Type I LacNAc (Gal-β1,3-GlcNAc) or Type II LacNAc (Gal-β1,4-GlcNAc) that can be further fucosylated and sialylated. To detect all these HMOs including their fucosylated and sialylated versions, we explored enzymatic incorporation of azido-fucose (N3-Fuc) by FUT2 or FUT3 directly, or through a replacement approach where existing fucose and sialic acid are removed with a specific glycosidase and replaced with an N3-Fuc. Specifically, AfcA, an α1,2-linkage specific fucosidase cloned from Aspergillus oryzae, was used to remove existing α1,2-Fuc. The substrate specificities and relative efficiencies of AfcA, FUT2 and FUT3 in terms of the usage of N3-Fuc were demonstrated on standard HMOs. FUT2 was finally selected for labeling and validated on HMOs isolated from human milk samples. Furthermore, using Cy5-labeled antibody glycan G2 as a gel control, the relative gel separation of an N3-Fuc labeled HMO was established, which could aid identification of the oligosaccharide. This strategy by N3-Fuc labeling and glycan electrophoresis expands the ability to profile HMOs and is complementary to traditional methods for HMO study.