Submitted to: Glycobiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/16/2025
Publication Date: 11/11/2025
Citation: Wu, Z.L., Naumann, T.A., Lu, S.Y., Skory, C.D. 2025. Detecting human milk oligosaccharides through enzymatic replacement with azido fucose. Glycobiology.https://doi.org/10.1093/glycob/cwaf069.
DOI: https://doi.org/10.1093/glycob/cwaf069

Interpretive Summary: Human milk oligosaccharides (HMOs) are complex sugars found in breast milk that play an important role in infant development. They promote the growth of beneficial bacteria in the gut, prevent growth of pathogens, and stimulate immune system development. ARS researchers in Peoria, Illinois, are investigating methods to manufacture HMOs from agricultural sugars so they can be used to supplement infant formulas. As part of this work, they previously developed a novel, easy to use procedure to analyze HMOs using fluorescent detection methods. This technique can quickly identify the presence of HMOs and provide useful information about their structure without the need for expensive analytical equipment. This new research further improves the method of detection to include HMOs that were not easily detected using the earlier technique. This work will facilitate the discovery of efficient production methods for HMOs and provide new value-added bioproducts for agricultural sugars.

Technical Abstract: Human milk oligosaccharides (HMOs) are complex sugars. These sugars possess prebiotic, immunomodulatory, and antagonistic properties towards pathogens and therefore are important for the health and well-being of newborn babies. Previously we reported glycan electrophoresis detection of HMOs terminated with beta 1,4-linked Gal residues (Type II LacNAc oligosaccharides) via enzymatic incorporation of azido sialic acid and fluorophore labeling by click chemistry. However, a significant portion of HMOs are terminated with beta 1,3-linked Gal residues (Type I LacNAc oligosaccharides). In addition, these Gal residues are frequently modified with alpha 1,2-linked fucose or alpha 2,3(6)-linked sialic acid residues that do not allow the incorporation of an azido sialic acid. To detect both Type I and Type II LacNAc containing HMOs and 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 by FUT2. Specifically, AfcA, an alpha 1,2-linkage specific fucosidase cloned from Aspergillus oryzae, was used to remove existing alpha 1,2-Fuc, enabling replacement with N3-Fuc by FUT2. The methods and the substrate specificities of AfcA,FUT2 and FUT3 in terms of the usage of N3-Fuc were demonstrated on standard HMOs. Furthermore, using Cy5-labeled antibody glycan G2 as a gel control, the relative gel separation of N3-Fuc labeled HMOs was established, which aids identification of the oligosaccharides. This strategy expands the ability to profile HMOs by glycan electrophoresis and is complementary to traditional methods for HMO study.