Labeling and Detecting Human Milk Oligosaccharides through ST3Gal3 Incorporation of Azido Sialic Acid and Gel Electrophoresis

Human milk oligosaccharides (HMOs) are complex sugars present in human milk. These sugars possess prebiotic, immunomodulatory, and antagonistic properties towards pathogens and therefore are important for the health and well-being of newborn babies. The backbone of an HMO contains either Type I LacNAc (Gal-β-1,3-GlcNAc) or Type II LacNAc (Gal-β-1,4-GlcNAc). Fucosylation and sialylation of these disaccharides lead to different varieties of glycan epitopes, that may convey different biological functions. Previously, we described one-pot labeling strategy for detecting HMOs with azido-fucose using the combination of neuraminidase and fucosyltransferase. However, the method does not allow us to distinguish these two basic types of glycans. Here we describe a complementary method for HMO detection involving the incorporation of N3-Neu5Ac using the combination of a fucosidase and ST3Gal3 or ST6Gal1. While ST3Gal3 recognizes both types of disaccharides, ST6Gal1 specifically recognizes Type II LacNAc, therefore allowing these two types of disaccharides to be distinguished. Both enzymes achieved visible labeling of LNnO, a linear octasaccharide, at a minimal concentration of 10 nM, but ST3Gal3 showed at least 2-fold higher activity than ST6Gal1. ST3Gal3 therefore is more active and has broader specificity than the commonly used ST6Gal1 for attaching N3-Neu5Ac to target glycans. The methods were further illustrated on assigning isomeric specific HMOs (e.g. LNT vs. LNnT) and identifying fucosylated HMOs (e.g. LNFP I vs. LNDFH III) in fractionated human milk whey samples based on labeling sensitivity to enzyme treatment and gel mobility of the labeled bands together with data obtained from MALDI mass spectrometry.