Structural determinants of site-specific Core Fucosylation in human transferrin: insights from crystallography and...

Core fucosylation of N-glycans, catalyzed by fucosyltransferase 8 (FUT8), plays a crucial role in various biological processes; however, the structural factors that determine which glycans are site-specifically modified by FUT8 remain poorly understood. Human transferrin carries two N-glycans at Asn432 and Asn630, of which only that at Asn630 is core-fucosylated in serum and cerebrospinal fluids. Here we compared the structural features of the two glycosylation sites on human transferrin as a model protein using crystallographic data and molecular dynamics (MD) simulations. Based on 13 crystal structures of transferrin with sufficient resolution, the solvent accessibility of the two glycosylation sites, Asn432 and Asn630, is 94 ± 17 Å2 and 120 ± 23 Å2, respectively, suggesting that Asn630 exhibits higher solvent accessibility. MD simulations of a glycosylated transferrin model based on AlphaFold predictions revealed that GlcNAc-1, the specific target of FUT8, is consistently more solvent-exposed at Asn630 than at Asn432, while root mean square fluctuation values showed that the glycan at Asn630 exhibits greater flexibility across all sugar residues. Hydrogen bond analysis further indicated that the glycan at Asn432 forms multiple stable interactions with the protein surface, whereas the Asn630 glycan shows minimal contact. These findings suggest that increased solvent accessibility, enhanced flexibility, and reduced structural constraints at Asn630 facilitate access by FUT8, enabling site-specific core fucosylation of transferrin.