Terminal sialic acids on CD44 N-glycans can block hyaluronan binding by forming competing intramolecular contacts with arginine sidechains.

Specific sugar residues and their linkages form the basis of molecular recognition for interactions of glycoproteins with other biomolecules. Seemingly small changes, like the addition of a single monosaccharide in the covalently attached glycan component of glycoproteins, can greatly affect these interactions. For instance, the sialic acid capping of glycans affects protein-ligand binding involved in cell-cell and cell-matrix interactions. CD44 is a single-pass transmembrane glycoprotein whose binding with its carbohydrate ligand hyaluronan (HA), an extracellular matrix component, mediates processes such as leukocyte homing, cell adhesion, and tumor metastasis. This binding is highly regulated by glycosylation of the N-terminal extracellular hyaluronan-binding domain (HABD); specifically, sialic acid capped N-glycans of HABD inhibit ligand binding. However, the molecular mechanism behind this sialic acid mediated regulation has remained unknown. Two of the five N-glycosyation sites of HABD have been previously identified as having the greatest inhibitory effect on HA binding, but only if the glycans contain terminal sialic acid residues. These two sites, Asn25 and Asn120, were chosen for in silico glycosylation in this study. Here, from extensive standard molecular dynamics simulations and biased simulations, we propose a molecular mechanism for this behavior based on spontaneously-formed charge-paired hydrogen bonding interactions between the negatively-charged sialic acid residues and positively-charged Arg sidechains known to be critically important for binding to HA, which itself is negatively charged. Such intramolecular hydrogen bonds would preclude associations critical to hyaluronan binding. This observation suggests how CD44 and related glycoprotein binding is regulated by sialylation as cellular environments fluctuate.