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

Abstract

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.

The challenges of glycan recognition with natural and artificial receptors

“…In this regard, positively-charged arginine residues are often involved in sialic acid interaction processes. 81 Examples of intermolecular contacts between lectins and glycans at the binding site can be found in a study reporting the investigation of the complexation of Siglec-8 lectin and its target, 6 0 -sulfo sialyl Lewis X (6 0 S sLe x ) ( Fig. 4).…”

Atomistic fingerprint of hyaluronan–CD44 binding

“…Finally, Faller et al glycosylated HABD in silico at two N-glycosylation sites (N25 and N120) to probe the effect of N-glycans on the function of HABD [ 31 ]. They concluded that the negatively-charged sialic acids on the termini of the N-glycans charge paired with basic residues important for HA binding, such as R41 or R154.…”

“…They concluded that the negatively-charged sialic acids on the termini of the N-glycans charge paired with basic residues important for HA binding, such as R41 or R154. These sialic acids could thereby impede the binding of HA by competing for the same binding sites [ 31 ].…”

Insight in Adhesion Protein Sialylation and Microgravity Dependent Cell Adhesion—An Omics Network Approach

“…The CD44 antigen bears terminal SAs at the tips of its glycans [96]. If CD44 N-glycans bear terminal SAs, hyaluronan binding is blocked as competing intramolecular contacts of sialic acid with arginine side chains are formed [97]. The sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid blocks the recognition of hyaluronic acid, showing that sialylation negatively regulates CD44 activity [98].…”

“…The sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid blocks the recognition of hyaluronic acid, showing that sialylation negatively regulates CD44 activity [98]. Activation of sialidase by TNFalpha via MAPK38 initiates binding of CD44 to hyaluronic acid [97]. In colorectal cancer, metastasis to the liver occurs more frequently when CD44 is expressed on the surface together with sialyl Lewis(a) antigens [99].…”