Single-Molecule Unbinding Forces between the Polysaccharide Hyaluronan and Its Binding Proteins

Abstract

The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidin⋅biotin bond. Implications for the molecular mechanism of unbinding of HA⋅hyaladherin bonds under force are discussed, which underpin the mechanical properties of HA⋅hyaladherin complexes and HA-rich extracellular matrices.

Hyaluronan biology: A complex balancing act of structure, function, location and context

“…It is still unclear how HA fragment size influences receptor activation. Sophisticated experiments suggest that tertiary or quaternary interactions of HA receptors with the HA matrix may be invoked to explain these interactions [108][109][110]. These studies pave the way for future studies.…”

Leucocyte Trafficking via the Lymphatic Vasculature— Mechanisms and Consequences

“…This role as lubricant is supported by physicochemical studies of LYVE-1 HA binding mechanics at the single molecule level using atomic force microscopy, which indicate the individual interactions are weak and that they rupture collectively under the low forces experienced in interstitial flow [see (19)]. It contrasts markedly with the behavior of CD44, a receptor tuned for leucocyte capture in post-capillary venules, which forms bonds that are stronger and detach sequentially in a Velcro (hook and loop) like fashion in response to the higher forces experienced in blood flow (108, 109). Nevertheless, the transit of cells through lymphatic endothelium must involve traction, and if this is not provided by LYVE-1, then it is likely that DCs use both HA and integrin-based adhesion either on different faces of the cell, or in sequential fashion during diapedesis.…”

Ligand Binding and Signaling of HARE/Stabilin-2

“…Both TSG-6 and 190HARE had a mean rupture force with HA at 24 and 25 picoNewtons (pN), respectively, further suggesting that both proteins interact with HA in a similar fashion. Comparatively, HA rupture forces were 34 pN for CD44, 37 pN for versican, and more than 52 pN for aggrecan X-Link domains [39]. Although the binding affinity of HA for 190HARE is in the low nanomolar range, the bond strength with which it binds is one of the weakest among the hyalectin family.…”