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

Fouzia Bano,Markku I Tammi,David W Kang,Edward N Harris,Ralf P Richter

doi:10.1016/j.bpj.2018.05.014

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.

Highlights

Hyaluronan (HA) is an abundant and vital element of the extracellular matrix in all vertebrates
The cellular uptake of HA via endocytosis is likely to expose the bonds between HA and its receptors (such as the HA receptor for endocytosis, HARE, called stabilin-2 [15]) to mechanical stress because of the packing constraints that are associated with the large size and flexibility of HA
AG1,link protein (LP), versican G1 domain (VG1), and TSG6_LM were immobilized via their biotin tags on dense monolayers of streptavidin formed on goldsupported biotin-displaying monolayers of thiol-terminated oligo(ethylene glycols) (OEGs) on gold surfaces

Summary

Introduction

Hyaluronan (HA) is an abundant and vital element of the extracellular matrix in all vertebrates. Large supramolecular complexes made from HA and aggrecan, a proteoglycan with a molecular structure akin to that of a bottle brush, make a vital contribution to the integrity and biomechanical properties of cartilage that are crucial for joint function [6,7,8] In this scenario, the G1 domain on the N-terminus of aggrecan binds to HA, where this interaction is stabilized by cartilage link protein (LP), which simultaneously binds HA and aggrecan. Complexes of HA with versican, another proteoglycan with a bottle-brush-like structure, contribute to the elasticity of blood vessel walls, and mechanical strain has been observed to modulate versican expression and organization by vascular smooth muscle cells [9] In these and other contexts, the protein tumor necrosis factor-stimulated gene 6 (TSG-6) is thought to promote the dynamic remodeling of HA-rich matrices under inflammatory conditions, e.g., by SMFS of HA-Hyaladherin Interactions cross-linking HA [1,10]. The cellular uptake of HA via endocytosis is likely to expose the bonds between HA and its receptors (such as the HA receptor for endocytosis, HARE, called stabilin-2 [15]) to mechanical stress because of the packing constraints that are associated with the large size and flexibility of HA

Methods

Results

Discussion

Conclusion