A single molecule assay to probe monovalent and multivalent bonds between hyaluronan and its key leukocyte receptor CD44 under force.

Fouzia Bano,David G Jackson,Suneale Banerji,Ralf P Richter,Mark Howarth

doi:10.1038/srep34176

Abstract

Glycosaminoglycans (GAGs), a category of linear, anionic polysaccharides, are ubiquitous in the extracellular space, and important extrinsic regulators of cell function. Despite the recognized significance of mechanical stimuli in cellular communication, however, only few single molecule methods are currently available to study how monovalent and multivalent GAG·protein bonds respond to directed mechanical forces. Here, we have devised such a method, by combining purpose-designed surfaces that afford immobilization of GAGs and receptors at controlled nanoscale organizations with single molecule force spectroscopy (SMFS). We apply the method to study the interaction of the GAG polymer hyaluronan (HA) with CD44, its receptor in vascular endothelium. Individual bonds between HA and CD44 are remarkably resistant to rupture under force in comparison to their low binding affinity. Multiple bonds along a single HA chain rupture sequentially and independently under load. We also demonstrate how strong non-covalent bonds, which are versatile for controlled protein and GAG immobilization, can be effectively used as molecular anchors in SMFS. We thus establish a versatile method for analyzing the nanomechanics of GAG·protein interactions at the level of single GAG chains, which provides new molecular-level insight into the role of mechanical forces in the assembly and function of GAG-rich extracellular matrices.

Highlights

Glycosaminoglycans (GAGs), a category of linear, anionic polysaccharides, are ubiquitous in the extracellular space, and important extrinsic regulators of cell function
It serves as a flexible linker to discriminate specific interactions from undesired non-specific interactions that may occur when the atomic force microscopy (AFM) tip is close to the surface[26]
Focusing on the fundamentally important interaction between HA and its primary cell surface receptor CD44, we have demonstrated how GAG·protein binding can be analyzed at the level of a single GAG chain in a well-defined system that preserves the native orientation of the receptor, but which enables important parameters such as its density and lateral mobility in the membrane to be varied

Summary

Introduction

Glycosaminoglycans (GAGs), a category of linear, anionic polysaccharides, are ubiquitous in the extracellular space, and important extrinsic regulators of cell function. Inflammation, where mechanical forces are of vital importance in regulating adhesive interactions between PSGL-1 on the leukocyte surface and selectins on the blood vessel endothelium and the ensuing cell adhesion and rolling[10,11]. In this particular scenario, interactions between HA and CD44 experience the shear stress of the blood flow[4,7,12]. On the most basic level, individual bonds need to be probed, and to better understand complexity in real biological systems, it must be determined how these elementary interactions are integrated into multivalent supramolecular systems This is important for GAGs because the polysaccharide chains can bind multiple proteins simultaneously. One of the main challenges to obtaining physiologically meaningful data using SMFS is achieving a well defined spatial arrangement of the studied molecules in their native orientation, while at the same time permitting the controlled application of the necessary tensile forces

Methods

Results

Conclusion