A single molecule assay to probe monovalent and multivalent bonds between hyaluronan and its key leukocyte receptor CD44 under force.
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.
CIC biomaGUNE, Paseo Miramon 182, 20009 Donostia-San Sebastian, Spain.
Fouzia
F
Bano
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX39DS, UK.
Suneale
S
Banerji
Department of Biochemistry, University of Oxford, Oxford, OX13QU, UK.
Mark
M
Howarth
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX39DS, UK.
David G
DG
Jackson
CIC biomaGUNE, Paseo Miramon 182, 20009 Donostia-San Sebastian, Spain.|Université Grenoble Alpes - CNRS, Laboratoire Interdisciplinaire de Physique (LIPhy), BP 87, 38402 Saint Martin d'Hères, France.|University of Leeds, School of Biomedical Sciences and School of Physics and Astronomy, Leeds, LS2 9JT, UK.
Ralf P
RP
Richter
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