Single-Molecule Mechanical Measurements of the Hyaluronan-Aggrecan Bottlebrush

Abstract

Large bottlebrush complexes formed from the polysaccharide hyaluronan (HA) and the proteoglycan aggrecan contribute to cartilage compression resistance and are necessary for healthy joint function. A variety of mechanical forces act on these complexes in the cartilage extracellular matrix, motivating the need for a quantitative description of their structure and mechanical response. Studies using electron microscopy have imaged the HA-aggrecan brush but require adsorption to a surface, dramatically altering the complex from its native conformation. We use magnetic tweezers force spectroscopy to measure changes in extension and mechanical response of an HA chain as aggrecan monomers bind and form a bottlebrush. This technique directly measures changes undergone by a single complex with time and under varying solution conditions. Upon addition of aggrecan, we find a large swelling effect manifests when the HA chain is under little to no tension (stretching forces less than ~1 pN). Theory of polymer brushes and models of side-chain-induced internal tension show our data are consistent with a polydisperse aggrecan population, attributable to varying degrees of glycosylation. Our results provide a mechanistic picture of how flexibility and size of HA and aggrecan lead to the brush architecture and mechanical properties of this important component of cartilage.