Ionic Effects and Self-assembly in the Solution of the Biopolymer Aggrecan

Introduction Aggrecan is a biologically important high molecular weight (1x10 < M < 3x10) proteoglycan. It possesses a bottlebrush structure, consisting of an extended protein core, to which many chondroitin sulfate and keratan sulfate (sulfated polysaccharide) chains are attached. In the presence of hyaluronic acid and a link protein, aggrecan molecules self-assemble into a supermolecular structure with as many as 100 (or more) aggrecan monomers condensed on a filament of hyaluronan. This complex, when interspersed in a collagen matrix, forms an interpenetrating network that gives cartilage its resilience and its ability to withstand compressive load with minimal deformation. It is noteworthy that degradative changes in the size and structure of aggrecan become more progressive with age. This process can be potentially deleterious to articular cartilage function as it reduces the charges on the aggrecan molecules and affects the mechanical properties of their assemblies. While recent research has focused mainly on the genetic and biochemical alterations associated with cartilage degeneration, relatively little is known about the physical properties that govern the interactions among the macromolecules that constitute the cartilage matrix itself. These interactions determine its osmotic resistance to compressive load. In the natural environment of aggrecan, in addition to other biological molecules, monoand divalent ions are present. Since the negatively charged aggrecan molecules bind cations, it is essential to know how cations affect the structural properties of this system. Calcium ions are particularly important as they play a critical role in skeletal metabolism. We investigated the effect of sodium and calcium ions on the organization of the aggrecan molecules in near physiological salt solutions by small angle neutron scattering (SANS) and small angle X-ray scattering (SAXS). We also measured the osmotic pressure of the aggrecan solutions in the presence of monoand divalent ions. Scattering and osmotic pressure measurements were made on solutions of aggrecan from bovine cartilage (Sigma) prepared in water containing 100 mM NaCl and various concentrations of CaCl2. In aqueous solutions of synthetic and natural polyelectrolytes small amounts of multivalent ions generally induce major changes in the molecular conformation, followed by phase separation. Here we report an unexpected finding for the biological polyelectrolyte aggrecan: addition of calcium chloride, even at concentrations as high as 200 mM, fails to precipitate this molecule, and does not even cause detectable changes in its conformation.