Studies on the role disulfide bond exchange plays in the aggregation of aggrecan with hyaluronic acid

Aggrecan is assembled in the extracellular matrix as an aggregate with hyaluronic acid and link protein. In older and osteoarthritic tissue, in the absence of link protein, aggrecan shows a time-dependent, post-secretion increase in affinity for hyaluronic acid converting from a low to a high affinity form. Because the rate of the switch increases with increased pH, disulfide bond rearrangement has been proposed. Aggrecan D1 preparations purified under argon routinely contained 0.3 to 0.5 mole of thiol/mole aggrecan that required at least 2.5 M guanidine hydrochloride (Gdn-HCl) to be present, but could be modified with hydrophobic thiol modification agents in the absence of Gdn-HCl. From multiple preparations of bovine and rat aggrecan D1 fractions, two tryptic peptides could be purified and sequenced; one isolated from the central domain of biglycan, and the other from the A loop of the N-terminal domain of aggrecan. Quantitation of the thiol content of highly purified aggrecan showed that aggrecan contains 0.05 mole free thiol/mole aggrecan. The cysteine residue in aggrecan was shown to be in a hydrophobic environment. The evidence for this was obtained by fluorescent quenching, and denaturation studies of AEDANS-labeled aggrecan. These results may represent the trapping of an intermediate species of aggrecan involved in disulfide bond rearrangement prior to binding hyaluronic acid.