IDENTIFICATION OF HYALURONAN-BINDING DOMAINS OF AGGRECAN

Aggrecan, a large cartilage proteoglycan, interacts with hyaluronan (H A), to form aggregates which function to resist compression in joints. The N-terminal region of aggrecan contains two structurally related g lobular domains, G(1) and G(2) separated by IGD domain. The G(1) domai n consists of three subdomains, A, B, and B', structural features char acteristic to many other HA-binding proteoglycans. Here, we studied th e interaction of aggrecan domains with HA using recombinant proteins e xpressed in 293 cells, an embryonal kidney cell line. Deglycosylation of the recombinant aggrecan fragment reduced the HA binding activity. We found that both the B and B' subdomains were required for HA bindin g and that a single module of A, B, or B' was unable to bind HA. The A subdomain increased the HA binding activity of the B-B' region. The G (2) domain had no HA binding activity confirming previous reports. Stu dies of HA-binding properties using a BIAcore(TM) biosensor system rev ealed that the K-D of recombinant aggrecan fragment (AgW) consisting o f G(1), IGD, and G(2) was 0.226 mu M, whereas the K-D of another HA-bi nding protein, native bovine link protein, is 0.089 mu M. In contrast, Ag-Mut11 which lacked subdomain A showed little HA binding activity. AgMut12 consisting of only B-B' had a 3.4-fold lower affinity and AgMu t13 containing A-B-B' was 1.5-fold lower than AgW. These results sugge st that carbohydrates are essential for high level aggrecan binding to HA and that the A subdomain of aggrecan functions in a cooperative ma nner with subdomains B and B'.