Identification of hyaluronan-binding domains of aggrecan

Abstract

Aggrecan, a large cartilage proteoglycan, interacts with hyaluronan (HA), to form aggregates which function to resist compression in joints. The N-terminal region of aggrecan contains two structurally related globular domains, G 1 and G 2 separated by IGD domain. The G 1 domain consists of three subdomains, A, B, and B', structural features characteristic to many other HA-binding proteoglycans. Here, we studied the interaction of aggrecan domains with HA using recombinant proteins expressed 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 binding 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. Studies of HA-binding properties using a BIAcore(TM) biosensor system revealed that the K(D) of recombinant aggrecan fragment (AgW) consisting of G 1, IGD, and G 2 was 0.226 μM, whereas the K(D) of another HA-binding protein, native bovine link protein, is 0.089 μM. In contrast, AgMut11 which lacked subdomain A showed little HA binding activity. AgMut12 consisting of only B-B' had a 3.4-fold lower affinity and AgMut13 containing A-B-B' was 1.5-fold lower than AgW. These results suggest that carbohydrates are essential for high level aggrecan binding to HA and that the A subdomain of aggrecan functions in a cooperative manner with subdomains B and B'.