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'.