Identification of hyaluronan-binding domains of aggrecan.
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, G1 and G2 separated by IGD domain. The G1 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 G2 domain had no HA binding activity confirming previous reports. Studies of HA-binding properties using a BIAcoreTM biosensor system revealed that the KD of recombinant aggrecan fragment (AgW) consisting of G1, IGD, and G2 was 0.226 microM, whereas the KD of another HA-binding protein, native bovine link protein, is 0.089 microM. 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'.
Watanabe
H
H
Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892-4370, USA.
Cheung
S C
SC
Itano
N
N
Kimata
K
K
Yamada
Y
Y
D053674:Aggrecans; D002460:Cell Line; D016326:Extracellular Matrix Proteins; D006031:Glycosylation; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism*; D037181:Lectins, C-Type; D011485:Protein Binding; D011509:Proteoglycans / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D053674:Aggrecans; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D011994:Recombinant Proteins; D006820:Hyaluronic Acid