Watanabe H, Cheung S C, Itano N, Kimata K, Yamada Y
Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892-4370, USA.
J Biol Chem. 1997 Oct 31;272(44):28057-65. doi: 10.1074/jbc.272.44.28057.
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'.
聚集蛋白聚糖是一种大型软骨蛋白聚糖，它与透明质酸（HA）相互作用，形成具有抵抗关节压缩功能的聚集体。聚集蛋白聚糖的N端区域包含两个结构相关的球状结构域，即G1和G2，它们被IGD结构域隔开。G1结构域由三个亚结构域A、B和B'组成，这是许多其他HA结合蛋白聚糖的典型结构特征。在此，我们使用在胚胎肾细胞系293细胞中表达的重组蛋白研究了聚集蛋白聚糖结构域与HA的相互作用。重组聚集蛋白聚糖片段的去糖基化降低了HA结合活性。我们发现B和B'亚结构域都是HA结合所必需的，且单个A、B或B'模块无法结合HA。A亚结构域增强了B - B'区域的HA结合活性。G2结构域没有HA结合活性，这与先前的报道一致。使用BIAcoreTM生物传感器系统对HA结合特性的研究表明，由G1、IGD和G2组成的重组聚集蛋白聚糖片段（AgW）的解离常数（KD）为0.226微摩尔，而另一种HA结合蛋白天然牛连接蛋白的KD为0.089微摩尔。相比之下，缺少亚结构域A的AgMut11几乎没有HA结合活性。仅由B - B'组成的AgMut12的亲和力低3.4倍，而包含A - B - B'的AgMut13比AgW低1.5倍。这些结果表明，碳水化合物对于聚集蛋白聚糖与HA的高水平结合至关重要，且聚集蛋白聚糖的A亚结构域与B和B'亚结构域协同发挥作用。
