Rauch U, Clement A, Retzler C, Fröhlich L, Fässler R, Göhring W, Faissner A
Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany.
J Biol Chem. 1997 Oct 24;272(43):26905-12. doi: 10.1074/jbc.272.43.26905.
Neurocan is a member of the aggrecan family of proteoglycans which are characterized by NH2-terminal domains binding hyaluronan, and COOH-terminal domains containing C-type lectin-like modules. To detect and enhance the affinity for complementary ligands of neurocan, the COOH-terminal neurocan domain was fused with the NH2-terminal region of tenascin-C, which contains the hexamerization domain of this extracellular matrix glycoprotein. The fusion protein was designed to contain the last downstream glycosaminoglycan attachment site and was expressed as a proteoglycan. In ligand overlay blots carried out with brain extracts, it recognized tenascin-C. The interaction was abolished by the addition of EDTA, or TNfn4,5, a bacterially expressed tenascin-C fragment comprising the fourth and fifth fibronectin type III module. The fusion protein directly reacted with this fragment in ligand blot and enzyme-linked immunosorbent assay procedures. Both tenascin-C and TNfn4,5 were retained on Sepharose 4B-linked carboxyl-terminal neurocan domains, which in BIAcore binding studies yielded a KD value of 17 nM for purified tenascin-C. We conclude that a divalent cation-dependent interaction between the COOH-terminal domain of neurocan and those fibronectin type III repeats is substantially involved in the binding of neurocan to tenascin-C.
神经黏蛋白是蛋白聚糖聚集蛋白聚糖家族的成员，其特征在于具有结合透明质酸的NH2末端结构域以及含有C型凝集素样模块的COOH末端结构域。为了检测并增强神经黏蛋白对互补配体的亲和力，将神经黏蛋白的COOH末端结构域与腱生蛋白-C的NH2末端区域融合，该区域包含这种细胞外基质糖蛋白的六聚化结构域。设计融合蛋白使其包含最后一个下游糖胺聚糖附着位点，并表达为蛋白聚糖。在用脑提取物进行的配体覆盖印迹中，它识别腱生蛋白-C。加入EDTA或TNfn4,5（一种细菌表达的包含第四和第五个纤连蛋白III型模块的腱生蛋白-C片段）后，这种相互作用被消除。在配体印迹和酶联免疫吸附测定程序中，融合蛋白与该片段直接反应。腱生蛋白-C和TNfn4,5都保留在与琼脂糖4B连接的神经黏蛋白COOH末端结构域上，在BIAcore结合研究中，纯化的腱生蛋白-C的KD值为17 nM。我们得出结论，神经黏蛋白的COOH末端结构域与那些纤连蛋白III型重复序列之间的二价阳离子依赖性相互作用在很大程度上参与了神经黏蛋白与腱生蛋白-C的结合。
