Glycosylation-independent binding to extracellular domains 11-13 of mannose-6-phosphate/insulin-like growth factor-2 receptor mediates the effects of soluble CREG on the phenotypic modulation of vascular smooth muscle cells
Glycosylation-independent binding to extracellular domains 11-13 of mannose-6-phosphate/insulin-like growth factor-2 receptor mediates the effects of soluble CREG on the phenotypic modulation of vascular smooth muscle cells
复制标题
与 6-磷酸甘露糖/胰岛素样生长因子 2 受体胞外域 11-13 的糖基化独立结合介导可溶性 CREG 对血管平滑肌细胞表型调节的影响
DOI：
10.1016/j.yjmcc.2010.12.013
复制
发表时间：
2011-04-01
影响因子：
5
通讯作者：
Li, Shaohua
中科院分区：
文献类型：
--
作者：
Han, Yaling;Luan, Bo;Li, Shaohua
El a刺激基因的细胞抑制因子（CREG）的生物效应依赖于其n-糖基化和与甘露糖-6-磷酸/胰岛素样生长因子-2受体（M6P/IGF2R）的结合。本研究旨在探讨其结合的详细模式和特定位点，以及这种结合在血管平滑肌细胞（SMCs）表型调节中的功能相关性。在HEK293细胞中表达和分离了野生型和糖基化突变型人CREG （wtCREG和mCREG）蛋白。用CREG敲低的SMCs评价其生物活性。当将wtCREG和mCREG添加到培养基中时，它们都能阻止CREG敲除的SMCs的细胞周期进程。体外结合实验显示，CREG分别以糖基化依赖和独立的方式结合M6P/IGF2R胞外结构域7-10和11-13。利用可溶性M6P/IGF2R片段和M6P/IGF2R中和抗体进一步阻断实验表明，与结构域11-13和7-10的结合足以使CREG调节SMC增殖。这些数据表明，可溶性CREG蛋白可以通过不依赖糖基化结合细胞表面M6P/IGF2R的胞外结构域11-13来发挥其生物学功能，从而为CREG调节SMC从收缩到增殖的表型转换提供了新的见解。(C) 2010 Elsevier Ltd.版权所有。
The bio-effects of cellular repressor of El A-stimulated genes (CREG) have been proposed to depend on its N-glycosylation and binding to mannose-6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R). The present study aimed to investigate the detailed mode and specific sites for their binding and the functional relevance of this binding in the phenotypic modulation of vascular smooth muscle cells (SMCs). Wild-type and glycosylation mutant human CREG (wtCREG and mCREG) proteins were expressed and isolated from HEK293 cells. CREG knocked-down SMCs were used to evaluate their biological activity. Both wtCREG and mCREG arrest cell cycle progression of CREG knocked-down SMCs when added to the culture medium. In vitro binding assay revealed that CREG bound to M6P/IGF2R extracellular domains 7-10 and 11-13 in a glycosylation-dependent and -independent manner, respectively. Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody suggest that the binding to domains 11-13, as well as to 7-10, is adequate for CREG to modulate SMC proliferation. These data suggest that soluble CREG protein can exert its biological function via glycosylation-independent binding to the extracellular domains 11-13 of cell surface M6P/IGF2R, and thereby provide novel insights into CREG modulation of SMC phenotypic switching from contractile to proliferative. (C) 2010 Elsevier Ltd. All rights reserved.