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Identification of biologically active domains of neuroglycan C that can be applicable to repair of brain injuries

鉴定可用于修复脑损伤的神经聚糖 C 的生物活性结构域

基本信息

批准号：
14370449
负责人：
OOHIRA Atsuhiko
金额：
$ 6.78万
依托单位：
Institute for Developmental Research, Aichi Human Service Center
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2005
项目状态：
已结题

项目摘要

The purpose of this project is identification of biologically active domains of neuroglycan C (NGC), a brain-specific transmembrane chondroitin sulfate proteoglycan. The major results obtained are as follows.1. A single EGF-like module exists in the ectodomain of the NGC core protein. A recombinant peptide containing the EGF-like module promoted phosphorylation of EGF-receptors, ErbB2 and ErbB3, and proliferation of cell lines that express all of four EGF-receptors, indicating that NGC is a novel member of the EGF family.2. A recombinant NGC ectodomain promoted neurite elongation from neocortical neurons in culture. Inhibitors of phosphatidylinositol 3-kinase and of protein kinase C prevented NGC-mediated neurite elongation. The active sites for promotion of neurite elongation existed on the acidic domain and the EGF-like domain of the NGC ectodomain.3. NGC bore achondroitin sulfate (CS) side chain only at Ser-123 on the core protein. NGC isolated from postnatal day-10 rat brains had a relatively high content of E-disaccharide units, an oversulfated CS disaccharide unit, compared with neurocan and phosphacan.4. CS-E, a commercial preparation of highly sulfated CS, promoted FGF-2-mediated proliferation of neural stem cells. To identify the minimum size of CS-E-derived oligosaccharides that have a promotive activity for neural stem cell growth, CS-E was partially degraded, and activities of the degradation products with different molecular sizes were determined. A fraction of 3 kDa (〜12 sugars) showed a strong activity for promotion of neural stem cell proliferation. Quartz-crystal microbalance method revealed that both the 3 kDa fraction and a hexasulfated hexasaccharide had a high affinity to FGF-2,but the latter did not have the promotive activity for cell proliferation.Thus, the EGF-like domain of the NGC core protein and a 3 kDa oligosaccharide derived from E-unit-rich CS have properties that can be applicable to repair of brain injuries.

本项目的目的是鉴定神经多糖C(NGC)的生物活性结构域，NGC是一种大脑特异的跨膜硫酸软骨素蛋白多糖。取得的主要结果如下：1.研究成果。在NGC核心蛋白的胞外区中存在一个单一的EGF样模块。含有类EGF模块的重组肽可促进EGF受体ErbB2和ErbB3的磷酸化，并促进表达全部四种EGF受体的细胞系的增殖，表明NGC是EGF家族的一个新成员。重组NGC胞外区可促进培养的新皮质神经元突起的伸长。磷脂酰肌醇3-激酶和蛋白激酶C的抑制剂可阻止NGC介导的轴突延长。促进轴突伸长的活性部位位于NGC外膜结构域的酸性结构域和EGF样结构域上。NGC只在核心蛋白的Ser-123处含有硫酸软骨素(CS)侧链。从出生后第10天的大鼠脑中分离的NGC中，E-二糖单位的含量相对较高，这是一种过度硫酸盐化的CS二糖单位，与神经聚糖和磷酸多糖相比。CS-E是一种商品化的高度硫酸盐化的CS，可促进成纤维细胞生长因子-2介导的神经干细胞的增殖。为了确定具有促进神经干细胞生长活性的CS-E衍生低聚糖的最小尺寸，对CS-E进行了部分降解，并测定了不同分子尺寸的降解产物的活性。3 kDa(~12糖)组分具有较强的促进神经干细胞增殖的活性。石英晶体微天平分析表明，NGC核心蛋白的EGF样结构域和富含E单元的CS衍生的3 kDa低聚糖具有脑损伤修复的特性。