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

本项目的目的是鉴定神经聚糖C （NGC）的生物活性域，NGC是脑特异性的跨膜硫酸软骨素蛋白多糖。得到的主要结果如下：1。一个单一的egf样模块存在于NGC核心蛋白的外域。含有EGF样模块的重组肽促进了EGF受体ErbB2和ErbB3的磷酸化，以及表达所有四种EGF受体的细胞系的增殖，这表明NGC是EGF家族的新成员。重组NGC外结构域促进培养的新皮层神经元的神经突伸长。磷脂酰肌醇3-激酶和蛋白激酶C抑制剂可阻止ngc介导的神经突伸长。2 .促进神经突伸长的活性位点存在于NGC外域的酸性区域和egf样区域。NGC仅在核心蛋白Ser-123处含有硫酸软骨素（achondroitin sulfate， CS）侧链。从出生第10天的大鼠脑中分离的NGC与神经聚糖和磷聚糖相比，e -双糖单位（一种过硫酸化的CS双糖单位）的含量相对较高。CS- e是一种高硫酸盐CS的商业化制剂，可促进fgf -2介导的神经干细胞增殖。为了确定具有促进神经干细胞生长活性的CS-E来源低聚糖的最小尺寸，我们对CS-E进行了部分降解，并测定了不同分子大小的降解产物的活性。3 kDa的一小部分（约12个糖）显示出强烈的促进神经干细胞增殖的活性。石英晶体微平衡法显示，3kda片段和六硫化六糖对FGF-2均具有高亲和力，但后者不具有促进细胞增殖的活性。因此，NGC核心蛋白的egf样结构域和来自富含e -unit的CS的3kda低聚糖具有可用于脑损伤修复的特性。

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.