Analysis of Signal Transduction Machineries Involving Two Types of NELL Proteins in the Differentiation Process of Neural Crest-derived Cells

NELL蛋白（NELL1,NELL2）是我们在1999年发现的具有独特分子结构的蛋白。从N-末端到c -末端，在这两种蛋白中都发现了信号肽，血栓反应蛋白1-N末端结构域，egf样基序和vwf衍生基序。这两个140-kDa的蛋白在氨基酸序列水平上有55%的相似性，作为分泌蛋白合成，形成约400-kDa的三聚体蛋白。通过我们前期的研究，由于NELL1 mRNA在颅盖增生患者颅骨缝合线处显著表达，最终证明NELL1是一种新的骨形态发生蛋白（BMP）。与传统的bmp（如BMP2、BMP4、BMP7）不同，NELL1蛋白只诱导成骨细胞的骨形成，而不影响胚胎背腹轴的测定。在本研究中，我们建立了利用昆虫细胞系统（连续、稳定、无血清）连续合成NELL蛋白的方法，并成功制备了大量纯化的NELL蛋白。这些NELL蛋白有助于我们通过表达克隆技术鉴定作为NELL特异性受体候选者的新分子，并通过x射线晶体学技术分析其3d结构。此外，我们成功建立了nell1过表达转基因小鼠和nell2偏转小鼠，以分析NELL蛋白的生理作用。

NELL proteins (NELL1,NELL2), found in 1999 by us, possess a unique molecular organization. From N- to C-terminal, a signal peptide, thrombospondin 1-N terminal domain, EGF-like motifs, and vWF-derived motifs are found in both proteins. These Two 140-kDa proteins show about 55% similarity in the amino acid sequence level, are synthesized as a secreted protein, and form a about 400-kDa homotrimeric proteins. By our previous studies, since NELL1 mRNA was significantly expressed at the suture of craniocynostosis patients' skull, the NELL1 was finally demonstrated as a novel bone morphogenic protein(BMP). Unlike the conventional BMPs (e.x., BMP2,BMP4,BMP7), the NELL1 protein showed exclusively the induction of bone formation in osteoblast cells and not affected the determination of dorsal-ventral axes in the embryos.In this study, we established the continuous synthesis of NELL proteins using insect cell system (continuous, stable, and serum-free) and succeeded in the preparation of large amount of pure NELL proteins. These NELL proteins facilitated us to identify the novel molecules as candidates of NELL-specific receptors by using expression cloning techniques and analyze the 3D-structure by using an X-ray crystallographic technique. Furthermore, we succeeded in the establishing NELL1-overexpressing transgenic mice and NELL2-deflcieat mice for analyzing the physiological roles of NELL proteins.