Lierheimer R, Kunz B, Vogt L, Savoca R, Brodbeck U, Sonderegger P
Institute of Biochemistry, University of Zürich, Switzerland.
Eur J Biochem. 1997 Jan 15;243(1-2):502-10. doi: 10.1111/j.1432-1033.1997.0502a.x.
Axonin-1, a member of the immunoglobulin/fibronectin type-III family of cell-adhesion molecules, occurs both as a glycosylphosphatidylinositol-(glycosylPtdIns)-anchored membrane-bound and a soluble form. In vivo observations show that the major part of axonin-1 is found in the soluble fraction and that soluble axonin-1 perturbs neurite fasciculation and pathfinding in the developing chicken embryo. This has prompted further investigations into the mechanism of the axonin-1 release. We demonstrate here that axonin-1 released from dorsal root ganglion neurons contains ethanolamine and inositol, components of the glycosylPtdIns anchor. Secreted axonin-1 does not exhibit the cross-reacting determinant epitope, an indication that the cleavage of the anchor is not mediated by a phosphatidylinositol-specific phospholipase C. Treatment of dorsal root ganglion neurons with 1,10-phenanthroline, an inhibitor of glycosylPtdIns-specific phospholipase D, reduces the release of axonin-1 by 56%. Moreover, glycosylPtdIns-specific phospholipase D activity was detected in dorsal root ganglion neurons and brain. These results suggest that axonin-1 is released from the membrane by an endogenously expressed glycosylPtdIns-specific phospholipase D in vivo. With domain-swaping experiments between axonin-1 and its non-released relative F11, deletion mutants and monoclonal antibodies, we demonstrate that the fourth fibronectin type-III-like domain of axonin-1 is required for the generation of the soluble form of axonin-1.
轴突蛋白-1是免疫球蛋白/纤连蛋白III型细胞粘附分子家族的成员，以糖基磷脂酰肌醇（糖基PtdIns）锚定的膜结合形式和可溶性形式存在。体内观察表明，轴突蛋白-1的主要部分存在于可溶部分，并且可溶性轴突蛋白-1会干扰发育中的鸡胚中的神经突束状化和路径寻找。这促使人们进一步研究轴突蛋白-1释放的机制。我们在此证明，从背根神经节神经元释放的轴突蛋白-1含有乙醇胺和肌醇，它们是糖基PtdIns锚的组成成分。分泌的轴突蛋白-1不表现出交叉反应决定簇表位，这表明锚的切割不是由磷脂酰肌醇特异性磷脂酶C介导的。用糖基PtdIns特异性磷脂酶D的抑制剂1,10-菲咯啉处理背根神经节神经元，可使轴突蛋白-1的释放减少56%。此外，在背根神经节神经元和脑中检测到了糖基PtdIns特异性磷脂酶D活性。这些结果表明，轴突蛋白-1在体内是由内源性表达的糖基PtdIns特异性磷脂酶D从膜上释放的。通过轴突蛋白-1与其非释放相关物F11之间的结构域交换实验、缺失突变体和单克隆抗体，我们证明轴突蛋白-1的第四个纤连蛋白III型样结构域是产生轴突蛋白-1可溶性形式所必需的。
