Suter U, Angst C, Tien C L, Drinkwater C C, Lindsay R M, Shooter E M
Department of Neurobiology, Stanford University School of Medicine, California 94305-5401.
J Neurosci. 1992 Jan;12(1):306-18. doi: 10.1523/JNEUROSCI.12-01-00306.1992.
Despite their extensive sequence identities at the amino acid level (approximately 55%), NGF and brain-derived neurotrophic factor (BDNF) display distinct neuronal specificity toward neurons of both the PNS and CNS. To explore which region(s) within these neurotrophic factors might determine their differential actions on various subpopulations of peripheral neurons, a systematic series (homolog-scanning mutagenesis) of chimeric NGF/BDNF molecules was prepared using PCR overlap-extension techniques. After expression in COS-7 cells, the chimeric proteins were tested for their biological activities in neurite outgrowth and neuronal survival assays. This approach led to the functional expression of 12 NGF/BDNF chimeras. Surprisingly, despite replacing successive amino acid segments throughout the entire length of NGF with the corresponding parts of BDNF, all chimeras displayed full NGF-like activity in bioassays carried out with PC12 cells, embryonic chick dorsal root ganglion explants, sympathetic ganglion explants, and dissociated cultures of dorsal root ganglion neurons. Most of the chimeras additionally showed BDNF-like activity as defined by neurite outgrowth on chick nodose ganglion explants. However, none of the chimeras supported the survival of dissociated nodose ganglion neurons. Our results suggest that NGF and BDNF must share very similar higher-order protein structures, and we propose that the overall structure or conformation of NGF, in contrast to short amino acid "active-site" segments, may determine its exact neuronal specificity.
尽管神经生长因子（NGF）和脑源性神经营养因子（BDNF）在氨基酸水平上具有广泛的序列同一性（约55%），但它们对周围神经系统（PNS）和中枢神经系统（CNS）的神经元表现出不同的神经元特异性。为了探究这些神经营养因子中的哪些区域可能决定它们对周围神经元不同亚群的差异作用，使用聚合酶链反应（PCR）重叠延伸技术制备了一系列系统性的嵌合NGF/BDNF分子（同源扫描诱变）。在COS-7细胞中表达后，对嵌合蛋白进行神经突生长和神经元存活测定，以检测其生物学活性。这种方法导致了12种NGF/BDNF嵌合体的功能性表达。令人惊讶的是，尽管用BDNF的相应部分替换了NGF全长中的连续氨基酸片段，但在用PC12细胞、胚胎鸡背根神经节外植体、交感神经节外植体以及背根神经节神经元的解离培养物进行的生物测定中，所有嵌合体都表现出完全的NGF样活性。大多数嵌合体在鸡结节神经节外植体上的神经突生长所定义的情况下还表现出BDNF样活性。然而，没有一种嵌合体能够支持解离的结节神经节神经元的存活。我们的结果表明，NGF和BDNF必须共享非常相似的高级蛋白质结构，并且我们提出，与短的氨基酸“活性位点”片段相比，NGF的整体结构或构象可能决定其确切的神经元特异性。
