IDENTIFICATION OF PROSAPOSIN AS A NEUROTROPHIC FACTOR
IDENTIFICATION OF PROSAPOSIN AS A NEUROTROPHIC FACTOR
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DOI：
10.1073/pnas.91.20.9593
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发表时间：
1994-09-27
影响因子：
11.1
通讯作者：
KISHIMOTO, Y
中科院分区：
文献类型：
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作者：
OBRIEN, JS;CARSON, GS;KISHIMOTO, Y
Prosaposin是一种神经营养因子，可刺激小鼠神经母细胞瘤（NS20Y）细胞的神经突生长和人神经母细胞瘤（SK-N-MC）细胞的胆碱乙酰转移酶（ChAT）活性。对四种天然存在的皂素进行了活性测试，这四种皂素是由皂素的蛋白水解加工得到的。皂苷C是有活性的，而皂苷A、B和D作为神经营养因子是无活性的。剂量-反应曲线表明，纳摩尔浓度的丙苷和皂苷C刺激神经突生长，增加ChAT活性。皂苷和皂苷C发挥活性的机制独立于神经生长因子、脑源性神经营养因子和神经营养因子3。利用皂苷C作为配体的结合实验得到了两个饱和的结合常数，高亲和力（K-d = 19 pM）和低亲和力（K-d = 1 nM）常数，每个NS20Y细胞分别有2000和15000个位点。磷酸化刺激实验表明，用皂素或皂素C短暂处理可增强多种蛋白质的磷酸化，其中一些蛋白质含有磷酸化的酪氨酸。由于这两种细胞系也受到睫状神经营养因子（CNTF）和丙皂苷的刺激，因此利用抗gp130单克隆抗体来测试抑制作用，该抗体特异性抑制CNTF的刺激；该抗体不抑制皂素或皂素C的刺激。这些结果表明，皂素和皂素C是一种神经营养因子，通过与高亲和力受体结合，诱导蛋白磷酸化，从而启动信号转导。
Prosaposin was identified as a neurotrophic factor stimulating neurite outgrowth in murine neuroblastoma (NS20Y) cells and choline acetyltransferase (ChAT) activity in human neuroblastoma (SK-N-MC) cells. The four naturally occurring saposins, which are derived by proteolytic processing of prosaposin, were tested for activity. Saposin C was found to be active, whereas saposins A, B, and D were inactive as neurotrophic factors. Dose-response curves demonstrated that nanomolar concentrations of prosaposin and saposin C stimulated neurite outgrowth and increased ChAT activity. Prosaposin and saposin C exerted activity by a mechanism independent of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3. Binding assays utilizing saposin C as a ligand gave two saturable binding constants, a high-affinity (K-d = 19 pM) and a low-affinity (K-d = 1 nM) constant, with 2000 and 15,000 sites per NS20Y cell, respectively. Phosphorylation stimulation experiments demonstrated that brief treatment with prosaposin or saposin C enhanced phosphorylation of a variety of proteins, some of which contained phosphorylated tyrosine(s). Since both cell lines were also stimulated by ciliary neurotrophic factor (CNTF) as well as prosaposin, inhibition was tested by utilizing an anti-gp130 monoclonal antibody, which specifically inhibited CNTF stimulation; this antibody did not inhibit prosaposin or saposin C stimulation. These results indicate that prosaposin and saposin C are neurotrophic factors which initiate signal transduction by binding to a high-affinity receptor that induces protein phosphorylation.