Oxidized galectin-1 promotes axonal regeneration in peripheral nerves but does not possess lectin properties
Oxidized galectin-1 promotes axonal regeneration in peripheral nerves but does not possess lectin properties
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DOI：
10.1046/j.1432-1327.2000.01311.x
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发表时间：
2000-05-01
期刊：
影响因子：
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通讯作者：
Kadoya, T
中科院分区：
文献类型：
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作者：
Inagaki, Y;Sohma, Y;Kadoya, T
Galectin-1 最近被确定为调节轴突切除术后周围神经初始轴突生长的因子。尽管半乳糖凝集素-1是一种众所周知的β-半乳糖苷结合凝集素，但其作为凝集素促进轴突再生的潜力尚未见报道。明确轴突切除术后周围神经的初始修复过程至关重要。因此，我们着手研究半乳糖凝集素-1的结构和轴突再生促进活性之间的关系。在非还原条件下纯化分泌到转染COS1细胞(rhGAL-1/COS1)的培养上清液中的重组人半乳糖凝集素-1，并进行结构分析。 rhGAL-1/COS1 肽片段的质谱分析表明，分泌蛋白以包含三个分子内二硫键（Cys2-Cys130、Cys16-Cys88 和 Cys42-Cys60）的氧化形式存在。重组人半乳糖凝集素-1 (rhGAL-1) 和所有六个半胱氨酸残基均被丝氨酸取代的半乳糖凝集素-1 突变体 (CSGAL-1) 在大肠杆菌中表达并纯化，用于进一步分析；纯化的 rhGAL-1 进行氧化，诱导出与 rhGAL-1/COS1 中观察到的相同模式的二硫键。氧化的rhGAL-1增强了成年大鼠背根神经节外植体与相关神经残端(5.0-5000 pg.mL(-1))横断神经部位的轴突再生，但缺乏凝集素活性。相反，CSGAL-1 诱导兔红细胞血凝，但缺乏轴突再生促进活性。这些结果表明，半乳糖凝集素-1仅以含有三个分子内二硫键的氧化形式促进轴突再生，而不以表现出凝集素活性的还原形式促进轴突再生。
Galectin-1 has recently been identified as a factor that regulates initial axonal growth in peripheral nerves after axotomy. Although galectin-1 is a well-known beta-galactoside-binding lectin, its potential to promote axonal regeneration as a lectin has not been reported. It is essential that the process of initial repair in peripheral nerves after axotomy is well clarified. We therefore undertook to investigate the relation between the structure and axonal regeneration-promoting activity of galectin-1. Recombinant human galectin-1 secreted into the culture supernatant of transfected COS1 cells (rhGAL-1/COS1) was purified under nonreducing conditions and subjected to structural analysis. Mass spectrometric analysis of peptide fragments from rhGAL-1/COS1 revealed that the secreted protein exists as an oxidized form containing three intramolecular disulfide bonds (Cys2-Cys130, Cys16-Cys88 and Cys42-Cys60). Recombinant human galectin-1 (rhGAL-1) and a galectin-1 mutant in which all six cysteine residues were replaced by serine (CSGAL-1) were expressed in and purified from Escherichia coli for further analysis; the purified rhGAL-1 was subjected to oxidation, which induced the same pattern of disulfide linkages as that observed in rhGAL-1/COS1. Oxidized rhGAL-1 enhanced axonal regeneration from the transected nerve sites of adult rat dorsal root ganglion explants with associated nerve stumps (5.0-5000 pg.mL(-1)), but it lacked lectin activity. In contrast, CSGAL-1 induced hemagglutination of rabbit erythrocytes but lacked axonal regeneration-promoting activity. These results indicate that galectin-1 promotes axonal regeneration only in the oxidized form containing three intramolecular disulfide bonds, not in the reduced form which exhibits lectin activity.