Identification of receptors for phosphacan which inhibit axonal regeneration

采用阴离子交换柱层析和凝胶过滤柱层析分离纯化高纯度的重组磷酸多糖。在培养的背根神经节神经元中，增加重组磷聚糖的梯度诱导营养不良的终球，这是再生停止轴突的形态标志。应用电子显微镜和免疫细胞化学方法，在营养不良的终球中发现了自噬小体，提示自噬是营养不良终球形成的重要细胞事件。表面等离子体共振分析表明，磷酸多糖与硫酸软骨素的受体PTPsigma和LAR强烈结合。令人惊讶的是，磷酸多糖与PTPsigma或LAR之间的相互作用是两相的，不仅涉及硫酸软骨素，还涉及硫酸角蛋白。另一种受体酪氨酸磷酸酶的成员PTPzeta也参与了轴突再生的抑制。

Highly purified recombinant phosphacan was purepared using anion exchange and gel filtration chromatography. Increasing gradient of recombinant phosphacan induced dystrophic endball, a morphological marker for regeneration stopping axons, to cultured dorsal root ganglion neurons. Using electron microscope and immunocytochemistry, autophagosomes were found in dystrophic endball, suggesting that autophagy is an essential cellular event for formation of dystrophic endball.Surface plasmon resonance assay revealed that phosphacan strongly bound to PTPsigma and LAR which are known as receptors for chondroitin sulfate. Surprisingly, interaction between phosphacan and PTPsigma or LAR was two-phase manner, and not only chondroitin sulfate but also keratan sulfate were involved in interaction. Free keratan sulfate sugar also bound to PTPsigma and LAR.PTPzeta, another member of receptors tyrosine phosphatase, was also identified to be involved in inhibition of axonal regeneration.