Identification of receptors for phosphacan which inhibit axonal regeneration

采用阴离子交换和凝胶过滤层析法制备了高纯度的重组磷酸根蛋白。重组磷酸腺苷梯度增加诱导培养的背根神经节神经元营养不良终球（轴突再生停止的形态学标志）。电镜和免疫细胞化学观察发现，在营养不良终球中存在自噬体，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，磷酸腺苷能与硫酸软骨素受体PTPsigma和LAR强结合。令人惊讶的是，磷聚糖与PTPsigma或LAR的相互作用是两相的，不仅硫酸软骨素参与了相互作用，硫酸角蛋白也参与了相互作用。游离硫酸角蛋白糖也与PTPsigma和LAR结合。PTPzeta，受体酪氨酸磷酸酶的另一个成员，也被确定参与抑制轴突再生。

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.