Identification of receptors for phosphacan which inhibit axonal regeneration

用阴离子交换和凝胶过滤层析法纯化了高纯度的重组磷酸聚糖。增加梯度的重组磷酸蛋白聚糖诱导营养不良的endball，再生停止轴突的形态学标记，培养背根神经节神经元。电镜和免疫细胞化学结果显示，在营养不良的终球中发现了自噬体，表明自噬是营养不良终球形成的一个重要的细胞事件。表面等离子体共振分析显示磷酸根聚糖与硫酸软骨素受体PTPsigma和LAR有很强的结合。令人惊讶的是，phosphacan与PTPsigma或LAR之间的相互作用是两相方式，并且不仅硫酸软骨素而且硫酸角质素参与相互作用。游离硫酸角质素糖也与PTPsigma和LAR结合。PTPzeta是酪氨酸磷酸酶受体的另一个成员，也被鉴定参与轴突再生的抑制。

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.