Analysis of the functional roles of brain-specific 6B4 proteoglycan

6B4 蛋白聚糖/磷酸聚糖是一种脑特异性硫酸软骨素蛋白聚糖，对应于受体样蛋白酪氨酸磷酸酶 PTPzeta/RPTPbeta 的胞外结构域。 6B4 蛋白聚糖和 PTPzeta 是通过选择性剪接产生的。在这项研究中，我鉴定了PTPzeta的配体分子，并分析了PTPzeta的功能作用。使用 6B4 蛋白聚糖-Sepharose 从脑微粒体组分的 CHAPS 提取物中纯化出 18 kDa 6B4 蛋白聚糖结合蛋白。 N 端测序鉴定该蛋白为多效素/肝素结合生长相关分子 (HB-GAM)。 6B4 蛋白多糖-多效蛋白结合的 Scatchard 分析揭示了低 (Kd = 3 nM) 和高 (Kd = 0.25 nM) 亲和力结合位点。蛋白多糖的软骨素酶 ABC 消化将结合亲和力降低至单一值 (Kd = 13 nM)，而不改变结合位点的数量，表明存在具有不同硫酸软骨素结构的两个蛋白多糖亚群。硫酸软骨素 C 而硫酸软骨素 A 不抑制多效蛋白-6B4 蛋白聚糖的结合这一事实也支持了这一点。添加到培养基中的抗 6B4 蛋白聚糖抗体、硫酸软骨素 C 和钒酸钠（一种蛋白酪氨酸磷酸酶抑制剂）可抑制多效蛋白诱导的神经突延伸和神经元迁移。这些结果表明多效蛋白是 PTPzeta 的功能性配体。

6B4 proteoglycan/phosphacan is a brain-specific chondroitin sulfate proteoglycan, which corresponds to the extracellular domain of a receptor-like protein tyrosine phosphatase, PTPzeta/RPTPbeta. 6B4 proteoglycan and PTPzeta are generated by alternative splicing. In this study, I identified a ligand molecule of PTPzeta, and analyzed the functional roles of PTPzeta. A 18 kDa 6B4 proteoglycan-binding protein was purified from the CHAPS extract of brain microsomal fractions using 6B4 proteoglycan-Sepharose. N-terminal sequencing identified this protein as pleiotrophin/heparin-binding growth-associated molecule (HB-GAM). Scatchard analysis of 6B4 proteoglycan- pleiotrophin binding revealed low (Kd = 3 nM) and high (Kd = 0.25 nM) affinity binding sites. Chondroitinase ABC digestion of the proteoglycan decreased the binding affinities to s single value (Kd = 13 nM) without changing the number of binding sites, suggesting the presence of two subpopulations of the proteoglycan with different chondroitin sulfate structures. This was supported by the fact that chondroitin sulfate C but not chondroitin sulfate A inhibited the pleiotrophin-6B4 proteoglycan binding. Anti-6B4 proteoglycan antibody, chondroitin sulfate C and sodium vanadate, a protein tyrosine phosphatase inhibitor, added to the culture medium suppressed pleiotrophin-induced neurite extension and neuronal migration. These results indicated that pleiotrophin is a functional ligand of PTPzeta.