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

脑特异性6B4蛋白多糖的功能作用分析

• 批准号: 08680775
• 负责人: MAEDA Nobuaki
• 金额: $ 1.47万
• 依托单位: (National Institute for Basic Biology) Okazaki National Research Institutes
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08680775/
• 关键词: proteoglycan; 6B4 proteoglycan; PTPzeta; pleiotrophin; neurite extension; neuronal migration; プレイオトロフィン

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.

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 蛋白聚糖-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 而不是硫酸软骨素 A 抑制多效蛋白-6B4 蛋白聚糖的结合，这一事实支持了这一点。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。

项目成果

Shintani Takafumi: "Characterization of rat receptor-like protein tyrosine phosphatase γ isoforms." Biochemical and Biophysical Research Communication. 230. 419-425 (1997)

Shintani Takafumi：“大鼠受体样蛋白酪氨酸磷酸酶 γ 亚型的表征。”生物化学和生物物理研究通讯。230. 419-425 (1997)

Maeda Nobuaki et al.: "6B4 proteoglycan/phasphacan is a repulsine substratum bect promotes morphological differentiation of cortical neurons." Develapment. 122. 647-658 (1996)

Maeda Nobuaki 等人：“6B4 蛋白聚糖/相蛋白聚糖是一种排斥基质，可促进皮质神经元的形态分化。”

Nishiwaki Taeko et al.: "Characterization and develvpmental regulation of proteoglyian-type protein tyrosine phosphatase ζ/RPTPβ isoforms." Journal of Biochemistry. 123(in press). (1998)

Nishiwaki Taeko 等人：“蛋白聚糖型蛋白酪氨酸磷酸酶 δ/RPTPβ 亚型的表征和发育调节”，《生物化学杂志》123（出版中）。

前田信明: "軸索湯同の制御因子としてのプロテオグリカン" 生体の科学. 48・6. 534-548 (1997)

Nobuaki Maeda：“蛋白多糖作为轴突调节的调节剂”生物科学48・6（1997）。

Maeda Nobuaki et al.: "6B4 proteoglycan/phesphacan,an extracellular variant of receptar-like protein tyrosine phosphatase ζ/RPTPβ,binds pleiotrophin/heparin binding growth-associated molecnle(HB・GAM)" Journal of Bioloyical Chemistry. 271. 21446-21452 (199

Maeda Nobuaki 等人：“6B4 蛋白聚糖/phesphacan，受体样蛋白酪氨酸磷酸酶 ζ/RPTPβ 的细胞外变体，结合多效素/肝素结合生长相关分子 (HB·GAM)”《生物化学杂志》271。21446-。 21452 (199