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的细胞外域。 6B4蛋白聚糖和ptpzeta是通过替代剪接产生的。在这项研究中，我确定了PTPZETA的配体分子，并分析了Ptpzeta的功能作用。使用6B4蛋白聚糖 - 塞芬罗斯从脑微粒体馏分的CHAP提取物中纯化了18 kDa 6B4蛋白聚糖结合蛋白。 N末端测序将该蛋白鉴定为多叶酸蛋白/肝素结合生长相关的分子（HB-GAM）。 SCATCHARD分析6B4蛋白聚糖 - 多叶酸蛋白结合显示较低（KD = 3 nm）和高（KD = 0.25 nm）亲和力结合位点。蛋白聚糖的软骨素酶ABC消化降低了与S单个值（KD = 13 nm）的结合亲和力，而无需改变结合位点的数量，这表明存在具有不同软骨素硫酸盐结构的蛋白聚糖的两个亚群。这是由硫酸软骨素C而非硫酸软骨素A抑制了多叶替诺蛋白6B4蛋白聚糖结合的事实支持的。抗蛋白酪氨酸磷酸酶抑制剂的抗6B4蛋白聚糖抗体，硫酸软骨C和钒酸钠抑制剂抑制了多叶酸蛋白蛋白诱导的神经蛋白延伸和神经元迁移。这些结果表明，pleiotiotrophin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）。

Nishizuka Masako et al.: "Cell purface-associated extracellular distribution of a neural preteaglycon,6B4 proteoghycon/phosphacan/in the olbactory epithelium,olfactory nerve" Neuroscience Research. 24. 345-355 (1996)

Nishizuka Masako 等人：“神经前茶糖苷、6B4 蛋白聚糖/磷酸聚糖/在嗅觉上皮、嗅神经中的细胞质相关的细胞外分布”神经科学研究。