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

6B4蛋白多糖/磷酸多糖是一种脑特异性硫酸软骨素蛋白多糖，与受体样蛋白酪氨酸磷酸酶PTPzeta/RPTPbeta的胞外结构域相对应。6B4蛋白多糖和PTPzeta通过选择性剪接产生。在本研究中，我鉴定了PTPzeta的一个配体分子，并分析了PTPzeta的功能作用。用6B4蛋白多糖-琼脂糖凝胶从脑微粒体CHAPS提取液中纯化出一种18 kDa的6B4蛋白多糖结合蛋白。N-末端测序证实该蛋白为多营养素/肝素结合生长相关分子(HB-GAM)。Scatchard分析显示，6B4蛋白多糖与多营养素结合的亲和力低(Kd=3 nM)和高(Kd=0.25 nM)。软骨素酶ABC消化降低了与S单值(Kd=13 nM)的结合亲和力，但不改变结合位点数，提示蛋白多糖存在两个亚群，具有不同的硫酸软骨素结构。支持这一事实的事实是，硫酸软骨素C而不是硫酸软骨素A抑制了多营养素-6B4蛋白多糖的结合。在培养液中加入抗6B4蛋白多糖抗体、硫酸软骨素C和蛋白酪氨酸磷酸酶抑制剂钒酸钠，可抑制多营养素诱导的神经突起延伸和神经元迁移。这些结果表明，多营养素是PTPzeta的一种功能配体。

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.