6B4 proteoglycan/phosphacan, an extracellular variant of receptor-like protein-tyrosine phosphatase zeta/RPTP beta, binds pleiotrophin/heparin-binding growth-associated molecule (HB-GAM)
6B4 proteoglycan/phosphacan, an extracellular variant of receptor-like protein-tyrosine phosphatase zeta/RPTP beta, binds pleiotrophin/heparin-binding growth-associated molecule (HB-GAM)
复制标题
DOI：
10.1074/jbc.271.35.21446
复制
发表时间：
1996-08-30
影响因子：
4.8
通讯作者：
Noda, M
中科院分区：
文献类型：
--
作者：
Maeda, N;Nishiwaki, T;Noda, M
脑中主要的硫酸软骨素蛋白多糖，6B4蛋白多糖/磷酸，对应于受体样蛋白酪氨酸磷酸酶的胞外区域，PTP Zeta/RPTPβ。在这里，我们从大鼠脑中纯化并鉴定了6B4蛋白多糖结合蛋白。从CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic酸中提取的脑微粒体部分，用6B4蛋白多糖-琼脂糖特异性地分离出18-28-和40-kDa的蛋白质。N-末端氨基酸序列分析表明，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(K-d=3 nM)和高(K-d=0.25 nM)。蛋白多糖的软骨素酶ABC消化将结合亲和力降低到一个单一值(K-d=13 nM)，而不改变结合位点数。这表明蛋白多糖存在两个亚群，具有不同的硫酸软骨素结构。肝素能有效地抑制6B4蛋白多糖与多效营养素的结合(IC50=3.5 ng/ml)。硫酸乙酰肝素和硫酸软骨素C的抑制作用中等(IC50分别为150和400 ng/ml)，而硫酸软骨素A和硫酸角蛋白的抑制作用较差(IC50和GT；100µg/ml)。免疫荧光和免疫印迹分析表明，6B4蛋白多糖和PTP Zeta均位于皮质神经元上。在培养液中加入抗6B4蛋白多糖抗体可抑制多营养素诱导的皮质神经元突起生长。这些结果表明，多营养素的作用需要6B4蛋白多糖和多营养素之间的相互作用，而6B4蛋白多糖上的硫酸软骨素链在其结合中起着调节作用。
A major chondroitin sulfate proteoglycan in the brain, 6B4 proteoglycan/phosphacan, corresponds to the extracellular region of a receptor-like protein-tyrosine phosphatase, PTP zeta/RPTP beta. Here, we purified and characterized 6B4 proteoglycan-binding proteins from rat brain. From the CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) extract of brain microsomal fractions, 18- 28-, and 40-kDa proteins were specifically isolated using 6B4 proteoglycan-Sepharose. N-terminal amino acid sequencing identified the 18-kDa protein as pleiotrophin/heparin-binding growth-associated molecule (HB-GAM). Scatchard analysis of 6B4 proteoglycan-pleiotrophin binding revealed low (K-d = 3 nM) and high (K-d = 0.25 nM) affinity binding sites. Chondroitinase ABC digestion of the proteoglycan decreased the binding affinities to a single value (K-d = 13 nM) without changing the number of binding sites. This suggested the presence of two subpopulations of the proteoglycan with different chondroitin sulfate structures. Heparin potently inhibited binding of 6B4 proteoglycan to pleiotrophin (IC50 = 3.5 ng/ml). Heparan sulfate and chondroitin sulfate C inhibited moderately (IC50 = 150 and 400 ng/ml, respectively), but, in contrast, chondroitin sulfate A and keratan sulfate were poor inhibitors (IC50 > 100 mu g/ml). Immunofluorescence and immunoblotting analyses indicated that both 6B4 proteoglycan and PTP zeta are located on cortical neurons. Anti-6B4 proteoglycan antibody added to the culture medium suppressed pleiotrophin-induced neurite outgrowth of cortical neurons. These results suggested that interaction between 6B4 proteoglycan and pleiotrophin is required for the action of pleiotrophin, and chondroitin sulfate chains on 6B4 proteoglycan play regulatory roles in its binding.