Composition of perineuronal net extracellular matrix in rat brain - A different disaccharide composition for the net-associated proteoglycans
Composition of perineuronal net extracellular matrix in rat brain - A different disaccharide composition for the net-associated proteoglycans
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DOI：
10.1074/jbc.m600544200
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发表时间：
2006-06-30
影响因子：
4.8
通讯作者：
Fawcett, James W.
中科院分区：
文献类型：
--
作者：
Deepa, Sarama Sathyaseelan;Carulli, Daniela;Fawcett, James W.
我们开发了一种方法来差异提取广泛存在于中枢神经系统（CNS）基质中的硫酸软骨素蛋白聚糖（CSPG）和存在于神经元周网（PNN）凝聚基质中的CSPG。用Tris缓冲盐水（TBS）、含TBS的洗涤剂、1 M NaCl和6 M尿素依次提取成年大鼠脑。用这些缓冲液提取组织切片显示，在前三种缓冲液中提取了弥散和膜结合的CSPG，但PNN相关的CSPG保留并且仅通过6 M尿素去除。大多数CSPG在一定程度上用所有缓冲液提取，其中神经聚糖、短蛋白聚糖、聚集蛋白聚糖和多功能蛋白聚糖特别与稳定的尿素可提取的PNN相关。在出生后第7-14天发现仅在尿素缓冲液中可提取的稳定复合物中的CSPG与PNN形成一致。二糖组成分析表明，与细胞外基质（ECM）密切相关的PG的糖胺聚糖（GAG）组成不同。与其他缓冲液提取物相比，CS/硫酸皮肤素（DS）-GAG的非硫酸化、6-O-硫酸化、2，6-O-二硫酸化和4，6-O-二硫酸化二糖的含量较高，硫酸乙酰肝素（HS）-GAG的尿素提取物中6-O-硫酸化、2-N-、6-O-二硫酸化、2-O-、2-N-二硫酸化和2-O-、2-N-、6-O-三硫酸化二糖的含量较高。用软骨素酶ABC和透明质酸酶进行的地高辛标记表明，聚集蛋白聚糖、多功能蛋白聚糖、神经聚糖、短蛋白聚糖和磷酸蛋白聚糖通过与透明质酸（HA）结合而保留在PNN中。脑和脊髓ECM相对于CSPG的比较表明CNS的两个部分中的PNN具有相同的组成。
We developed a method to extract differentially chondroitin sulfate proteoglycans (CSPGs) that are diffusely present in the central nervous system (CNS) matrix and CSPGs that are present in the condensed matrix of perineuronal nets (PNNs). Adult rat brain was sequentially extracted with Tris-buffered saline (TBS), TBS-containing detergent, 1 M NaCl, and 6 M urea. Extracting tissue sections with these buffers showed that the diffuse and membrane-bound CSPGs were extracted in the first three buffers, but PNN- associated CSPGs remained and were only removed by 6 M urea. Most of the CSPGs were extracted to some degree with all the buffers, with neurocan, brevican, aggrecan, and versican particularly associated with the stable urea-extractable PNNs. The CSPGs in stable complexes only extractable in urea buffer are found from postnatal day 7-14 coinciding with PNN formation. Disaccharide composition analysis indicated a different glycosaminoglycan (GAG) composition for PGs strongly associated with extracellular matrix (ECM). For CS/dermatan sulfate (DS)-GAG the content of nonsulfated, 6-O-sulfated, 2,6-O-disulfated, and 4,6-O-disulfated disaccharides were higher and for heparan sulfate (HS)-GAG, the content of 6-O-sulfated, 2-N-,6-O-disulfated, 2-O-,2-N-disulfated, and 2-O-, 2-N-, 6-O-trisulfated disaccharides were higher in urea extract compared with other buffer extracts. Digestions with chondroitinase ABC and hyaluronidase indicated that aggrecan, versican, neurocan, brevican, and phosphacan are retained in PNNs through binding to hyaluronan (HA). A comparison of the brain and spinal cord ECM with respect to CSPGs indicated that the PNNs in both parts of the CNS have the same composition.