Brandan E, Inestrosa N C
J Neurobiol. 1987 May;18(3):271-82. doi: 10.1002/neu.480180303.
We have previously shown that asymmetric collagen-tailed acetylcholinesterase (AChE) is anchored to the extracellular matrix (ECM) by heparan sulfate proteoglycans (HSPGs). Here we present our studies on the characterization of such PGs from the ECM of rat skeletal muscles. After radiolabeling with 35SO4 for 24h, PGs were extracted from the muscle ECM with 4.0 M guanidine-HCl containing protease inhibitors. PGs were subsequently isolated using sequential DEAE-Sephacel chromatography, digestion with chondroitinase ABC, and Sepharose CL-4B. Two different hydrodynamic size species of HSPGs were found. One type had a Mr of 4-6 X 10(5) (Kav = 0.25) as estimated by gel chromatography in the presence of 1% SDS and accounted for 75% of the total HSPGs. The other HSPG had a Mr 1.5-2.5 X 10(5) (Kav = 0.41). The glycosaminoglycan (GAG) side chains (Mr 20,000 and 12,000) were found composed only of heparan sulfate as determined by nitrous acid oxidation and heparitinase treatment. The large-sized HSPG, which is concentrated in synaptic regions, contains only GAG chains of Mr 20,000, suggesting that each HSPG contains only one kind of heparan sulfate chain in its structure. Our results definitively establish by biochemical criteria that the basement membrane of mammalian skeletal muscle contains HSPGs, the likely matrix receptor for the immobilization of the asymmetric collagen-tailed AChE at the neuromuscular junction.
我们之前已经表明，不对称胶原尾型乙酰胆碱酯酶（AChE）通过硫酸乙酰肝素蛋白聚糖（HSPGs）锚定在细胞外基质（ECM）上。在此，我们展示了对来自大鼠骨骼肌ECM的此类蛋白聚糖的特性研究。用35SO4进行24小时放射性标记后，用含有蛋白酶抑制剂的4.0 M盐酸胍从肌肉ECM中提取蛋白聚糖。随后通过连续的DEAE - Sephacel色谱、用软骨素酶ABC消化以及Sepharose CL - 4B分离蛋白聚糖。发现了两种不同流体动力学大小的HSPGs。一种类型在1% SDS存在下通过凝胶色谱估计其Mr为4 - 6×10(5)（Kav = 0.25），占总HSPGs的75%。另一种HSPG的Mr为1.5 - 2.5×10(5)（Kav = 0.41）。通过亚硝酸氧化和乙酰肝素酶处理确定，糖胺聚糖（GAG）侧链（Mr分别为20,000和12,000）仅由硫酸乙酰肝素组成。集中在突触区域的大尺寸HSPG仅包含Mr为20,000的GAG链，这表明每个HSPG在其结构中仅包含一种硫酸乙酰肝素链。我们的结果通过生化标准明确证实，哺乳动物骨骼肌的基底膜含有HSPGs，它可能是在神经肌肉接头处固定不对称胶原尾型AChE的基质受体。
