Maeda N, Hamanaka H, Oohira A, Noda M
Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki, Japan.
Neuroscience. 1995 Jul;67(1):23-35. doi: 10.1016/0306-4522(94)00069-h.
A large brain-specific chondroitin sulfate proteoglycan, identified with monoclonal antibody 6B4 (6B4 proteoglycan/phosphacan), was isolated from rat brain. Soluble proteoglycans in the phosphate-buffered saline extract from 20-day-old rat whole brain were fractionated by anion exchange chromatography and CsCl density gradient centrifugation. 6B4 proteoglycan was further purified by gel filtration and additional ion exchange chromatography. The molecular mass of 6B4 proteoglycan shifted from 800 to 300 x 10(3) mol. wt after chondroitinase ABC digestion. The core protein was substituted with chondroitin sulfate chains with an average molecular weight of 21,000, keratan sulfate and HNK-1 carbohydrates. Glycosidase digestion of 6B4 proteoglycan with O-glycanase, N-glycanase, endo-beta-galactosidase, or keratanase did not remove the HNK-1 epitopes. The expression of 6B4 proteoglycan was developmentally regulated in the rat cerebral cortex; appearing first at embryonic day 14, peaking at postnatal day 0, and persisting throughout adulthood at a lower level. Immunohistochemical analysis indicated that 6B4 proteoglycan was distributed along the radial glial fibers and on the migrating neurons in the embryonal rar cerebrum. The radial glial fibers were stained intensely all along their length, but the neurons in the cortical plate were not stained in contrast to the moderate staining of the migrating neurons in the intermediate zone and the subplate. From postnatal day 5 to postnatal day 20, 6B4 proteoglycan was present throughout the cortex. After postnatal day 30, staining of the neuropil was weakened, and the expression of 6B4 proteoglycan was restricted around subsets of neurons. The positive neurons were mostly non-pyramidal cells (> 95%) and were relatively concentrated in layers IV and VI of the primary somatosensory cortex. Immunohistochemical analysis of the dissociated cortical neurons indicated that 6B4 proteoglycan was distributed on the cell bodies and neurites. 6B4 proteoglycan strikingly promoted neurite extension of cortical neurons from embryonic day-16 rat embryos when coated on coverslips as a substrate. 6B4 proteoglycan is a brain-specific chondroitin sulfate proteoglycan which carries keratan sulfate and HNK-1 carbohydrates. The spatiotemporal expression profile and effects on the dissociated cerebral neurons suggest that 6B4 proteoglycan plays important roles in the migration and differentiation of neurons in the immature cortex, and also in the maintenance of subsets of neurons in the mature cortex.
一种用单克隆抗体6B4鉴定的大脑特异性硫酸软骨素蛋白聚糖（6B4蛋白聚糖/磷蛋白聚糖）从大鼠脑中分离出来。通过阴离子交换色谱法和CsCl密度梯度离心法对20日龄大鼠全脑磷酸盐缓冲盐水提取物中的可溶性蛋白聚糖进行分级分离。6B4蛋白聚糖通过凝胶过滤和额外的离子交换色谱法进一步纯化。经软骨素酶ABC消化后，6B4蛋白聚糖的分子量从800×10³道尔顿变为300×10³道尔顿。核心蛋白被平均分子量为21,000的硫酸软骨素链、硫酸角质素和HNK-1碳水化合物取代。用O-聚糖酶、N-聚糖酶、内切β-半乳糖苷酶或角质素酶对6B4蛋白聚糖进行糖苷酶消化并未去除HNK-1表位。6B4蛋白聚糖在大鼠大脑皮质中的表达受发育调控；在胚胎第14天首次出现，在出生后第0天达到峰值，并在成年期持续存在于较低水平。免疫组织化学分析表明，6B4蛋白聚糖沿放射状胶质纤维分布，并存在于胚胎大鼠大脑中的迁移神经元上。放射状胶质纤维在其全长上均被强烈染色，但与中间带和板下层中迁移神经元的中度染色形成对比的是，皮质板中的神经元未被染色。从出生后第5天到出生后第20天，6B4蛋白聚糖存在于整个皮质中。出生后第30天之后，神经毡的染色减弱，6B4蛋白聚糖的表达局限于神经元亚群周围。阳性神经元大多是非锥体细胞（>95%），相对集中在初级体感皮质的IV层和VI层。对解离的皮质神经元进行免疫组织化学分析表明，6B4蛋白聚糖分布在细胞体和神经突上。当作为底物包被在盖玻片上时，6B4蛋白聚糖显著促进了来自胚胎第16天大鼠胚胎的皮质神经元的神经突延伸。6B4蛋白聚糖是一种大脑特异性硫酸软骨素蛋白聚糖，携带硫酸角质素和HNK-1碳水化合物。其时空表达谱以及对解离的大脑神经元的影响表明，6B4蛋白聚糖在未成熟皮质中神经元的迁移和分化以及成熟皮质中神经元亚群的维持中发挥重要作用。
