Ripellino J A, Margolis R U, Margolis R K
Department of Pharmacology, New York University Medical Center, New York 10016.
J Cell Biol. 1989 May;108(5):1899-907. doi: 10.1083/jcb.108.5.1899.
The 1C6 monoclonal antibody to the hyaluronic acid-binding region weakly stained a 65-kD component in immunoblots of the chondroitin sulfate proteoglycans of brain, and the 8A4 monoclonal antibody, which recognizes two epitopes in the polypeptide portion of link protein, produced strong staining of a 45-kD component present in the brain proteoglycans. These antibodies were utilized to examine the localization of hyaluronic acid-binding region and link protein epitopes in rat cerebellum. Like the chondroitin sulfate proteoglycans themselves and hyaluronic acid, hyaluronic acid-binding region and link protein immunoreactivity changed from a predominantly extracellular to an intracellular (cytoplasmic and intra-axonal) location during the first postnatal month of brain development. The cell types which showed staining of hyaluronic acid-binding region and link protein, such as granule cells and their axons (the parallel fibers), astrocytes, and certain myelinated fibers, were generally the same as those previously found to contain chondroitin sulfate proteoglycans and hyaluronic acid. Prominent staining of some cell nuclei was also observed. In agreement with earlier conclusions concerning the localization of hyaluronic acid and chondroitin sulfate proteoglycans, there was no intracellular staining of Purkinje cells or nerve endings or staining of certain other structures, such as oligodendroglia and synaptic vesicles. The similar localizations and coordinate developmental changes of chondroitin sulfate proteoglycans, hyaluronic acid, hyaluronic acid-binding region, and link protein add further support to previous evidence for the unusual cytoplasmic localization of these proteoglycans in mature brain. Our results also suggest that much of the chondroitin sulfate proteoglycan of brain may exist in the form of aggregates with hyaluronic acid.
针对透明质酸结合区域的1C6单克隆抗体在脑硫酸软骨素蛋白聚糖的免疫印迹中对一个65-kD的成分进行弱阳性染色，而识别连接蛋白多肽部分两个表位的8A4单克隆抗体对脑蛋白聚糖中存在的一个45-kD的成分产生强阳性染色。这些抗体被用于检测大鼠小脑透明质酸结合区域和连接蛋白表位的定位。与硫酸软骨素蛋白聚糖本身和透明质酸一样，在出生后第一个月的脑发育过程中，透明质酸结合区域和连接蛋白的免疫反应性从主要位于细胞外转变为细胞内（细胞质和轴突内）定位。显示透明质酸结合区域和连接蛋白染色的细胞类型，如颗粒细胞及其轴突（平行纤维）、星形胶质细胞和某些有髓纤维，通常与先前发现含有硫酸软骨素蛋白聚糖和透明质酸的细胞类型相同。还观察到一些细胞核有明显染色。与先前关于透明质酸和硫酸软骨素蛋白聚糖定位的结论一致，浦肯野细胞或神经末梢没有细胞内染色，某些其他结构，如少突胶质细胞和突触小泡也没有染色。硫酸软骨素蛋白聚糖、透明质酸、透明质酸结合区域和连接蛋白的相似定位以及协调的发育变化，进一步支持了先前关于这些蛋白聚糖在成熟脑中异常细胞质定位的证据。我们的结果还表明，脑中的许多硫酸软骨素蛋白聚糖可能以与透明质酸聚集的形式存在。
