Gowda D C, Goossen B, Margolis R K, Margolis R U
Department of Pharmacology, New York University Medical Center, New York 10016.
J Biol Chem. 1989 Jul 5;264(19):11436-43.
We have isolated and characterized the cell-associated and secreted proteoglycans synthesized by a clonal line of rat adrenal medullary PC12 pheochromocytoma cells, which have been extensively employed for the study of a wide variety of neurobiological processes. Chondroitin sulfate accounts for 70-80% of the [35S] sulfate-labeled proteoglycans present in PC12 cells and secreted into the medium. Two major chondroitin sulfate proteoglycans were detected with molecular sizes of 45,000-100,000 and 120,000-190,000, comprising 14- and 105-kDa core proteins and one or two chondroitin sulfate chains with an average molecular size of 34 kDa. In contrast to the chondroitin sulfate proteoglycans, one major heparan sulfate proteoglycan accounts for most of the remaining 20-30% of the [35S] sulfate-labeled proteoglycans present in the PC12 cells and medium. It has a molecular size of 95,000-170,000, comprising a 65-kDa core protein and two to six 16-kDa heparan sulfate chains. Both the chondroitin sulfate and heparan sulfate proteoglycans also contain O-glycosidically linked oligosaccharides (25-28% of the total oligosaccharides) and predominantly tri- and tetraantennary N-glycosidic oligosaccharides. Proteoglycans produced by the original clone of PC12 cells were compared with those of two other PC12 cell lines (B2 and F3) that differ from the original clone in morphology, adhesive properties, and response to nerve growth factor. Although the F3 cells (a mutant line derived from B2 and reported to lack a cell surface heparan sulfate proteoglycan) do not contain a large molecular size heparan sulfate proteoglycan species, there was no significant difference between the B2 and F3 cells in the percentage of total heparan sulfate released by mild trypsinization, and both the B2 and F3 cells synthesized cell-associated and secreted chondroitin sulfate and heparan sulfate proteoglycans having properties very similar to those of the original PC12 cell line but with a reversed ratio (35:65) of chondroitin sulfate to heparan sulfate.
我们已经分离并鉴定了由大鼠肾上腺髓质嗜铬细胞瘤PC12细胞克隆系合成的细胞相关和分泌型蛋白聚糖，该细胞系已被广泛用于多种神经生物学过程的研究。硫酸软骨素占PC12细胞中存在并分泌到培养基中的[35S]硫酸盐标记蛋白聚糖的70-80%。检测到两种主要的硫酸软骨素蛋白聚糖，分子大小分别为45,000-100,000和120,000-190,000，包含14 kDa和105 kDa的核心蛋白以及一或两条平均分子大小为34 kDa的硫酸软骨素链。与硫酸软骨素蛋白聚糖不同，一种主要的硫酸乙酰肝素蛋白聚糖占PC12细胞和培养基中剩余20-30%的[35S]硫酸盐标记蛋白聚糖的大部分。其分子大小为95,000-170,000，包含一个65 kDa的核心蛋白和两到六条16 kDa的硫酸乙酰肝素链。硫酸软骨素和硫酸乙酰肝素蛋白聚糖还含有O-糖苷键连接的寡糖（占总寡糖的25-28%）以及主要为三分支和四分支的N-糖苷键寡糖。将原始PC12细胞克隆产生的蛋白聚糖与另外两种PC12细胞系（B2和F3）的蛋白聚糖进行了比较，这两种细胞系在形态、黏附特性和对神经生长因子的反应方面与原始克隆不同。尽管F3细胞（一种源自B2且据报道缺乏细胞表面硫酸乙酰肝素蛋白聚糖的突变系）不含有大分子大小的硫酸乙酰肝素蛋白聚糖种类，但B2和F3细胞经轻度胰蛋白酶消化后释放的总硫酸乙酰肝素百分比没有显著差异，并且B2和F3细胞都合成了细胞相关和分泌型的硫酸软骨素和硫酸乙酰肝素蛋白聚糖，其性质与原始PC12细胞系非常相似，但硫酸软骨素与硫酸乙酰肝素的比例相反（35:65）。
