McQuillan D J, Midura R J, Hascall V C, Yanagishita M
Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Australia.
Biochem J. 1992 Jul 1;285 ( Pt 1)(Pt 1):25-33. doi: 10.1042/bj2850025.
The heparan sulphate (HS) proteoglycans associated with the cell layer of a rat osteosarcoma cell line [UMR 106-01 (BSP)] were compared with similar cell-associated proteoglycans from other cells, and their interaction with the plasma membrane was studied. HS proteoglycans were metabolically labelled by incubation of cell cultures with [3H]glucosamine or [3H]leucine and [35S]sulphate. HS proteoglycan core protein preparation generated by heparitinase digestion of the major species from UMR 106-01 (BSP) cells co-migrated on PAGE with identical preparations from ovarian granulosa cells and parathyroid cells (at approximately 70 kDa). The hydrophobic nature of the major HS proteoglycans from these diverse cell lines, based on elution position from octyl-Sepharose, were also comparable. Linkages of the HS proteoglycan to the cell membrane were investigated by labelling plasma-membrane preparations with a lipid soluble photoactivatable reagent, 3-(trifluoromethyl)-3- (m-[125I]iodophenyl)diazirine (TID), which selectively labels plasma-membrane-spanning peptide domains. Purified HS proteoglycan from UMR 106-01 (BSP) cells was shown to be accessible to the [125I]TID, and the core protein portion of the molecule was labelled, confirming its close association with the plasma membrane. Approx. 36% of 35S-labelled HS proteoglycans were released from the cell surface by phospholipase C (Bacillus thuringiensis), which specifically cleaves phosphatidylinositol-linked proteins. In the presence of insulin, the metabolism of the phospholipase C-sensitive population was unaltered; however, release of the phospholipase C-insensitive population into the medium was increased. These data indicate that a subpopulation of HS proteoglycans are covalently bound to the plasma membrane by a glycosylphosphatidylinositol structure, with the remainder representing those species directly inserted into the plasma membrane via a hydrophobic peptide domain. These observations are similar to those reported for ovarian granulosa cells [Yanagishita & McQuillan (1989) J. Biol. Chem. 264 17551-17558], and thus may represent a general phenomenon for many cell types.
将大鼠骨肉瘤细胞系[UMR 106 - 01 (BSP)]细胞层相关的硫酸乙酰肝素（HS）蛋白聚糖与其他细胞中类似的细胞相关蛋白聚糖进行比较，并研究它们与质膜的相互作用。通过用[³H]葡糖胺或[³H]亮氨酸以及[³⁵S]硫酸盐孵育细胞培养物对HS蛋白聚糖进行代谢标记。用来自UMR 106 - 01 (BSP)细胞的主要种类经肝素酶消化产生的HS蛋白聚糖核心蛋白制剂，在聚丙烯酰胺凝胶电泳（PAGE）上与来自卵巢颗粒细胞和甲状旁腺细胞的相同制剂共同迁移（约70 kDa）。基于从辛基 - 琼脂糖的洗脱位置，这些不同细胞系中主要HS蛋白聚糖的疏水性也具有可比性。通过用脂溶性光活化试剂3 -（三氟甲基）- 3 -（间 - [¹²⁵I]碘苯基）二氮杂环丙烷（TID）标记质膜制剂来研究HS蛋白聚糖与细胞膜的连接，TID可选择性标记跨质膜的肽结构域。来自UMR 106 - 01 (BSP)细胞的纯化HS蛋白聚糖显示可被[¹²⁵I]TID标记，并且分子的核心蛋白部分被标记，证实其与质膜紧密相关。约36%的³⁵S标记的HS蛋白聚糖通过磷脂酶C（苏云金芽孢杆菌）从细胞表面释放，磷脂酶C可特异性切割磷脂酰肌醇连接的蛋白。在胰岛素存在下，对磷脂酶C敏感群体的代谢未改变；然而，对磷脂酶C不敏感群体释放到培养基中的量增加。这些数据表明，HS蛋白聚糖的一个亚群通过糖基磷脂酰肌醇结构与质膜共价结合，其余部分代表那些通过疏水肽结构域直接插入质膜的种类。这些观察结果与报道的卵巢颗粒细胞的观察结果相似[柳下田和麦奎兰（1989年）《生物化学杂志》264 17551 - 17558]，因此可能代表许多细胞类型的普遍现象。
