Kammer G M, Walter E I, Medof M E
Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH 44106.
J Immunol. 1988 Nov 1;141(9):2924-8.
Recent studies have identified cell-associated proteins that are membrane anchored by glycosyl-inositol-phospholipid structures but the biologic implications of this mode of membrane attachment are incompletely understood. Among proteins anchored in this way is the decay-accelerating factor (DAF), a complement (C) regulatory factor that functions on blood cell surfaces to prevent autologous C attack. As one approach to investigate the functional consequences of glycosyl-inositol-phospholipid-anchoring of DAF in T lymphocytes, the effects of crosslinking surface DAF molecules were compared to those of crosslinking conventionally by anchored cluster of differentiation (CD) proteins. Upon incubation with anti-DAF mAb and anti-murine IgG, DAF re-distributed to a pole of the cell with a t1/2 at 37 degrees C of 4.4 min as compared to t1/2 of 3.5 to 7 min for CD3, CD4, and CD8. Re-distribution of DAF occurred independently of CD2, CD3, CD4, or CD8. Anti-DAF immunoprecipitates of membrane extracts of cells chemically cross-linked with dithiobis(succinimidylpropionate) contained only monomeric DAF. Immunofluorescent staining demonstrated clustered actin, tubulin, and vimentin beneath the capped DAF protein. Pre-treatment of cells with colchicine or 8-azidoadenosine 3',5'-cyclic phosphate, but not lumicolchicine, resulted in reduction of the t1/2 for DAF to 1 to 2.6 min. Conversely, treatment of cells with cytochalasins B or D completely blocked DAF capping. The results indicate that, upon cross-linking, glycosyl-inositol-phospholipid-anchored DAF molecules undergo capping similar to conventionally anchored CD molecules and that DAF capping is associated with cytoskeletal reorganization.
最近的研究已鉴定出通过糖基-肌醇-磷脂结构锚定在细胞膜上的细胞相关蛋白，但这种膜附着方式的生物学意义尚未完全明确。以这种方式锚定的蛋白中有衰变加速因子（DAF），它是一种补体（C）调节因子，在血细胞表面发挥作用以防止自体C攻击。作为研究T淋巴细胞中DAF糖基-肌醇-磷脂锚定功能后果的一种方法，将交联表面DAF分子的效果与通过传统的分化簇（CD）蛋白锚定进行交联的效果进行了比较。用抗DAF单克隆抗体和抗鼠IgG孵育后，DAF重新分布到细胞的一极，在37℃下的半衰期为4.4分钟，而CD3、CD4和CD8的半衰期为3.5至7分钟。DAF的重新分布独立于CD2、CD3、CD4或CD8发生。用二硫代双（琥珀酰亚胺丙酸酯）化学交联的细胞的膜提取物的抗DAF免疫沉淀物仅含有单体DAF。免疫荧光染色显示在帽状DAF蛋白下方有聚集的肌动蛋白、微管蛋白和波形蛋白。用秋水仙碱或8-叠氮腺苷3'，5'-环磷酸酯预处理细胞，但不是光秋水仙碱，导致DAF的半衰期降至1至2.6分钟。相反，用细胞松弛素B或D处理细胞完全阻断了DAF帽化。结果表明，交联后，糖基-肌醇-磷脂锚定的DAF分子经历类似于传统锚定的CD分子的帽化，并且DAF帽化与细胞骨架重组有关。
