Gane P, Le Van Kim C, Bony V, El Nemer W, Mouro I, Nicolas V, Colin Y, Cartron J P
INSERM U76, Institut National de la Transfusion Sanguine, Paris, France.
Br J Haematol. 2001 Jun;113(3):680-8. doi: 10.1046/j.1365-2141.2001.02757.x.
The linkage between blood group-related cell surface proteins and the detergent-insoluble material (DIM) was estimated by flow cytometry using a panel of specific monoclonal antibodies (mAbs) as a comparison of the antibody-binding capacity of intact and Triton-X100-treated cells. Studies were performed with K562 cells expressing endogenous or recombinant proteins and with human erythroid progenitors during their proliferation and differentiation in vitro. Glycophorin C (GPC) was found to be Triton-insoluble in both cellular models. When expressed (erythroid progenitors), Band 3 remained Triton-insoluble. Glycophorin A (GPA), however, behaved as Triton-soluble or insoluble according to the absence (K562) or the presence (erythroid progenitors) of Band 3 respectively. Comparison of the cellular models regarding the proteins that compose the Rh complex also indicated that Rh(D), RhAG and CD47 were resistant to Triton extraction in cells lacking Band 3. Similarly, RhAG and CD47 remained predominantly Triton-insoluble in K562 cells and early progenitors before Rh and Band 3 expression. Further analysis showed that the Kell protein was DIM-associated. In contrast, CD99 and DARC (Fy) proteins were not, or were very poorly, DIM-associated. Additionally, the adhesion molecules CD44 and Lu were completely or partially resistant to detergent extraction respectively. Deletion of the Lu cytoplasmic tail or its replacement by the cytoplasmic domain of GPC resulted in significant increase or decrease of the Triton solubility of the transfected proteins respectively. These data suggest that Triton insolubility of Lu results in part from direct attachment of its cytoplasmic tail with the cytoskeleton. We assume that this method should provide a useful tool to map interaction sites localized in the cytoplasmic domain of recombinant transmembrane proteins.
通过流式细胞术，使用一组特异性单克隆抗体（mAb）来估计血型相关细胞表面蛋白与去污剂不溶性物质（DIM）之间的联系，以此比较完整细胞和经Triton-X100处理的细胞的抗体结合能力。研究在表达内源性或重组蛋白的K562细胞以及体外增殖和分化过程中的人类红系祖细胞上进行。发现血型糖蛋白C（GPC）在两种细胞模型中均不溶于Triton。当表达时（红系祖细胞），带3蛋白仍不溶于Triton。然而，血型糖蛋白A（GPA）根据是否存在带3蛋白分别表现为可溶于或不溶于Triton（分别在K562细胞中不存在带3蛋白，在红系祖细胞中存在带3蛋白）。关于组成Rh复合物的蛋白质的细胞模型比较还表明，在缺乏带3蛋白的细胞中，Rh（D）、RhAG和CD47对Triton提取具有抗性。同样，在Rh和带3蛋白表达之前，RhAG和CD47在K562细胞和早期祖细胞中仍主要不溶于Triton。进一步分析表明，Kell蛋白与DIM相关。相比之下, CD99和DARC（Fy）蛋白与DIM不相关或相关性很差。此外，粘附分子CD44和Lu分别完全或部分抵抗去污剂提取。删除Lu的细胞质尾巴或将其替换为GPC的细胞质结构域分别导致转染蛋白的Triton溶解度显著增加或降低。这些数据表明，Lu不溶于Triton部分是由于其细胞质尾巴与细胞骨架的直接附着。我们认为这种方法应该为定位重组跨膜蛋白细胞质结构域中的相互作用位点提供一个有用的工具。
