Beaulieu E, Demeule M, Pouliot J F, Averill-Bates D A, Murphy G F, Béliveau R
Laboratoire de Membranologie, Université du Québec à Montréal, Canada.
Biochim Biophys Acta. 1995 Jan 26;1233(1):27-32. doi: 10.1016/0005-2736(94)00239-l.
P-glycoprotein (P-gp), an active efflux pump of antitumor drugs, is strongly expressed in endothelial cells of the blood brain barrier (BBB). Two proteins (155 and 190 kDa) were detected by Western blot analysis of beef and rat capillaries with the monoclonal antibody (MAb) C219. In order to characterize the nature of these proteins, their profile of solubilization by different detergents was established and compared with that of P-gp from the CHRC5 tumoral cell line. The 155 kDa protein (p155) of capillaries and the P-gp of CHRC5 cells were well solubilized by deoxycholate and Elugent, whereas the 190 kDa kDa protein (p190) was only solubilized by sodium dodecylsulfate (SDS). Both proteins have different patterns of extraction by Triton X-114, p155 partitioning as a membrane protein, while p190 was insoluble. Deglycosylation of capillary proteins resulted in a 27-28 kDa decrease in the apparent molecular weight of p155, similar to that observed for the P-gp of CHRC5 cells, but a decrease of only 7-8 for p190. Only p155 was immunoprecipitated by MAb C219. These results suggest that only p155 is the P-gp in BBB and that MAb C219 cross-reacts with a 190 kDa MDR-unrelated glycosylated protein. Consequently, the use of this antibody, which is frequently used to detect P-gp in tumors, could be a pitfall of immunohistochemistry screening for cancer tissues and lead to false positive in the diagnosis of MDR.
P-糖蛋白（P-gp）是一种抗肿瘤药物的活性外排泵，在血脑屏障（BBB）的内皮细胞中强烈表达。用单克隆抗体（MAb）C219对牛肉和大鼠毛细血管进行蛋白质印迹分析，检测到两种蛋白质（155 kDa和190 kDa）。为了表征这些蛋白质的性质，确定了它们在不同去污剂作用下的溶解情况，并与CHRC5肿瘤细胞系中的P-gp进行了比较。毛细血管的155 kDa蛋白（p155）和CHRC5细胞的P-gp能被脱氧胆酸盐和Elugent很好地溶解，而190 kDa蛋白（p190）仅能被十二烷基硫酸钠（SDS）溶解。两种蛋白质在Triton X-114作用下的提取模式不同，p155作为膜蛋白分配，而p190不溶。毛细血管蛋白的去糖基化导致p155的表观分子量降低27 - 28 kDa，与CHRC5细胞的P-gp情况相似，但p190仅降低7 - 8 kDa。只有p155能被MAb C219免疫沉淀。这些结果表明，只有p155是血脑屏障中的P-gp，且MAb C219与一种190 kDa的多药耐药无关糖基化蛋白发生交叉反应。因此，这种常用于检测肿瘤中P-gp的抗体，可能是癌症组织免疫组化筛查中的一个陷阱，并导致多药耐药诊断出现假阳性。
