P-GLYCOPROTEIN OF BLOOD-BRAIN-BARRIER - CROSS-REACTIVITY OF MAB-C219 WITH A 190-KDA PROTEIN IN BOVINE AND RAT ISOLATED BRAIN CAPILLARIES

P-glycoprotein (P-gp), an active efflux pump of antitumor drugs, is st rongly expressed in endothelial cells of the blood brain barrier (BBB) . Two proteins (155 and 190 kDa) were detected by Western blot analysi s 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 CH(R)C5 tumoral cell line. The 155 kDa pro tein (p155) of capillaries and the P-gp of CH(R)C5 cells were well sol ubilized by deoxycholate and Elugent, whereas the 190 kDa protein (p19 0) was only solubilized by sodium dodecylsulfate (SDS). Both proteins have different patterns of extraction by Triton X-114, p155 partitioni ng as a membrane protein, while p190 was insoluble. Deglycosylation of capillary proteins resulted in a 27-28 kDa decrease in the apparent m olecular weight of p155, similar to that observed for the P-gp of CH(R )C5 cells, but a decrease of only 7-8 for p190. Only p155 was immunopr ecipitated 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 immun ohistochemistry screening for cancer tissues and lead to false positiv e in the diagnosis of MDR.