The overproduction of P-glycoprotein in cancer cells is associated with multidrug resistance during chemotherapy by acting as an ATP-driven efflux pump to remove anticancer agents (Simon and Schindler, 1994). P- glycoprotein (P-gp represents the human MDR1 gene product) appears also in high concentration in capillaries that line the blood-brain barrier (Schinkel et al., 1994). It is, therefore, an important regulator of both foreign and man-made toxins in the human body. The P-glycoprotein polypeptide has been studied extensively for its ability to bind and export a wide variety of drugs. From these studies it has been shown that P-gp is heavily N-linked glycosylated. Little is known, however, of the role this extensive and variable glycosylation plays in the targeting, structuring, and function of the various forms of P-gp found in the different human cancer cell types. We have been successful in purifying P-gp from human KB-A1 HeLa cells using preparative electrophoresis; specifically, Bio Rad's new Prep Cell. We propose to purify P-glycoprotein from KB-A1 HeLa cells using our Prep Cell protocol to identify and sequence the oligosaccharides found on isolated human P- gp molecules, including intermediate forms of the protein. The P-gp oligosaccharides will be sequenced using the anew FACE (Fluorophore Assisted Carbohydrate Electrophoresis) Imaging System from Glyko Biology. These specific P-glycoprotein sugar sequences will be cataloged and used to study the role P-glycoprotein carbohydrates play in human multidrug resistance.
| Greer, D A; Ivey, S (2007) Distinct N-glycan glycosylation of P-glycoprotein isolated from the human uterine sarcoma cell line MES-SA/Dx5. Biochim Biophys Acta 1770:1275-82 |
