Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells

Abstract

An antipeptide antibody (P7) to P-glycoprotein has been produced by immunizing rabbits with a synthetic peptide. Antibody P7 is directed against the amino-terminal region of P170 (residues 28-35). The antibody immunoprecipitates a 170-kDa P-glycoprotein from extracts of drug-resistant KB-V1 cells that is not present in the drug-sensitive cell line KB-3-1. Antibody P7 was used to quantitate the amount of P-glycoprotein present in drug-resistant KB lines at various levels of resistance and to demonstrate the presence of P-glycoprotein in NIH 3T3 cells transfected with a cloned MDR1 cDNA or human genomic DNA encoding MDR1. Pulse-chase labeling experiments demonstrated that P-glycoprotein is synthesized as a 140-kDa precursor which is slowly converted over 2-4 h to a 170-kDa glycoprotein. Tunicamycin treatment blocks the conversion of the precursor to the mature form, and removal of N-linked oligosaccharides with Endo F reduces the relative molecular weight of P-glycoprotein to 140K. The mobility of mature P-glycoprotein is unaffected by treatment with neuraminidase and Endo H. These data indicate that P-glycoprotein is N-glycosylated and contains little or no neuraminic acid. P-Glycoprotein is also phosphorylated, and the extent of phosphate incorporated is proportional to the amount of protein present in drug-resistant cells.

Inhibition of the Multidrug Resistance P-Glycoprotein: Time for a Change of Strategy?

“…The seminal manuscript that gave P-gp its name relied on detecting the protein in drug-resistant CHO cell membranes by virtue of its glycosylation status (Juliano and Ling, 1976). Further studies on its glycosylation status indicated that P-gp was synthesized as a 140-kDa precursor that was glycosylated over a 2-to 4-hour period to its fully mature form that migrated as a broad band at 180 kDa in SDS-PAGE (Richert et al, 1988;Yoshimura et al, 1989). Two investigations demonstrated that in the drug-resistant human KB cell line, P-gp was markedly stable, with a half-life greater than 24 hours (Richert et al, 1988;Yoshimura et al, 1989).…”

Characterization of Phosphorylation-defective Mutants of Human P-glycoprotein Expressed in Mammalian Cells

“…1-3). P-glycoprotein was described as a phosphoglycoprotein (9,10), and several studies have corroborated that both native and recombinant P-glycoproteins are phosphorylated in vivo (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Numerous studies have been conducted to address the importance of phosphorylation for the multidrug transporter activity of P-glycoprotein.…”

The ABC of glycosylation

“…In addition to these concepts, we think that post-translational modifications of ABC transporters, specifically glycosylation, is an important field of reasearch 6 through which new insights into the function of ABC transporters in tumorigenesis should be forthcoming. That glycosylation defects on ABC transporters, in particular ABCC1 (also known as MRP1), have consequences has been provided by a study showing the mislocalization of multidrug transporters in cisplatin-resistant cancer cell lines 7 .…”