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

Biochemistry. 1988 Oct 4;27(20):7607-13. doi: 10.1021/bi00420a006.

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.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Animals
  • Antibodies
  • Drug Resistance
  • Glycosylation
  • Humans
  • KB Cells / drug effects
  • KB Cells / metabolism*
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / immunology
  • Membrane Glycoproteins / metabolism*
  • Phosphorylation
  • Transfection

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Antibodies
  • Membrane Glycoproteins