Functional characterization of glycosylation-deficient human P-glycoprotein using a vaccinia virus expression system

J Membr Biol. 2000 Feb 1;173(3):203-14. doi: 10.1007/s002320001020.


P-glycoprotein (P-gp), the product of human MDR1 gene, which functions as an ATP-dependent drug efflux pump, is N-linked glycosylated at asparagine residues 91, 94, and 99 located within the first extracellular loop. We report here the biochemical characterization of glycosylation-deficient (Gly(-)) P-gp using a vaccinia virus based transient expression system. The staining of HeLa cells expressing Gly(-) P-gp (91, 94, and 99N-->Q), with P-gp specific monoclonal antibodies, MRK-16, UIC2 and 4E3 revealed a 40 to 50% lower cell-surface expression of mutant P-gp compared to the wild-type protein. The transport function of Gly(-) P-gp, assessed using a variety of fluorescent compounds indicated that the substrate specificity of the pump was not affected by the lack of glycosylation. Additional mutants, Gly(-) D (91, 94, 99N-->D) and Gly(-) Delta (91, 94, 99 N deleted) were generated to verify that the reduced cell surface expression, as well as total expression, were not a result of the glutamine substitutions. Gly(-) D and Gly(-) Delta Pgps were also expressed to the same level as the Gly(-) mutant protein. (35)S-Methionine/cysteine pulse-chase studies revealed a reduced incorporation of (35)S-methionine/cysteine in full length Gly(-) P-gp compared to wild-type protein, but the half-life ( approximately 3 hr) of mutant P-gp was essentially unaltered. Since treatment with proteasome inhibitors (MG-132, lactacystin) increased only the intracellular level of nascent, mutant P-gp, the decreased incorporation of (35)S-methionine/cysteine in Gly(-) P-gp appears to be due to degradation of improperly folded mutant protein by the proteasome and endoplasmic reticulum-associated proteases. These results demonstrate that the unglycosylated protein, although expressed at lower levels at the cell surface, is functional and suitable for structural studies.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism*
  • Acetylcysteine / analogs & derivatives
  • Acetylcysteine / pharmacology
  • Adenosine Triphosphate / metabolism
  • Azides / metabolism
  • Cysteine Endopeptidases / metabolism
  • Cysteine Proteinase Inhibitors / pharmacology
  • Flow Cytometry
  • Glycosylation
  • HeLa Cells
  • Humans
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Multienzyme Complexes / metabolism
  • Mutation
  • Photoaffinity Labels / metabolism
  • Prazosin / analogs & derivatives
  • Prazosin / metabolism
  • Proteasome Endopeptidase Complex
  • Transfection
  • Trypsin
  • Vaccinia virus / genetics


  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Azides
  • Cysteine Proteinase Inhibitors
  • Membrane Glycoproteins
  • Multienzyme Complexes
  • Photoaffinity Labels
  • lactacystin
  • Adenosine Triphosphate
  • azidoprazosin
  • Trypsin
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex
  • Acetylcysteine
  • Prazosin