title: Functional characterization of glycosylation-deficient human P-glycoprotein using a vaccinia virus expression system.
abstract: 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.
authors.affiliation: Laboratory of Cell Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Gribar
authors.forename: M
authors.initials: M
authors.lastname: Ramachandra
authors.forename: C A
authors.initials: CA
authors.lastname: Hrycyna
authors.forename: S
authors.initials: S
authors.lastname: Dey
authors.forename: S V
authors.initials: SV
authors.lastname: Ambudkar
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D019920:Photoaffinity Labels / Q000378:metabolism; D011224:Prazosin / Q000031:analogs & derivatives / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D014162:Transfection; D014357:Trypsin; D014616:Vaccinia virus / Q000235:genetics
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D015853:Cysteine Proteinase Inhibitors; D008562:Membrane Glycoproteins; D009097:Multienzyme Complexes; D019920:Photoaffinity Labels; C067713:lactacystin; D000255:Adenosine Triphosphate; C041552:azidoprazosin; D014357:Trypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine; D011224:Prazosin