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

P-glycoprotein (P-gp), the product of human MDR1 gene, which functions as a n ATP-dependent drug efflux pump, is N-linked glycosylated at asparagine re sidues 91, 94, and 99 located within the first extracellular loop. We repor t here the biochemical characterization of glycosylation-deficient (Gly(-)) P-gp using a vaccinia virus based transient expression system. The stainin g of HeLa cells expressing Gly(-) P-gp (91, 94, and 99N-->Q), with P-gp spe cific monoclonal antibodies, MRK-16, UIC2 and 4E3 revealed a 40 to 50% lowe r 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 fluores cent compounds indicated that the substrate specificity of the pump was not affected by the lack of glycosylation. Additional mutants, Gly(-) D (91, 9 4, 99N-->D) and Gly(-) Delta (91, 94, 99 N deleted) were generated to verif y that the reduced cell surface expression, as well as total expression, we re not a result of the glutamine substitutions. Gly(-) D and Gly(-) Delta P gps were also expressed to the same level as the Gly(-) mutant protein. S-3 5-Methionine/cysteine pulse-chase studies revealed a reduced incorporation of S-35-methionine/cysteine in full length Gly- P-gp compared to wild-type protein, but the half-life (similar to 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 decre ased incorporation of S-35-methionine/cysteine in Gly(-) P-gp appears to be due to degradation of improperly folded mutant protein by the proteasome a nd endoplasmic reticulum-associated proteases, These results demonstrate th at the unglycosylated protein, although expressed at lower levels at the ce ll surface, is functional and suitable for structural studies.