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Functional Characterization of Glycosylation-Deficient Human P-Glycoprotein Using A Vaccinia Virus Expression System

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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 GlyΔ (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 GlyΔ Pgps were also expressed to the same level as the Gly mutant protein. 35S-Methionine/cysteine pulse-chase studies revealed a reduced incorporation of 35S-methionine/cysteine in full length Gly P-gp compared to wild-type protein, but the half-life (∼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 35S-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.

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Received: 28 July 1999/Revised: 20 October 1999

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Gribar, J., Ramachandra, M., Hrycyna, C. et al. Functional Characterization of Glycosylation-Deficient Human P-Glycoprotein Using A Vaccinia Virus Expression System. J. Membrane Biol. 173, 203–214 (2000). https://doi.org/10.1007/s002320001020

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  • DOI: https://doi.org/10.1007/s002320001020

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