Purification and characterization of N-glycosylation mutant mouse and human P-glycoproteins expressed in Pichia pastoris cells.
P-glycoprotein confers multidrug resistance in mammalian cells and basic structure-function studies of it are germane to anti-cancer and anti-AIDS therapy. Pure, detergent-soluble mouse MDR3 and human MDR1 P-glycoproteins have recently been obtained in sufficient quantity for high-resolution structure analysis after expression in Pichia pastoris (N. Lerner-Marmarosh et al. (1999) J. Biol. Chem. 274, 34711-34718). The degree of glycosylation of these preparations was unknown, and was of relevance for crystallization studies. Therefore mutant proteins in which the N-glycosylation sites were eliminated (Asn --> Gln in mouse MDR3 Pgp, Asn --> Gln or Ala in human MDR1 Pgp) were expressed in P. pastoris and purified to homogeneity. Yields of mutant Pgp were the same as for parent wild-type proteins. Nucleotide-binding and catalytic (ATPase) characteristics were completely normal in the mutant proteins. Mass spectrometry indicated that mutant and wild-type proteins did not differ significantly in mass, demonstrating that the wild-type proteins contain no N-glycosylation.
Urbatsch
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Department of Biochemistry and Biophysics, University of Rochester Medical Center, New York 14642, USA.
Wilke-Mounts
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Gimi
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Senior
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D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D002847:Chromatography, Agarose; D018076:DNA, Complementary / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D013058:Mass Spectrometry; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019920:Photoaffinity Labels / Q000494:pharmacology; D010843:Pichia / Q000737:chemistry* / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014665:Vasodilator Agents / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D018435:ATP Binding Cassette Transporter, Subfamily B; D018528:ATP-Binding Cassette Transporters; D018076:DNA, Complementary; D019920:Photoaffinity Labels; D014665:Vasodilator Agents; D005973:Glutamine; D001216:Asparagine; C095122:multidrug resistance protein 3; D014700:Verapamil; D000251:Adenosine Triphosphatases; D000409:Alanine; D004229:Dithiothreitol