Purification and characterization of N-glycosylation mutant mouse and human P-glycoproteins expressed in Pichia pastoris cells

Arch Biochem Biophys. 2001 Apr 1;388(1):171-7. doi: 10.1006/abbi.2001.2299.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B / genetics
  • ATP Binding Cassette Transporter, Subfamily B / metabolism
  • ATP-Binding Cassette Transporters / genetics
  • ATP-Binding Cassette Transporters / metabolism
  • Adenosine Triphosphatases / metabolism
  • Alanine / chemistry
  • Animals
  • Asparagine / chemistry
  • Binding Sites
  • Catalysis
  • Chromatography, Agarose
  • DNA, Complementary / metabolism
  • Dithiothreitol / pharmacology
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • Glutamine / chemistry
  • Glycosylation
  • Humans
  • Inhibitory Concentration 50
  • Kinetics
  • Mass Spectrometry
  • Mice
  • Mutagenesis, Site-Directed
  • Mutation
  • Photoaffinity Labels / pharmacology
  • Pichia / chemistry*
  • Pichia / genetics*
  • Plasmids / metabolism
  • Protein Binding
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Time Factors
  • Vasodilator Agents / pharmacology
  • Verapamil / pharmacology

Substances

  • ATP Binding Cassette Transporter, Subfamily B
  • ATP-Binding Cassette Transporters
  • DNA, Complementary
  • Photoaffinity Labels
  • Vasodilator Agents
  • Glutamine
  • Asparagine
  • multidrug resistance protein 3
  • Verapamil
  • Adenosine Triphosphatases
  • Alanine
  • Dithiothreitol