title: Disruption of N-linked glycosylation enhances ubiquitin-mediated proteasomal degradation of the human ATP-binding cassette transporter ABCG2.
abstract: The human ATP-binding cassette (ABC) transporter, ABCG2 (BCRP/MXR/ABCP), is a plasma membrane protein containing intramolecular and intermolecular disulfide bonds and an N-linked glycan at Asn596. We have recently reported that the intramolecular disulfide bond is a critical checkpoint for determining the degradation fates of ABCG2. In the present study, we aimed to analyze quantitatively the impact of the N-linked glycan on the protein stability of ABCG2. For this purpose, we incorporated one single copy of ABCG2 cDNA into a designated site of genomic DNA in Flp-In-293 cells to stably express ABCG2 or its variant proteins. When ABCG2 wild type-expressing cells were incubated with various N-linked glycosylation inhibitors, tunicamycin profoundly suppressed the protein expression level of ABCG2 and, accordingly, reduced the ABCG2-mediated cellular resistance to the cancer chemotherapeutic SN-38. When Asn596 was converted to Gln596, the resulting variant protein was not glycosylated, and its protein level was about one-third of the wild type level in Flp-In-293 cells. Treatment with MG132, a proteasome inhibitor, increased the level of the variant protein. Immunoblotting with anti-ubiquitin IgG1k after immunoprecipitation of ABCG2 revealed that the N596Q protein was ubiquitinated at levels that were significantly enhanced by treatment with MG132. Immunofluorescence microscopy demonstrated that treatment with MG132 increased the level of ABCG2 N596Q protein both in intracellular compartments and in the plasma membrane. In conclusion, we propose that the N-linked glycan at Asn596 is important for stabilizing de novo-synthesized ABCG2 and that disruption of this linkage results in protein destabilization and enhanced ubiquitin-mediated proteasomal degradation.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Kanako
authors.initials: K
authors.lastname: Wakabayashi-Nakao
authors.forename: Ai
authors.initials: A
authors.lastname: Tamura
authors.forename: Yu
authors.initials: Y
authors.lastname: Toyoda
authors.forename: Shoko
authors.initials: S
authors.lastname: Koshiba
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ishikawa
mesh_terms: D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001216:Asparagine / Q000378:metabolism; D002460:Cell Line; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007212:Indolizines / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D016415:Sequence Alignment; D014415:Tunicamycin / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism*; D054836:Indolizidines
chemical_list: D017485:1-Deoxynojirimycin; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D001216:Asparagine; D005944:Glucosamine; D007212:Indolizines; D007976:Leupeptins; D018942:Macrolides; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009363:Neoplasm Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D046988:Proteasome Endopeptidase Complex; D014415:Tunicamycin; D025801:Ubiquitin; C116614:ABCG2 protein, human; C501633:deoxynojirimycine; C040929:bafilomycin A1; C037806:castanospermine; D054836:Indolizidines; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde