tailieunhanh - Báo cáo khoa học: Disruption of N-linked glycosylation enhances ubiquitin-mediated proteasomal degradation of the human ATP-binding cassette transporter ABCG2

The human ATP-binding cassette (ABC) transporter, ABCG2 (BCRP⁄ MXR⁄ABCP), is a plasma membrane protein containing intramolecular and intermolecular disulfide bonds and anN-linked glycan at Asn596. We have recently reported that the intramolecular disulfide bond is a critical checkpoint for determining the degradation fates of ABCG2. | ỊFEBS Journal Disruption of N-linked glycosylation enhances ubiquitin-mediated proteasomal degradation of the human ATP-binding cassette transporter ABCG2 Hiroshi Nakagawa Kanako Wakabayashi-Nakao Ai Tamura Yu Toyoda Shoko Koshiba and Toshihisa Ishikawa Department of Biomolecular Engineering Graduate Schoolof Bioscience and Biotechnology Tokyo Institute of Technology Midori-ku Yokohama Japan Keywords ABC transporter ABCG2 N-linked glycosylation proteasome ubiquitin Correspondence Toshihisa Ishikawa Omics Science Center RIKEN Yokohama Institute 1-7-22 Suehiro-cho Tsurumi-ku Yokohama 230-0045 Japan Fax 81 45 503 9216 Tel 81 45 503 9222 or 9179 E-mail toshi-i@ Received 2 May 2009 revised 7 September 2009 accepted 5 October 2009 doi 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 .

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