ABCMdb

PMID: 19909340

Nakagawa H, Wakabayashi-Nakao K, Tamura A, Toyoda Y, Koshiba S, Ishikawa T
Disruption of N-linked glycosylation enhances ubiquitin-mediated proteasomal degradation of the human ATP-binding cassette transporter ABCG2.
FEBS J. 2009 Dec;276(24):7237-52. Epub ., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCG2 p.Asn596Gln 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. Login to comment 8 ABCG2 p.Asn596Gln Immunofluorescence microscopy demonstrated that treatment with MG132 increased the level of ABCG2 N596Q protein both in intracellular compartments and in the plasma membrane. Login to comment 10 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln Structured digital abstract l MINT-7265522, MINT-7265536: ABCG2 (uniprotkb:Q9UNQ0) physically interacts (MI:0915) with Ubiquitin (uniprotkb:P62988) by anti bait coimmunoprecipitation (MI:0006) Abbreviations ABC, ATP-binding cassette; ABCG2 N596Q, ABCG2 variant in which Asn596 was substituted by Gln596; ABCP, placenta-specific ABC transporter; BCRP, breast cancer resistance protein; BMA, bafilomycin A1; DMEM, Dulbecco`s modified Eagle`s medium; Endo H, Endoglycosidase H; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; Flp-In-293/ABCG2 N596Q cells, Flp-In-293 cells expressing ABCG2 N596Q variant; Flp-In-293/ABCG2 WT cells, Flp-In-293 cells expressing ABCG2 WT; Flp-In-293/Mock cells, Flp-In-293 cells transfected with pcDNA/FRT Mock vector; FRT, Flp recombination target; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; MXR, mitoxantrone resistance-associated protein; NaCl / Pi, phosphate-buffered saline; PNGase F, N-glycosidase F; TM5, transmembrane domain 5; TM6, transmembrane domain 6; TTBS, Tris-buffered saline containing 0.05% (v / v) Tween 20. Login to comment 22 ABCG2 p.Gln141Lys ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn In fact, the protein expression levels of ABCG2 SNP variants (Q141K, F208S and S441N) were significantly lower than that of the wild-type (WT) ABCG2 [23]. Login to comment 95 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln Characterization of human ABCG2 N596Q expressed in Flp-In-293 cells To examine in more detail the potential role of N-linked glycosylation in the stability and degradation of ABCG2 protein, we established the N596Q variant of human ABCG2, in which Asn596, the amino acid for N-linked glycosylation, was substituted by Gln596 and therefore N-linked glycosylation was predicted not to occur at all. Login to comment 98 ABCG2 p.Asn596Gln Comparison between human ABCG2 WT and N596Q expressed in Flp-In-293 cells. Login to comment 99 ABCG2 p.Asn596Gln (A) Apparent molecular weights (left panel) and sensitivities to glycosidases (right panel) of human ABCG2 WT and human ABCG2 N596Q. Login to comment 101 ABCG2 p.Asn596Gln ABCG2 WT and ABCG2 N596Q proteins in the resulting samples were analyzed as described above. Login to comment 102 ABCG2 p.Asn596Gln The relative molecular mass value of human ABCG2 N596Q was the same as that of nonglycosylated human ABCG2 WT (72 000). Login to comment 103 ABCG2 p.Asn596Gln (B) Protein and mRNA levels of human ABCG2 WT and N596Q expressed in Flp-In-293 cells. Login to comment 104 ABCG2 p.Asn596Gln The mRNA levels were analyzed by RT-PCR and by quantitative PCR with total RNA extracted from Flp-In-293 cells expressing ABCG2 WT or ABCG2 N596Q. Login to comment 108 ABCG2 p.Asn596Gln (C) The effect of cycloheximide on the protein stability of human ABCG2 WT and ABCG2 N596Q expressed in Flp-In-293 cells. Login to comment 109 ABCG2 p.Asn596Gln Cells expressing ABCG2 WT (circles) or ABCG2 N596Q (closed triangles) were incubated with 10 lM cycloheximide for 0, 2, 4 and 8 h. Login to comment 112 ABCG2 p.Asn596Gln The protein levels of ABCG2 WT or ABCG2 N596Q were calculated from the corresponding signal intensities and then normalized to the values observed at t = 0 h. Login to comment 116 ABCG2 p.Asn596Gln Flp-In-293 cells expressing ABCG2 WT (circles) or ABCG2 N596Q (closed triangles) and Flp-In-293 / Mock (squares) cells were cultured with different concentrations of SN-38 for 72 h. Login to comment 119 ABCG2 p.Asn596Gln of human ABCG2 N596Q (72 000) was smaller than that of WT ABCG2 (81 000). Login to comment 120 ABCG2 p.Asn596Gln The relative molecular mass of the N596Q variant was changed by neither Endoglycosidase H (Endo H) nor PNGase F treatments (Fig. 3A right panel). Login to comment 121 ABCG2 p.Asn596Gln These results confirm that human ABCG2 N596Q completely loses the N-linked glycan and that there are no other N-linked glycosylation sites in this protein. Login to comment 122 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln Immunoblot analysis revealed that the protein expression level of human ABCG2 N596Q in Flp-In-293 cells was about one-third of the expression level of ABCG2 WT protein (Fig. 3B left panel), whereas the mRNA expression levels of both ABCG2 WT and ABCG2 N596Q were almost identical (Fig. 3B right panel). Login to comment 123 ABCG2 p.Asn596Gln To examine the protein stability of the N596Q variant, we treated Flp-In-293 cells with 10 lm cycloheximide to inhibit de novo protein synthesis. Login to comment 124 ABCG2 p.Asn596Gln Immunoblotting (Fig. 3C) revealed that the amount of ABCG2 N596Q protein decreased faster than that of the ABCG2 WT protein after treatment with cycloheximide. Login to comment 126 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln Fig. 3D demonstrates the SN-38 resistance profiles of Flp-In-293 / Mock, Flp-In-293 / ABCG2 WT and Flp-In-293/ Flp-In-293 cells expressing ABCG2 N596Q variant (ABCG2 N596Q cells). Login to comment 128 ABCG2 p.Asn596Gln Flp-In-293 / ABCG2 N596Q cells were 2.5-fold more sensitive to SN-38 than were Flp-In-293 /ABCG2 cells. Login to comment 129 ABCG2 p.Asn596Gln The half-maximal inhibitory concentration (IC50) values were 8 and 20 nm for Flp-In-293 / ABCG2 N596Q cells and Flp-In-293 / ABCG2 cells, respectively (Fig. 3D). Login to comment 130 ABCG2 p.Asn596Gln Effects of bafilomycin A1 and MG132 on the expression level of human ABCG2 N596Q protein Protein degradation occurs in two major sites, namely lysosomes and proteasomes. Login to comment 132 ABCG2 p.Asn596Gln To elucidate the contribution of those pathways to the degradation and turnover of ABCG2 WT and N596Q proteins, we incubated Flp-In-293 cells expressing either WT protein or the variant lacking N-linked glycan in the presence of those inhibitors (2 lm MG132 or 10 nm BMA). Login to comment 134 ABCG2 p.Asn596Gln Figure 4A demonstrates the effect of MG132 and BMA on the protein levels of ABCG2 WT and N596Q. Login to comment 136 ABCG2 p.Asn596Gln A marked difference was observed between human ABCG2 WT and ABCG2 N596Q with respect to the effects of MG132 and BMA. Login to comment 138 ABCG2 p.Asn596Gln In the case of ABCG2 N596Q lacking N-linked glycan, however, the protein level was increased about twofold, not only by BMA but also by treatment with MG132. Login to comment 141 ABCG2 p.Asn596Gln To examine the effects of MG132 on homodimer formation of ABCG2 WT and N596Q, SDS/ PAGE was performed under nonreducing conditions (i.e. without 2-mercaptoethanol). Login to comment 142 ABCG2 p.Asn596Gln As demonstrated in Fig. 4B, the ABCG2 N596Q variant forms homodimers via a cysteinyl disulfide bond under standard conA B Fig. 4. Login to comment 143 ABCG2 p.Asn596Gln Effect of MG132 on the protein levels of human ABCG2 WT and ABCG2 N596Q. Login to comment 144 ABCG2 p.Asn596Gln Cell lysate samples (20 lg of protein) from Flp-In-293 / ABCG2 WT cells and Flp-In-293 / ABCG2 N596Q cells cultured with 10 nM BMA (A) or 2 lM MG132 (MG) (A and B) for 24 h were subjected to SDS / PAGE after treatment with (A) or without (B) 2-mercaptoethanol. Login to comment 150 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln It is important to note that MG132 enhanced the levels of ABCG2 WT and ABCG2 N596Q monomers, where the level of ABCG2 N596Q protein was much higher than the nonglycosylated monomer form of ABCG2 (Fig. 4B). Login to comment 151 ABCG2 p.Asn596Gln Thus, MG132 enhanced the levels of ABCG2 WT and ABCG2 N596Q monomers, which suggests that the proteasomal degradation pathway prefers monomeric forms of ABCG2. Login to comment 152 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln Effect of MG132 on the cellular localization of ABCG2 WT and ABCG2 N596Q It was of great interest to establish how the inhibition of proteasomal protein degradation by MG132 affects the cellular localization of ABCG2 WT and N596Q proteins. Login to comment 153 ABCG2 p.Asn596Gln Figure 5A depicts the immunofluorescence images of Flp-In-293 cells expressing ABCG2 WT or N596Q that were incubated with or without 2 lm MG132 for 24 h. Login to comment 158 ABCG2 p.Asn596Gln Immunofluorescence of the N596Q variant was relatively weaker at the plasma membrane as well as within intracellular compartments (Fig. 5A, panels e and g). Login to comment 160 ABCG2 p.Asn596Gln Furthermore, immunofluorescence with the 5D3 antibody revealed that the plasma membrane localization of ABCG2 N596Q was clearly enhanced by treatment with MG132 (Fig. 5A, compare panels g and h). Login to comment 165 ABCG2 p.Asn596Gln In accordance with the immunoblotting data shown in Fig. 4A, the inhibition of proteasomal protein degradation by MG132 significantly increased the levels of the ABCG2 N596Q protein within each Flp-In-293 cell. Login to comment 166 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln Effect of MG132 on the ubiquitination states of human ABCG2 N596Q To investigate the effect of MG132 on the ubiquitination states of human ABCG2 N596Q, the Flp-In-293 cells expressing human ABCG2 N596Q were incubated in the presence or absence of 2 lm MG132 for 24 h. Login to comment 167 ABCG2 p.Asn596Gln As shown in Fig. 6, a significant increase in the ubiquitinated form (arrowheads) of human ABCG2 N596Q was detected by immunoblotting with the anti-ubiqu- * * A a b c d e f g h B Fig. 5. Login to comment 168 ABCG2 p.Asn596Gln Immunocytochemical staining of Flp-In-293 cells expressing ABCG2 WT and N596Q proteins. Login to comment 191 ABCG2 p.Asn596Gln It is important to note that in those studies, ABCG2 WT, the R482 acquired mutant form, or their N596Q variants were expressed in mammalian cells (CHO9, MDCKII or HeLa) by transient transfection methods using the pcDNA3 vector or the vTF 7-3 vaccinia virus [28,29]. Login to comment 193 ABCG2 p.Asn596Gln In the present study, to examine the role of N-linked glycosylation at Asn596 on the protein stability of ABCG2, we used the Flp-In method to integrate cDNA of ABCG2 WT or cDNA of the ABCG2 N596Q variant into genomic DNA. Login to comment 196 ABCG2 p.Asn596Gln Effect of MG132 on ubiquitination of ABCG2 WT and N596Q. Login to comment 197 ABCG2 p.Asn596Gln After incubation with or without 2 lM MG132 for 24 h, cell lysate samples were prepared from Flp-In-293 / ABCG2 WT cells and Flp-In-293 / N596Q cells. Login to comment 202 ABCG2 p.Asn596Gln Flp-In-293 cells used in the present study showed equal mRNA levels for both human ABCG2 WT and the ABCG2 N596Q variant (Fig. 3B). Login to comment 212 ABCG2 p.Asn596Gln Those observations are in accordance with the effect of disruption of N-linked glycosylation on the protein level of ABCG2 N596Q and the cellular resistance to SN-38 (Figs. Login to comment 218 ABCG2 p.Asn596Gln Likewise, nonglycosylated and monomeric forms were observed for ABCG2 N596Q, when the cells were treated with MG132. Login to comment 232 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln The present study conveys experimental data demonstrating that the protein level of ABCG2 N596Q expressed in Flp-In-293 cells was significantly lower than that of ABCG2 WT (Fig. 3B), and that the N596Q variant protein was less stable than the WT protein when de novo protein synthesis was inhibited with cycloheximide (Fig. 3C). Login to comment 233 ABCG2 p.Asn596Gln Interestingly, the proteasome inhibitor, MG132, increased the expression level of human ABCG2 N596Q protein in Flp-In-293 cells, whereas it had little effect on the expression level of ABCG2 WT protein (Fig. 4A). Login to comment 235 ABCG2 p.Asn596Gln Because the expression level of human ABCG2 N596Q protein was increased by treatments with BMA and MG132 (Fig. 4A), the variant protein appears to be degraded via both the lysosomal and ubiquitin-mediated proteasomal proteolytic pathways. Login to comment 236 ABCG2 p.Asn596Gln Based on the results shown in Fig. 4, we assume that about half of the de novo synthesized N596Q variant proteins are sorted to the plasma membrane through the Golgi apparatus and then degraded in lysosomes, whereas the other half undergo ERAD (i.e. ubiquitin-mediated proteasomal proteolysis). Login to comment 256 ABCG2 p.Asn596Gln For this purpose, we prepared Flp-In-293 / ABCG2 N596Q cells, in addition to the Flp-In-293 / ABCG2 WT and Flp-In-293 / Mock cells that had been established in our previous studies [33]. Login to comment 258 ABCG2 p.Asn596Gln By contrast, Flp-In-293 / ABCG2 WT, Flp-In-293 / ABCG2 N596Q and Flp-In-293 / Mock cells were maintained in DMEM supplemented as described above, except that 100 lgÆmL)1 of hygromycin B was used instead of ZeocinÔ. Login to comment 259 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln Generation of the N596Q variant form of human ABCG2 The ABCG2-pcDNA5 / FRT vector, constructed in our previous study [33], was used as the template for generation of the N596Q variant form of human ABCG2. Login to comment 262 ABCG2 p.Asn596Gln The resulting sequence was examined to confirm the generation of the ABCG2 N596Q-pcDNA5 / FRT vector. Login to comment 263 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln The cells expressing human ABCG2 N596Q (Flp-In-293 / ABCG2 N596Q cells) were prepared as previously reported [33] by using ABCG2 N596Q-pcDNA5 / FRT. Login to comment 302 ABCG2 p.Asn596Gln Detection of mRNA by RT-PCR Total RNA was extracted using NucleoSpinÒ RNA II (Macherey-Nagel GmbH & Co. KG, Duren, Germany) from Flp-In-293 / ABCG2 WT and Flp-In-293 / ABCG2 N596Q cells, according to the manufacturer`s instructions. Login to comment 307 ABCG2 p.Asn596Gln Quantitative real-time PCR The RNA levels of ABCG2 and GAPDH in Flp-In-293 / ABCG2 WT cells and Flp-In-293 / ABCG2 N596Q cells were determined in a 7500 Fast Real Time-PCR System (Applied Biosystems) using TaqManÒ Fast Universal Master Mix (Applied Biosystems) and TaqManÒ probes (ABCG2, Hs00184979_m1; GAPDH, Hs99999905_m1) (Applied Biosystems). Login to comment