ABCMdb

ABCG2 p.Asn596Gln

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Publications

PMID: 15807535 [PubMed] Diop NK et al: "N-Linked glycosylation of the human ABC transporter ABCG2 on asparagine 596 is not essential for expression, transport activity, or trafficking to the plasma membrane."

No. Sentence Comment

5 Immunoblot and pulse-chase analyses revealed that the glycosylation-deficient ABCG2 (N596Q) variant and the glycosylated parent transporter are expressed equivalently at steady state and have similar half-lives. Login to comment
6 Cell surface analysis of ABCG2 expression showed comparable amounts of the N596Q variant present at the plasma membrane compared to the glycosylated ABCG2 protein. Login to comment
7 The ABCG2 (N596Q) variant is also functional, demonstrating rhodamine 123 transport in intact cells comparable to that in cells expressing glycosylated ABCG2. Login to comment
8 Furthermore, in crude membrane preparations, neither the basal nor the prazosin-stimulated (~2-fold) ATPase activities of ABCG2 (N596Q) were affected compared to glycosylated ABCG2. Login to comment
32 In this study, we identified Asn 596 as the single N-linked glycosylated amino acid in ABCG2 using chemical treatments with tunicamycin or PNGase F and site-directed mutagenesis, constructing and evaluating three N f Q variants (N418Q, N557Q, and N596Q). Login to comment
33 The glycosylation-deficient ABCG2 (N596Q) is expressed at similar cell surface and steady-state levels as the glycosylated protein and has a similar half-life. Login to comment
34 Furthermore, ABCG2 (N596Q) is functional for both basal and drug-stimulated ATPase activity as well as for drug transport in intact cells, comparable to glycosylated ABCG2. Login to comment
52 The following genetic variants were obtained: Asn 418 (N418Q), Asn 557 (N557Q), and Asn 596 (N596Q). Login to comment
59 A 70-80% confluent monolayer of HeLa cells was infected with the vTF 7-3 vaccinia virus (10 pfu/cell) and cotransfected with an appropriate amount of a pTM1 expression plasmid containing ABCG2 or the following ABCG2 variants: N418Q, N557Q, and N596Q. Login to comment
89 Twenty hours post-infection/transfection, HeLa cells plated in 35 mm dishes expressing ABCG2 or the glycosylation-deficient mutant ABCG2 (N596Q) were washed twice with 1× PBS and once with DMEM supplemented with FBS and Gln but without methionine and cysteine (DMEM [(-) Cys, (-) Met]) and then preincubated in 1 mL of DMEM [(-) Cys, (-) Met] for 5 min. Login to comment
105 HeLa cells expressing either ABCG2 or ABCG2 (N596Q) were harvested 17 h post-infection/transfection and resuspended in 1 mL of 1× PBS. Login to comment
136 (B) HeLa cells were co-transfected/infected with the pTM1 negative control plasmid, pTM1-ABCG2 or pTM1-ABCG2 (N596Q), in the absence of tunicamycin for 48 h. Crude membranes derived from these cells were subsequently treated with (+) or without (-) Endo H. Login to comment
153 Site-directed mutagenesis was performed on the human ABCG2 cDNA to generate three mutant variants of the half-transporter where the Asn residues were replaced by Gln residues: ABCG2 (N418Q), ABCG2 (N557Q), and ABCG2 (N596Q). Login to comment
157 However, ABCG2 (N596Q) migrated as a single species at ~60 kDa (Figure 4A, lane 7). Login to comment
161 Upon treatment with PNGase F, both ABCG2 variants N418Q (Figure 4A, lane 4) and N557Q (Figure 4A, lane 6) also migrate to the same lower molecular mass position (~60 kDa) as the PNGase F treated ABCG2 glycosylated protein and the untreated N596Q protein (Figure 4A, lanes 2, 4, 6, and 7). Login to comment
162 In addition, no further reduction in the molecular mass of ABCG2 (N596Q) was observed upon FIGURE 3: Cell surface localization and rhodamine 123 accumulation of ABCG2 in the presence and absence of tunicamycin. Login to comment
168 Furthermore, treatment with Endo H had no effect on the mobility of ABCG2 (N596Q), demonstrating that the protein does not possess even core glycosylation features (Figure 2B, lane 6). Login to comment
170 The Glycosylation-Deficient Mutant ABCG2 (N596Q) and Glycosylated ABCG2 Proteins Are Expressed EquiValently at Steady State and HaVe Similar Cellular Half-LiVes. Login to comment
171 To determine if ABCG2 and ABCG2 (N596Q) proteins were expressed at similar steady-state levels and have similar half-lives, pulse-chase experiments were performed in vaccinia virus transfected/infected HeLa cells. Login to comment
175 The nonglycosylated ABCG2 (N596Q) is only expressed as the 60 kDa species (Figure 5A, bottom panel). Login to comment
178 The Glycosylation-Deficient Mutant ABCG2 (N596Q) Is an ActiVe Drug-Stimulated ATPase. Login to comment
179 The basal and drug-stimulated ATPase activities of ABCG2 and the glycosylation-deficient ABCG2 (N596Q) variant were measured in crude membrane preparations prepared from transiently transfected/infected HeLa cells (Figure 6). Login to comment
181 The negative control membranes, containing the empty vector pTM1, show a basal activity of approximately 6.5 ( 1.6 nmol π min-1 mg-1 and FIGURE 4: Immunoblot detection of ABCG2 and the N418Q, N557Q, and N596Q ABCG2 variants. Login to comment
186 FIGURE 5: Synthesis and degradation of ABCG2 and glycosylation-deficient ABCG2 (N596Q) in transiently transfected HeLa cells. Login to comment
187 (A) ABCG2 and ABCG2 (N596Q) expressing HeLa cells were pulsed with a 35S-trans label in DMEM lacking cysteine and methionine. Login to comment
191 (B) Quantitation of 35S-trans label incorporation into ABCG2 and ABCG2 (N596Q). Login to comment
194 For ABCG2 (N596Q), only the signal from the ~60 kDa band was measured, as this variant is expressed as a single species. Login to comment
195 FIGURE 6: Drug-stimulated ATPase activity of ABCG2 and the glycosylation-deficient variant ABCG2 (N596Q). Login to comment
196 Vanadate-sensitive ATPase hydrolysis was measured as free phosphate release in crude membrane extracts (10 µg) from transiently transfected HeLa cells expressing either pTM1, ABCG2, or ABCG2 (N596Q) as described in Experimental Procedures. The assays were performed in both the absence (open bars) and presence (hatched bars) of 20 µM prazosin, resulting in basal and drug-stimulated activities, respectively. Login to comment
200 The glycosylation-deficient ABCG2 (N596Q) membranes have a basal activity of approximately 14.8 ( 2.1 nmol min-1 mg-1 and a prazosin-stimulated activity of 32.1 ( 4.0 nmol min-1 mg-1, also an approximate 2-fold stimulation (Figure 6). Login to comment
203 The Glycosylation-Deficient ABCG2 (N596Q) Protein Is Expressed at the Cell Surface and Is Functional for Rhodamine 123 Transport. Login to comment
204 To determine if the glycosylation-deficient ABCG2 (N596Q) has cell surface expression and transport properties that are different from the glycosylated protein, we performed cell surface analysis and a time course analysis of rhodamine 123 transport. Login to comment
207 Using the extracellular epitope-specific monoclonal antibody 5D3 and a FITC-conjugated anti-mouse secondary antibody, individual cells overexpressing glycosylated ABCG2 showed surface punctuate fluorescence similar to cells overexpressing the glycosylation-deficient ABCG2 (N596Q) protein; compare glycosylated ABCG2 expressing cells in the top panel of Figure 7B (right) to the cell expressing ABCG2 (N596Q) in the bottom left of the lower panel of Figure 7B (right). Login to comment
210 The ABCG2 (N596Q) mutant is capable of transporting rhodamine 123 to the same extent as the glycosylated protein after 40 min (Figures 8C,D). Login to comment
217 Our flow cytometric analyses showed cell surface expression for ABCG2-transfected cells treated with tunicamycin and for the glycosylation-deficient mutant ABCG2 (N596Q) that was nearly equivalent to the fully glycosylated ABCG2 protein. Login to comment
218 Confocal microscopy images of transiently transfected HeLa cells with ABCG2 or ABCG2 (N596Q) demonstrated the same results qualitatively (data not shown), with individual cells expressing the glycosylation-deficient ABCG2 (N596Q) showing a similar punctuate fluorescence pattern at the plasma membrane to cells expressing glycosylated ABCG2. Login to comment
220 According to the predicted model, N418 and N557 are located one to three residues from the FIGURE 7: Cell surface expression of ABCG2 in HeLa cells expressing ABCG2 or the glycosylation-deficient variant ABCG2 (N596Q). Login to comment
221 HeLa cells were cotransfected/infected with the empty vector pTM1 (shaded peak), ABCG2 ()), or ABCG2 (N596Q) (s) and incubated for 21 h at 32 °C. (A) Cell surface expression of ABCG2 was assessed by flow cytometry as described in Experimental Procedures using the ABCG2-specific monoclonal antibody 5D3. Login to comment
238 In contrast, we have shown in this study that the steady-state expression and half-life of the FIGURE 8: Time-dependent rhodamine 123 transport in HeLa cells expressing ABCG2 or the glycosylation-deficient variant ABCG2 (N596Q). Login to comment
239 HeLa cells were cotransfected/infected with the empty vector pTM1 (shaded peak), ABCG2 ()), or ABCG2 (N596Q) (s) and incubated for 21 h at 32 °C. Cellular rhodamine 123 accumulation was assessed as described in Experimental Procedures. Login to comment
243 glycosylation-deficient mutant ABCG2 (N596Q) protein are comparable to its glycosylation-competent counterpart. Login to comment
254 Furthermore, recent data from our laboratory show ABCG2 (N596Q) protein as a mixture of monomers and higher order oligomers, mostly dimers, on nonreducing SDS-PAGE gels (A. Bhatia and C. A. Hrycyna, unpublished data). Login to comment

PMID: 17027309 [PubMed] Li YF et al: "Towards understanding the mechanism of action of the multidrug resistance-linked half-ABC transporter ABCG2: a molecular modeling study."

No. Sentence Comment

182 In sf9 cell, it is expressed on cell surface, but with no ATPase activity [56] L554P TM5 Lowered drug resistance [42] N557D,E TM5 Functional [21] S566Aa ECL (between TM5 and 6) Lowered drug resistance for the cell line [42] N596Q Between TM5 and 6 N-glycosylation site [65] Y605Ca Loop between TM5 and 6 Lowered drug resistance for the cell line [42] D620N Loop between TM5 and 6 SNP polymorphism [22] H630E,L TM6 Functional [21] A632Va TM Lowered drug resistance for the cell line [42] a Mutants not well characterized. Login to comment

PMID: 18237272 [PubMed] Nakagawa H et al: "Ubiquitin-mediated proteasomal degradation of non-synonymous SNP variants of human ABC transporter ABCG2."

No. Sentence Comment

235 In fact, the ABCG2 N596Q protein was not N-glycosylated, but was expressed at lower levels (approximately half of the WT protein level) and localized in the plasma membrane of the cells (results not shown). Login to comment

PMID: 19111842 [PubMed] Wakabayashi-Nakao K et al: "Quality control of human ABCG2 protein in the endoplasmic reticulum: ubiquitination and proteasomal degradation."

No. Sentence Comment

903 It is important to note, however, that in those studies, ABCG2 WT [18], the R482 acquired mutant form [19], or their N596Q variants were expressed in CHO9, MDCKII [18], or HeLa cells [19] by transient transfection methods using the pcDNA3 vector or the vTF 73 vaccinia virus. Login to comment
905 To examine the role of N-linked glycosylation at Asn596 on the protein stability of ABCG2, we have used the Flp-In method to integrate cDNA of ABCG2 WT or the N596Q variant into genomic DNA (Fig. 3). Login to comment
907 The Flp-In-293 cells used in our recent study (Nakagawa et al. unpublished data) showed equal mRNA levels of human ABCG2 WT and the N596Q variant. Login to comment
929 The proteasome inhibitor MG132 increased the protein expression level of human ABCG2 N596Q expressed in Flp-In-293 cells, whereas it had little effect on the protein level of ABCG2 WT. Login to comment
931 Since the protein expression level of human ABCG2 N596Q was increased by treatments with bafilomycin A1 and MG132, the variant protein appears to be degraded via both the lysosomal and ubiquitin-mediated proteasomal proteolysis pathways. Login to comment
932 We assume that about half of the de novo synthesized N596Q variant proteins were sorted to the plasma membrane through the Golgi apparatus and then degraded in lysosomes, while the other half underwent ERAD, i.e., ubiquitin-mediated proteasomal proteolysis. Login to comment

PMID: 19827267 [PubMed] Ishikawa T et al: "Human ABC transporter ABCG2 in cancer chemotherapy and pharmacogenomics."

No. Sentence Comment

206 The proteasome inhibitor MG132 increased the protein expression level of human ABCG2 N596Q expressed in Flp-In-293 cells, whereas it had little effect on the protein level of ABCG2 WT (Nakagawa et al., 2009). Login to comment
214 Since the protein expression level of human ABCG2 N596Q was increased by treatments with bafilomycin A1 and MG132, the variant protein appears to be degraded via both the lysosomal and ubiquitin-mediated proteasomal proteolysis pathways (Nakagawa et al., 2009). Login to comment
215 It is assumed that about half of the de novo synthesized N596Q variant proteins were sorted to the plasma membrane through the Golgi apparatus and then degraded in lysosomes, while the other half underwent ER-associated degradation (ERAD), i.e., ubiquitin-mediated proteasomal proteolysis. Login to comment

PMID: 19909340 [PubMed] Nakagawa H et al: "Disruption of N-linked glycosylation enhances ubiquitin-mediated proteasomal degradation of the human ATP-binding cassette transporter ABCG2."

No. Sentence Comment

7 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 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 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
95 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 Comparison between human ABCG2 WT and N596Q expressed in Flp-In-293 cells. Login to comment
99 (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 WT and ABCG2 N596Q proteins in the resulting samples were analyzed as described above. Login to comment
102 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 (B) Protein and mRNA levels of human ABCG2 WT and N596Q expressed in Flp-In-293 cells. Login to comment
104 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 (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 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 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 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 of human ABCG2 N596Q (72 000) was smaller than that of WT ABCG2 (81 000). Login to comment
120 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 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 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 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 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 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 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 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 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 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 Figure 4A demonstrates the effect of MG132 and BMA on the protein levels of ABCG2 WT and N596Q. Login to comment
136 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 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 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 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 Effect of MG132 on the protein levels of human ABCG2 WT and ABCG2 N596Q. Login to comment
144 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 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 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 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 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 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 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 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 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 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 Immunocytochemical staining of Flp-In-293 cells expressing ABCG2 WT and N596Q proteins. Login to comment
191 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 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 Effect of MG132 on ubiquitination of ABCG2 WT and N596Q. Login to comment
197 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 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 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 Likewise, nonglycosylated and monomeric forms were observed for ABCG2 N596Q, when the cells were treated with MG132. Login to comment
232 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 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 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 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 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 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 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 The resulting sequence was examined to confirm the generation of the ABCG2 N596Q-pcDNA5 / FRT vector. Login to comment
263 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 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 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

PMID: 20812902 [PubMed] Ni Z et al: "Structure and function of the human breast cancer resistance protein (BCRP/ABCG2)."

No. Sentence Comment

307 Gln substitution of Asn596, but not the other two Asn residues (Asn418 and Asn557), produced a smaller molecular weight BCRP band on SDS-PAGE due to the lack of glycosylation [92-93]. Login to comment

PMID: 21184741 [PubMed] Sugiyama T et al: "Posttranslational negative regulation of glycosylated and non-glycosylated BCRP expression by Derlin-1."

No. Sentence Comment

5 On the other hand, protein expression of N596Q variant of BCRP, N-linked glycosylation-deficient mutant that preferentially undergoes ubiquitin-mediated ER-associated degradation (ERAD), was strongly suppressed by the overexpression of Derlin-1, whereas knockdown of Derlin-1 stabilized N596Q protein, suggesting a negative regulatory role of Derlin-1 for N596Q protein expression. Login to comment
28 For example, not wild-type (WT) but N596Q variant of human BCRP, in which N-linked glycosylation was predicted not to occur at all, was susceptible to ER-associated degradation (ERAD) [9]. Login to comment
31 In the present study, we first screened ER-localized E3 ubiquitin ligases and their co-factor that functions in the regulation of WT and N596Q variant of BCRP expression and identified Derlin-1, a member of a family of proteins that bears homology to yeast Der1p [12], as a negative regulator for both glycosylated and non-glycosylated BCRP expression. Login to comment
32 In addition, we demonstrated that the difference was observed between WT and N596Q variant of BCRP with respect to the mechanism underlying negative regulation of BCRP by Derlin-1. Login to comment
52 Screening of ER-localized factors that function in the regulation of WT and N596Q variant of BCRP expression Although the posttranslational modification and ERAD of BCRP are becoming of great interest [8], there is only limited information on the specific factors that are critical for the regulation of BCRP expression. Login to comment
53 In the present study, we focused on WT and N596Q variant of BCRP. Login to comment
57 Whereas N596Q BCRP, in which Asn596 is substituted by Gln596, preferentially undergoes ERAD because of the lack of N-linked glycosylation although its PM targeting and transporter activity are less affected if it is trafficked from the ER to PM [9]. Login to comment
58 We also checked the expression pattern of N596Q BCRP in HEK293 cells and confirmed the expression of non-glycosylated (Non-G) form of BCRP (Fig. 1A, lower pannel). Login to comment
62 Notably, none of WT and dominant-negative mutant form of E3 ligases did not robustly affect the expression level and pattern of both WT and N596Q BCRP (Fig. 1B-E), while overexpression of Derlin-1 strongly reduced 81 kDa band (possibly C-G form) and increased 72 kDa band (possibly S-G form) of WT BCRP (Fig. 2F). Login to comment
63 More interestingly, Derlin-1 overexpression strongly inhibited the expression level of N596Q BCRP (Fig. 2G). Login to comment
64 Thus, these data suggest that Derlin-1, but not other known E3 ligases tested, may be the critical molecule to control the expression of both WT and N596Q BCRP proteins. Login to comment
78 Derlin-1 facilitates ERAD of N596Q BCRP protein via reduction of its protein stability Since Derlin-1 overexpression suppressed expression of N596Q BCRP protein (Fig. 1G), we hypothesized that Derlin-1 could facilitate ERAD of N596Q BCRP protein. Login to comment
79 Treatment with MG-132, a proteasome inhibitor [21], caused increased expression of N596Q BCRP protein, which is consistent with the previous study that showed that N596Q BCRP is relatively susceptible to ERAD (Fig. 3A) [9]. Login to comment
80 Interestingly, knockdown of Derlin-1 significantly increased the expression of N596Q BCRP protein to a similar extent as MG-132 treatment (Fig. 3B and C), implying the possible involvement of Derlin-1 in the regulation of N596Q BCRP degradation. Login to comment
81 Accordingly, significant reduction of N596Q BCRP expression was observed in the presence of Derlin-1 after 6 h CHX chase period (Fig. 3D and E), suggesting that both endogenous and exogenous Derlin-1 could reduce stability of N596Q protein and may contribute to ERAD of N596Q BCRP. Login to comment
84 Based on our finding that Derlin-1 facilitates ERAD of non-glycosylated form of BCRP protein, N596Q BCRP, it is quite reasonable to think that Derlin-1 is also involved in tunicamycin-induced degradation of WT BCRP. Login to comment
86 Further, pretreatment with MG132 inhibited the effect of tunicamycin-dependent change A - -+ - +- -BCRP 72 81 (kDa) S-G Non-G C-G Non-G EndoH PNGaseF WT BCRP N596Q BCRP - -+ - +- -BCRP 72 81 (kDa) EndoH PNGaseF B C -actin WT C42S Rma1 pcDNA 3.1 -Rma1 -BCRP 72 81 (kDa) WT C42S Rma1 pcDNA 3.1 WT BCRP N596Q BCRP S-G C-G Non-G actin Exo Rma1 Endo Rma1 D E F G -BCRP 72 -actin -Derlin-1 HA-Derlin-1 N596Q BCRP +- actin Non-G Exo Derlin-1 Endo Derlin-1 (kDa) -actin WT R2M gp78 pEF6 myc/his -myc -BCRP 72 81 (kDa) WT R2M gp78 WT BCRP N596Q BCRP S-G C-G Non-G actin gp78 pEF6 myc/his -actin WTC329S HRD1 pcDNA6 myc/his -myc -BCRP 72 81 (kDa) WT C329S HRD1 WT BCRP N596Q BCRP S-G C-G Non-G actin HRD1 pcDNA6 myc/his -actin WTH260Q CHIP pcDNA 3.1 -myc -BCRP 72 81 (kDa) WT H260Q CHIP WT BCRP N596Q BCRP S-G C-G Non-G actin CHIP pcDNA 3.1 HA-Derlin-1 WT BCRP +- -BCRP 72 81 -actin (kDa) actin S-G C-G Non-G -Derlin-1 Exo Derlin-1 Endo Derlin-1 Fig. 1. Login to comment
87 Derlin-1 is the critical molecule to control the expression of both WT and N596Q BCRP proteins. Login to comment
88 (A) HEK293 cells were transfected with WT or N596Q variant BCRP. Login to comment
90 (B-E) WT or N596Q BCRP-transfected HEK293 cells were co-transfected with empty vectors or expression vectors that encode either WT or dominant-negative mutants of myc-tagged gp78 (B), Flag-tagged Rma1 (C), myc-tagged HRD1 (D) and myc-tagged CHIP (E). Login to comment
91 (F-G) Empty vector pcDNA3.1 or HA-tagged Derlin-1 was co-transfected with WT (F) or N596Q (G) BCRP in HEK293 cells. Login to comment
104 On the other hand, Derlin-1 is likely to be an ERAD mediator of N-linked glycosylation-deficient mutant N596Q BCRP and tunicamycin-induced non-glycosylated WT-BCRP proteins. Login to comment
127 A MG-132 N596Q BCRP +- -BCRP 72 -actin -Derlin-1 (kDa) actin Derlin-1 Non-G B si-Derlin-1 N596Q BCRP +- -BCRP 72 -actin -Derlin-1 (kDa) actin Derlin-1 Non-G C N596Q BCRP -actin 0 -Derlin-1 72 Non-G 3 6 pcDNA3.1 0 3 6 HA-Derlin-1 actin Exo Derlin-1 Endo Derlin-1 CHX chase (h) -BCRP (kDa) si-Derlin-1 N596Q BCRP +-D 0 25 50 75 100 125 150 175 0 3 6 (h) TotalBCRP%remaining pcDNA3.1 HA-Derlin-1 * CHX chase E RelativeQuantityof BCRPprotein 0 0.5 1 1.5 2 2.5 3 * Fig. 3. Login to comment
128 Derlin-1 facilitates ERAD of N596Q BCRP protein via reduction of its protein stability. Login to comment
129 (A) N596Q BCRP-transfected HEK293 cells were treated with 5 lM of MG132 for 24 h and lystaes were subjected to Westen blotting. Login to comment
130 (B and C) N596Q BCRP-transfected HEK293 cells were co-transfected with 50 nM of control (À) or specific siRNA for Derlin-1 (+), followed by Western blotting (B). Login to comment
133 (D, E) N596Q BCRP-transfected HEK293 cells were co-transfected with empty vector pcDNA3.1 or HA-tagged Derlin-1. Login to comment
135 Non-G N596Q BCRP intensity was measured to express total BCRP amounts (E). Login to comment
137 A -KDEL -Derlin-1 Derlin-1 72 81 S-G C-G Non-G -BCRP (kDa) 0 0.1 0.5 1 WT BCRP Tunicamycin -actin actin GRP94 GRP78 (µg/ml) -KDEL 72 81 S-G C-G -BCRP (kDa) -actin actin Non-G GRP94 GRP78 WT BCRP C - ++ - +- Tunicamycin MG-132 D -KDEL -Derlin-1 72 81 S-G C-G -BCRP (kDa) -actin actin Non-G Derlin-1 GRP94 GRP78 WT BCRP - ++ - +- Tunicamycin si-Derlin-1 Simply-glycosylated (S-G) BCRP ER Proteasome Degradation Non-glycosylated (Non-G) BCRP N596Q BCRP WT BCRP Golgi and plasma membrane Complex-glycosylated (C-G) BCRP -+ Derlin1 MG-132 - + Tunicamycin FE WT BCRP - ++ - +- Tunicamycin si-Derlin-1 * B RelativeQuantityofBCRP protein(C-G+Non-G) Tunicamycin (1 µg/ml) WT BCRP +- 0 0.2 0.4 0.6 0.8 1 1.2 * 0 0.2 0.4 0.6 0.8 1 1.2 1.4 RelativeQuantityofBCRP protein(C-G+Non-G) Fig. 4. Login to comment
148 (F) Schematic flow of Derlin1-dependent posttranslational regulation of glycosylated and non-glycosylated WT or N596Q BCRP. Login to comment
149 Derlin-1 physically interacts with WT BCRP and reduces its maturation, and Derlin-1 initiates the efficient ERAD of non-glycosylated WT or N596Q BCRP. Login to comment
151 MG-132 inhibits ERAD of Non-G WT-BCRP and N596Q BCRP. Login to comment
157 Notably, overexpression of WT form of Rma1 and HRD1 but not dominant negative form of Rma1 (C42S) and HRD1 (C329S) seemed to slightly decrease N596Q BCRP expression (Fig. 1C and D), implicating that Derlin-1 may work with Rma1 and/or HRD1 during non-glycosylated BCRP degradation. Login to comment
163 Further, we could also assess the impact of Der- lin-1 on the expression of other BCRP variants such as R383A, G553L and G553E variants, which are also shown to be impaired N-linked glycosylation like N596Q BCRP [27,28]. Login to comment
165 Overall, our data demonstrate that Derlin-1 interacts physically with WT BCRP and reduces its maturation, and Derlin-1 initiates the efficient ERAD of non-glycosylated WT and N596Q BCRP, indicating that Derlin-1 is the negative regulator for the expression of both glycosylated and non-glycosylated WT BCRP proteins. Login to comment

PMID: 21567408 [PubMed] Nakagawa H et al: "Ubiquitin-mediated proteasomal degradation of ABC transporters: a new aspect of genetic polymorphisms and clinical impacts."

No. Sentence Comment

99 However, it is important to note that in those studies, ABCG2 WT, the R482 acquired mutant form, or their N596Q variants were expressed in CHO9, MDCKII, or HeLa cells by Table 1. Login to comment
105 To quantitatively evaluate the role of N-linked glycosylation at Asn596 on the protein stability of ABCG2, we integrated cDNA of ABCG2 WT or the N596Q variant into genomic DNA by the Flp-InTM method. Login to comment
108 By fluorescence in situ hybridization (FISH) analysis, we have demonstrated that the FRT site resides at the telomeric region of one of chromosomes 12 in Flp-In-293 cells.84 Owing to the single copy cDNA integration into the FRT site, the Flp-In-293 cells showed equal mRNA levels of human ABCG2 WT and the N596Q variant.76 Disruption of N-linked glycosylation results in a reduced protein expression level of human ABCG2,76 whereas its sorting to the plasma membrane was not affected. Login to comment
109 Likewise, tunicamycin, an N-linked glycosylation inhibitor, strongly reduced the protein level of human ABCG2 WT expressed in Flp-In-293 cells.76 Therefore it is concluded that the N-linked glycan is necessary to achieve the maximal stabilization of de novo synthesized ABCG2 in the ER.76,77 The proteasome inhibitor MG132 increased the protein expression level of human ABCG2 N596Q expressed in Flp-In-293 cells, whereas it had little effect on the protein level of ABCG2 WT.76 These observations indicate that disruption of N-linked glycosylation leads to an increase of misfolded ABCG2 proteins and enhances their susceptibility to the ubiquitin-proteasome proteolytic pathway.76 Check Points for Quality Control of ABCG2 Protein in ER The N-linked glycosylated WT protein of ABCG2 is degraded in lysosomes (Fig. 5b).77,78 On the contrary, misfolded mutant proteins undergo ubiquitin-mediated protein degradation in proteasomes.77,78 As indicated in Figure 5a, we anticipate that, in the ER, there may be at least two check points to monitor the quality of the human ABCG2 protein.77,78 Namely, one check point is monitoring the intramolecular disulfide bond between Cys592 and Cys608, and the other is checking the N-linked glycan at Asn596 (Fig. 5a). Login to comment

PMID: 24130050 [PubMed] Hou H et al: "Tunicamycin potentiates cisplatin anticancer efficacy through the DPAGT1/Akt/ABCG2 pathway in mouse Xenograft models of human hepatocellular carcinoma."

No. Sentence Comment

63 The Asn596-encoding codon AAT was converted to CAA to generate the N596Q variant of ABCG2 using the megaprimer PCR method as described previously (27). Login to comment
64 The N596Q mutant primer is listed in Supplementary Table S2. Login to comment
88 To examine the potential role of NLG in stabilizing and degrading the ABCG2 protein, we established the N596Q variant of human ABCG2, in which Asn596, the amino acid for NLG, was substituted by Gln596. Login to comment
89 Both wild-type (WT) ABCG2 and the N596Q variant were stably transfected into Huh7 cells. Login to comment
90 A Western blot analysis revealed that the protein expression level of the human ABCG2 N596Q variant in Huh7 cells was much lower than that of WT ABCG2 (Fig. 1C), whereas the mRNA expression levels of the WT and N596Q variant ABCG2 were nearly identical (Fig. 1D). Login to comment
116 The shift in the ABCG2 molecular weight was detected by Western blotting. C, wild-type ABCG2 and the ABCG2 N596Q variant were stably transfected into Huh7 cells. Login to comment
117 ABCG2 expression was analyzed using Western blotting. D, ABCG2 mRNA levels were analyzed by real-time PCR with total RNA extracted from Huh7 cells expressing wild-type ABCG2 or the N596Q variant. Login to comment

PMID: 24777822 [PubMed] Jani M et al: "Structure and function of BCRP, a broad specificity transporter of xenobiotics and endobiotics."

No. Sentence Comment

81 Mutation of Asn596 to Gln resulted in a molecular weight shift of the BCRP band on a SDS-PAGE due to the absence of glycosylation (Diop and Hrycyna 2005). Login to comment

PMID: 26294421 [PubMed] Haider AJ et al: "Identification of residues in ABCG2 affecting protein trafficking and drug transport, using co-evolutionary analysis of ABCG sequences."

No. Sentence Comment

110 A further three control mutations were analysed, namely a substitution of lysine to alanine in the Walker-A motif (K86A), a substitution of glutamate to glutamine in the Walker-B motif (E211Q) and a substitution of asparagine to glutamine at the site of glycosylation (N596Q). Login to comment
129 His12-N596Q is a glycosylation defective form of ABCG2. Login to comment
133 His12-N596Q shows no change in molecular mass upon PNGaseF treatment. Login to comment