ABCMdb

PMID: 15807535

Diop NK, Hrycyna CA
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.
Biochemistry. 2005 Apr 12;44(14):5420-9., 2005-04-12 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln 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 19 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Arginine 482 (R482) has been reported in the literature as the wild-type form of ABCG2, while glycine 482 (R482G) and threonine 482 (R482T) arose as a result of drug selection (6-8). Login to comment 32 ABCG2 p.Asn596Gln ABCG2 p.Asn418Gln ABCG2 p.Asn557Gln 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 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln 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 51 ABCG2 p.Arg482Gly Single point mutations were introduced by sequence overlap extension (SOE) PCR using pTM1-ABCG2 (R482G) DNA as the expression vector template. Login to comment 52 ABCG2 p.Asn596Gln ABCG2 p.Asn418Gln ABCG2 p.Asn557Gln The following genetic variants were obtained: Asn 418 (N418Q), Asn 557 (N557Q), and Asn 596 (N596Q). Login to comment 59 ABCG2 p.Asn596Gln ABCG2 p.Asn418Gln ABCG2 p.Asn557Gln 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 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln 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 113 ABCG2 p.Arg482Gly The ABCG2 protein variant R482G was chosen for this study because it has broader substrate specificity than the wild-type ABCG2 (R482) and can transport the dye rhodamine 123, our model substrate (28). Login to comment 114 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly All N-glycosylation variants constructed and described here have ABCG2 (R482G) as the protein backbone, and in this study, the ABCG2 designation will refer to the ABCG2 (R482G) protein unless otherwise specified. Login to comment 120 ABCG2 p.Arg482Thr Similar migration patterns are observed for wild-type ABCG2 and the R482T variant (data not shown). Login to comment 136 ABCG2 p.Asn596Gln (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 ABCG2 p.Asn596Gln ABCG2 p.Asn418Gln ABCG2 p.Asn557Gln 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 156 ABCG2 p.Asn418Gln ABCG2 p.Asn557Gln ABCG2 (N418Q) and ABCG2 (N557Q) migrated as a range of bands between the 50 and 75 kDa markers and are indistinguishable from the glycosylated ABCG2 control (Figure 4A, lanes 1, 3, and 5). Login to comment 157 ABCG2 p.Asn596Gln However, ABCG2 (N596Q) migrated as a single species at ~60 kDa (Figure 4A, lane 7). Login to comment 158 ABCG2 p.Arg482Thr In the stable drug-selected cell line MCF-7/AdVp3000 that overexpresses ABCG2 (R482T), the protein also migrates as a single species but at a higher molecular mass closer to the 75 kDa marker (Figure 4B, lane 1). Login to comment 161 ABCG2 p.Asn596Gln ABCG2 p.Asn418Gln ABCG2 p.Asn557Gln 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 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln The nonglycosylated ABCG2 (N596Q) is only expressed as the 60 kDa species (Figure 5A, bottom panel). Login to comment 178 ABCG2 p.Asn596Gln The Glycosylation-Deficient Mutant ABCG2 (N596Q) Is an ActiVe Drug-Stimulated ATPase. Login to comment 179 ABCG2 p.Asn596Gln 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 180 ABCG2 p.Arg482Gly The drug prazosin (20 µM), a substrate for ABCG2 (R482G) (30), was used to stimulate the ATPase activity. Login to comment 181 ABCG2 p.Asn596Gln ABCG2 p.Asn418Gln ABCG2 p.Asn557Gln 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 183 ABCG2 p.Arg482Thr (B) Crude membranes derived from MCF-7/AdVp3000 cells (10 µg) overexpressing ABCG2 (R482T) were treated with (+) or without (-) PNGase F. Login to comment 186 ABCG2 p.Asn596Gln FIGURE 5: Synthesis and degradation of ABCG2 and glycosylation-deficient ABCG2 (N596Q) in transiently transfected HeLa cells. Login to comment 187 ABCG2 p.Asn596Gln (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 ABCG2 p.Asn596Gln (B) Quantitation of 35S-trans label incorporation into ABCG2 and ABCG2 (N596Q). Login to comment 194 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln FIGURE 6: Drug-stimulated ATPase activity of ABCG2 and the glycosylation-deficient variant ABCG2 (N596Q). Login to comment 196 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln The Glycosylation-Deficient ABCG2 (N596Q) Protein Is Expressed at the Cell Surface and Is Functional for Rhodamine 123 Transport. Login to comment 204 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln 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 214 ABCG2 p.Arg482Thr DISCUSSION Our studies demonstrate that ABCG2 transiently expressed in HeLa cells and ABCG2 (R482T) overexpressed in the drug-selected cell line, MCF-7/AdVp3000, are N-linked glycosylated. Login to comment 217 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln 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 ABCG2 p.Asn596Gln glycosylation-deficient mutant ABCG2 (N596Q) protein are comparable to its glycosylation-competent counterpart. Login to comment 254 ABCG2 p.Asn596Gln 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