ABCMdb

PMID: 18644784

Ozvegy-Laczka C, Laczko R, Hegedus C, Litman T, Varady G, Goda K, Hegedus T, Dokholyan NV, Sorrentino BP, Varadi A, Sarkadi B
Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter.
J Biol Chem. 2008 Sep 19;283(38):26059-70. Epub 2008 Jul 21., 2008-09-19 [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCG2 p.Cys603Ala When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S-S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. Login to comment 8 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala However, disruption of the intramolecular S-S bridge (in C592A, C608A, or C592A/C608A mutants) in this loop abolished 5D3 binding, whereas the function of the protein was preserved. Login to comment 28 ABCG2 p.Cys603Ala ABCG2 p.Cys603Ser ABCG2 p.Cys603Gly Interestingly, mutation of Cys-603 to Ala, Gly, or Ser does not remarkably influence the expression and functionality of the transporter (9-11). Login to comment 52 ABCG2 p.Arg482Gly Expression Vectors, Cell Lines, and Cell Culturing pCIN4 bicistronic mammalian expression vectors containing the cDNAs of ABCG2-R482G, or additional Cys to Ala mutations, were generated as described previously (10). Login to comment 55 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala To obtain a cell line showing higher ABCG2-C592A/C608A expression, HEK- C592A/C608A cells were sorted based on rhodamine123 extrusion capacity in a FACSAria flow cytometer. Login to comment 56 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala The sorted HEK- C592A/C608A cell line was used throughout this study. Login to comment 113 ABCG2 p.Arg482Gly To analyze whether there is a correlation between covalent dimer formation upon PFA fixation and increased 5D3 binding, and whether PFA cross-linking inhibits ABCG2 function, we treated intact HEK cells expressing ABCG2 (R482G) with increasing concentrations of PFA and analyzed 5D3 binding, ABCG2 function, and covalent dimer formation. Login to comment 114 ABCG2 p.Arg482Gly Throughout this study we used both the wild-type and the R482G mutant variant of ABCG2 in HEK cells, because the background of the cysteine mutations was this latter variant (10). Login to comment 115 ABCG2 p.Arg482Gly The R482G mutant also allowed the measurement of transport activity followed by rhodamine123 extrusion, characteristic of the mutant protein. Login to comment 116 ABCG2 p.Arg482Gly In all functional experiments the R482G protein variant showed the same behavior regarding 5D3 binding as the wild-type ABCG2, that is PFA or Ko143 caused a significant 5D3 shift (Fig. 1B). Login to comment 121 ABCG2 p.Arg482Gly Fig. 1C also shows rhodamine123 transport activity (activity factor) of the HEK-ABCG2-R482G cells treated with increasing concentrations of PFA. Login to comment 131 ABCG2 p.Arg482Gly The experiments shown in Fig. 2 were performed in HEK-ABCG2-R482G cells, but experiments repeated in both PLB985 and A431 cells, expressing the wild-type ABCG2 protein, provided the same results (data not shown). Login to comment 140 ABCG2 p.Arg482Gly HEK-ABCG2-R482G cells were lysed, dissolved in disaggregation buffer containing the reducing agent beta-mercaptoethanol or without it (as indicated on the figure), and subjected to 7.5% SDS-PAGE. ABCG2 in cells fixed with 0.5% PFA prior to cell lysis is also shown. Login to comment 144 ABCG2 p.Arg482Gly HEK293 cells transfected with empty pCIN4 or pCIN4-ABCG2(R482G) were labeled with Alexa647-conjugated 5D3. Login to comment 149 ABCG2 p.Arg482Gly Mock or pCIN4-ABCG2(R482G)-transfected HEK293 cells were fixed with increasing concentrations of PFA, washed, and then labeled with 5D3 antibody or mouse IgG2b (isotype control (IT)). Login to comment 168 ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Fig. 4A shows that all Cys-to-Ala mutants (except for C603A/ C608A that was expressed in very low amount and exclusively in an underglycosylated form) could be detected by Western blotting, using the ABCG2-specific BXP-21 antibody. Login to comment 169 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala The mutants C592A and C603A showed expression levels comparable to that of the wild-type ABCG2, whereas the amount of double mutant C592A/C608A or the triple Ala mutant proteins was about 50% of that seen for the wild-type ABCG2. Login to comment 171 ABCG2 p.Arg482Gly We analyzed the transport of four different fluorescent ABCG2 substrate compounds, MX, pheophorbide A, Hoechst 33342, and rhodamine123 (rhodamine123 is transported only by the R482G ABCG2 mutant). Login to comment 173 ABCG2 p.Arg482Gly ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Hoechst 33342 and pheophorbide A were transported by all of the mutants (except for C603A/C608A); however, the mitoxantrone transport capacity of the mutants, lacking the intramolecular or both disulfide bonds, was significantly weaker than that of the R482G or C603A variants. Login to comment 174 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala There was also a difference in rhodamine123 uptake, with the C592A and C592A/ C603A mutants showing practically no transport activity (Fig. 4B). Login to comment 176 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala When cells expressing the different Cys-to-Ala mutants were labeled with the 5D3 antibody, we found that only the C603A variant had a clearly detectable 5D3 labeling and the C592A/ C608A mutant showed some weak 5D3 binding capacity (Fig. 5A, upper panel). Login to comment 177 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Similar to that seen in the case of the wild-type ABCG2, PFA fixation (Fig. 5A, lower panel) or Ko143 treatment (not shown) of the cells expressing the C603A mutant and the double mutant C592A/C608A resulted in an increased 5D3 binding. Login to comment 180 ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Fig. 5B shows that all mutants could be detected by the BXP-21 antibody, recognizing an intracellular epitope of ABCG2, and all of them were present in the plasma membrane (except for the hardly expressed C603A/C608A double mutant, which was found, for the most part, intracellularly). Login to comment 181 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala However, 5D3 labeling analyzed by confocal microscopy gave the same result as the flow cytometry measurements, that is, only the cells expressing the wild-type ABCG2, C603A, and the C592A/ C608A variants (the latter one seen only at increased detector voltage) could bind the 5D3 antibody. Login to comment 184 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala We found that, in contrast to a 30-40% inhibition found in the case of the wild-type ABCG2, 5D3 did not influence the Hoechst 33342 transport activity of the C592A/C608A and C592A/C603A/C608A mutants (data not shown). Login to comment 185 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala All of these data strongly suggest that the ABCG2 mutant proteins lacking the cysteines required for intramolecular S-S bridge forma- TABLE 1 Effects of protein cross-linkers on 5D3 binding, transport activity, and covalent dimer formation of ABCG2 Cross-linker Side chains cross-linked Spacer arm length Increased 5D3 binding Inhibition of transport function Cross-linked ABCG2 on Western blot Å BM͓PEO͔3 SH2-H2 14.7 Yes Yes Yes BMPH CH3-SH2 8.1 Yes Yes No EDC COOH-NH2 0 Yes No No Sulfo-EGS NH2-NH2 16.1 No No Yes Sulfo-MBS NH2-SH2 9.9 Yes No Yes PMPI SH2-OH 8.7 Yes No Yes Interaction of ABCG2 with the 5D3 Monoclonal Antibody 26064 tion are expressed in comparable amounts, reach the cell surface, and work as active transporters in a manner similar to the wild-type ABCG2, but these variants (except for the C592A/ C608A mutant showing weak 5D3 binding) are unable to bind the 5D3 antibody. Login to comment 186 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Effect of DTT on 5D3 Labeling of the Cys-to-Ala Mutants-To test whether decreased 5D3 binding in ABCG2-expressing cells treated with DTT was due to the reduction of the extracellular cysteines, we also examined the effect of DTT on 5D3 labeling of the mutants C603A and C592A/C608A in native or PFA-fixed cells. Login to comment 187 ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala Fig. 6, A and B, shows that DTT is still effective in the reduction of 5D3 binding in the case of the C603A mutant but has practically no effect on 5D3 labeling of the C592A/C608A mutant. Login to comment 190 ABCG2 p.Arg482Gly HEK-ABCG2-R482G cells were incubated with different protein cross-linkers, washed, and then labeled with 5D3-Alexa647 (left panel) or incubated with 2 ␮M rhodamine123 (right panel) in the presence or absence of the inhibitor Ko143. Fluorescence was determined by flow cytometry. Login to comment 199 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala Interaction of ABCG2 with the 5D3 Monoclonal Antibody SEPTEMBER 19, 200•VOLUME 283•NUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 26065 in 5D3 binding that reached its minimum (almost the fluorescence of the background) at 10-50 mM DTT in ABCG2 and C603A-expressing cells, whereas DTT had no effect on labeling of the C592A/C608A double mutant (Fig. 6C). Login to comment 213 ABCG2 p.Arg482Gly In this study we have further analyzed how alterations in ABCG2 structure, covalent cross-linking, or changes in the S-S FIGURE3.EffectofDTTtreatmenton5D3binding.HEK293cellstransfected with empty pCIN4 (A) or pCIN4-ABCG2(R482G) (B) were incubated with or without 10 mM DTT, washed, and then labeled with 5D3 or mouse IgG2b and goat anti-mouse phycoerythrin-conjugated secondary antibody. Login to comment 218 ABCG2 p.Arg482Gly HEK cells were transfected with pCIN4 vectors encoding different Cys-to-Ala ABCG2 mutants or R482G (indicated as ABCG2). Login to comment 221 ABCG2 p.Arg482Gly B, mitoxantrone, pheophorbide A, rhodamine123, and Hoechst 33342 transport activity of Cys-to-Ala mutants. HEK cells expressing different ABCG2 mutants or R482G (indicated as ABCG2) were incubated with 5 ␮M mitoxantrone, 1 ␮M pheophorbide A, 2 ␮M rhodamine123, or 1 ␮M Hoechst 33342 in the absence or presence of 1 ␮M Ko143. Fluorescence of mitoxantrone, pheophorbide A, and rhodamine123 was detected in a FACSCalibur cytometer, and activity factors were calculated from mean fluorescence values as described under "Experimental Procedures." Login to comment 234 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala To find out which disulfide bridge is important for epitope formation, we analyzed different Cys-to-Ala mutants lacking the intermolecular (C603A), the intramolecular (C592A, C608A, C592A/C608A), or both kinds of (C592A/C603A, C603A/C608A, or C592A/ C603A/C608A) S-S bonds. Login to comment 235 ABCG2 p.Cys603Ala We found that the C603A mutant behaves similarly to ABCG2 having intact S-S bridges. Login to comment 236 ABCG2 p.Cys603Ala The C603A mutant, which can be expressed in an amount comparable to ABCG2, is found in the plasma membrane (Figs. Login to comment 245 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala Fluorescence was acquired using the same equipment settings, except the slide representing 5D3 labeling of C592A/C608A was taken at an increased detector voltage. Login to comment 249 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala The single mutants, lacking the intramolecular S-S bond, i.e. C592A, C608A, as well as the C592A/C603A/C608A variant, had clearly detectable expression levels, were present in the plasma membrane, and were functional for active transport with somewhat altered substrate specificities (Figs. 4 and 5). Login to comment 254 ABCG2 p.Cys592Ser ABCG2 p.Cys608Ser They found that C592S and C608S had impaired 5D3 binding; however, these two mutants showed very low expression levels in this study (9). Login to comment 255 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala In our experiments we were able to express C592A and C608A mutants in comparable levels to the wild-type ABCG2. Login to comment 265 ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala ABCG2 p.Cys608Ala Interaction of ABCG2 with the 5D3 Monoclonal Antibody 26068 ing; they detected no 5D3 labeling by confocal microscopy for C592A, C608A, and the C592A/C608A double mutant. Login to comment 267 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala However, in our hands the C592A/ C608A double mutant showed a weak 5D3 binding both in flow cytometry and confocal microscopy, and the 5D3 shift upon PFA or Ko143 treatment could also be observed (Fig. 5). Login to comment 268 ABCG2 p.Cys592Ala ABCG2 p.Cys608Ala DTT had no effect on the 5D3 labeling of the C592A/C608A variant (Fig. 6), and the excess amount of 5D3 did not inhibit the function of this mutant. Login to comment