ABCMdb

PMID: 23205634

Telbisz A, Ozvegy-Laczka C, Hegedus T, Varadi A, Sarkadi B
Effects of the lipid environment, cholesterol and bile acids on the function of the purified and reconstituted human ABCG2 protein.
Biochem J. 2013 Mar 1;450(2):387-95. doi: 10.1042/BJ20121485., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCG2 p.Arg482Gly Both wild-type ABCG2 and its R482G mutant variant require cholesterol for full activity, although they exhibit different cholesterol sensitivities. Login to comment 21 ABCG2 p.Arg482Gly We have reconstituted the purified wild-type and the ABCG2-R482G variant into liposomes with different lipid compositions, and evaluated the effects of mutations on the cholesterol and bile acid sensitivity of these transporter variants. Login to comment 29 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly The His6-tagged ABCG2-R482G mutant was created by cloning the PstI/SacI fragment of pAcUW21-L/ABCG2-R482G into the pAcUW21-L/His6-wtABCG2 vector. Login to comment 163 ABCG2 p.Arg482Gly Characterization of the purified ABCG2-R482G variant ABCG2 Arg482 variants have grossly altered substrate, cholesterol and bile acid interactions when expressed in Sf9 cells ([8,13,20] and &#b4; A. Telbisz and Cs. &#a8; Ozvegy-Laczka, unpublished work). Login to comment 164 ABCG2 p.Arg482Gly In the present study we also generated the N-terminally His6-tagged version of ABCG2-R482G and characterized its activity in reconstituted proteoliposomes. Login to comment 165 ABCG2 p.Arg482Gly The His6-ABCG2-R482G variant was successfully expressed in Sf9 cells, at a similar level to the wild-type ABCG2 protein (results not shown). Login to comment 167 ABCG2 p.Arg482Gly By examining the ATPase activity of the purified and reconstituted ABCG2-R482G protein, we used E. coli lipids and cholesterol-containing liposomes and the simplified reconstitution protocol. Login to comment 168 ABCG2 p.Arg482Gly As shown in Supplementary Figure S5 (at http://www.biochemj.org/bj/450/bj4500387add.htm), we found that, in accordance with results obtained in cholesterol-loaded Sf9 cell membrane preparations, the calculated turnover of the ATPase activity of the purified R482G variant was similar to or higher than that of the wild-type protein. Login to comment 170 ABCG2 p.Arg482Gly In contrast with the wild-type protein, the ATPase activity of the R482G variant could be significantly stimulated by elacridar or imatinib [21], inhibitors of the wild-type ABCG2, whereas quercetin or nilotinib had a similar stimulatory effect as in the case of wild-type ABCG2 (see Supplementary Figure S5). Login to comment 171 ABCG2 p.Arg482Gly Figure 6 summarizes the data obtained for the sterol modulation of the activity of the purified and reconstituted ABCG2-R482G protein. Login to comment 173 ABCG2 p.Arg482Gly As depicted in Figure 6(A), when reconstituted in E. coli lipids in the absence of cholesterol, the ATPase activity of the ABCG2-R482G protein was low and practically not stimulated by quercetin (or by other activating agents, results not shown). Login to comment 176 ABCG2 p.Arg482Gly We also compared the effects of various cholesterol concentrations in the reconstituting lipid mixture on the ATPase activities of the wild-type ABCG2 and the R482G variant (Figure 6B). Login to comment 178 ABCG2 p.Arg482Gly Although the wild-type protein requires 30-40 mol% of cholesterol to reach full drug-stimulated activity, the R482G variant is already maximally active at 20 mol% of cholesterol. Login to comment 179 ABCG2 p.Arg482Gly This difference may explain the findings in the insect cell membranes, where low levels of endogenous sterols may provide full activity for the R482G variant, but not for the wild-type ABCG2 protein. Login to comment 180 ABCG2 p.Arg482Gly The activity of the purified and reconstituted ABCG2-R482G protein was also modulated by the addition of bile acids (cholic acid, taurocholate or deoxycholate). Login to comment 181 ABCG2 p.Arg482Gly The results obtained in the presence of 1 mM cholic acid are presented on Figure 6(C), demonstrating the effect of bile acids on the R482G mutant of ABCG2. Login to comment 182 ABCG2 p.Arg482Gly The basal ATPase activity of the R482G variant reconstituted in brain lipid with 40 mol% cholesterol was reduced by cholic acid, although to a lesser extent than that of the wild-type protein. Login to comment 188 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly These Figure 6 Comparison of the sterol and bile acid dependence of the ATPase activity of the purified and reconstituted wild-type and R482G variant of ABCG2 (A) ATPase activity of ABCG2-R482G was determined in control (cont, pure E. coli lipid based) liposomes or in liposomes prepared from E. coli lipid with 20 or 40 mol% of the indicated sterol. Login to comment 191 ABCG2 p.Arg482Gly (B) Comparison of the ATPase activity of the wild-type (wt) and the ABCG2-R482G proteins reconstituted in E. coli lipid-based liposomes with increasing amounts of cholesterol. Login to comment 192 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly Basal (wild-type, Ƭf;; and R482G, Ⴢ) and drug-stimulated (5 bc;M quercetin: wild-type, Ⴡ; and R482G, Ƭa;) ATPase activities were examined. Login to comment 193 ABCG2 p.Arg482Gly (C) Effect of cholic acid on the ATPase activity of reconstituted wild-type ABCG2 and ABCG2-R482G protein. Login to comment 217 ABCG2 p.Arg482Gly An interesting finding, which could not be derived from studies on ABCG2 in cholesterol-loaded Sf9 cell membrane preparations, was the absolute cholesterol dependence of the activity of the ABCG2 protein, even in the case of the R482G variant, which was found to be insensitive to cholesterol loading of the Sf9 membranes [8]. Login to comment 218 ABCG2 p.Arg482Gly We suggest that the explanation for this discrepancy is that even very low membrane sterol levels may fully activate the R482G transporter variant in the Sf9 membrane. Login to comment 219 ABCG2 p.Arg482Gly Thus the R482G protein is not insensitive to cholesterol, but instead has a high affinity for cholesterol, exceeding that of the wild-type ABCG2 protein. Login to comment 229 ABCG2 p.Arg482Gly This basal activity is somewhat different in the R482G mutant variant, depending on the lipid environment and cholesterol, but may not be coupled to any transport activity. Login to comment