ABCMdb

ABCG2 p.Leu558Ala

None has been submitted yet.

Publications

PMID: 18094074 [PubMed] Velamakanni S et al: "A functional steroid-binding element in an ATP-binding cassette multidrug transporter."

No. Sentence Comment

28 Mutations in the ABCG2R482G gene were introduced using the forward primer 5b18;-TTT TTT CAC GTC TGT TGG TCA ATC TCA C-3b18; and the reverse primer 5b18;-ATT GAC CAA CAG ACG TGA AAA AAT CAT C-3b18; for G553R, the forward primer 5b18;-GAT GAT TTT TAT GGG TCT GTT GGT CAA TCT CAC-3b18; and reverse primer 5b18;-CCA ACA GAC CCA TAA AAA TCA TCA TAA ACA C-3b18; for S552M, and the forward primer 5b18;-GGT CTG GGG GTC AAT GGC ACA ACC ATT GCA TCT TGG-3b18; and reverse primer 5b18;- ATG GTT GTG CCA TTG ACC CCC AGA CCT GAA AAA ATC-3b18; for L555A L558A. Login to comment

PMID: 24384916 [PubMed] Telbisz A et al: "Regulation of the function of the human ABCG2 multidrug transporter by cholesterol and bile acids: effects of mutations in potential substrate and steroid binding sites."

No. Sentence Comment

5 When leucines in the potential steroid-binding element (SBE, aa 555-558) of ABCG2 were replaced by alanines, cholesterol dependence of ABCG2 activity was strongly reduced, although the L558A mutant variant when purified and reconstituted still required cholesterol for full activity. Login to comment
38 In our present study, we provide a detailed mutational analysis of the cholesterol-sensing capability of different ABCG2 R482 mutants as well as mutants carrying the L555A, L558A, or L555A/L558A point mutations. Login to comment
49 The steroid-binding element mutants were created by site-directed polymerase chain reaction (PCR) mutagenesis using the following complementary primer pairs: L555A: 59 T TCA GGT CTC GCG GTC AAT CT and 59AG ATT GAC CGC GAG ACC TGA A; L558A: 59 GT CTG TTG GTG AAT GCC ACA ACC ATT and 59 AAT GGT TGT GGC ATT CAC CAA CAG AC; L555/558A: 59 GT CTC GCG GTG AAT GCC ACA ACC ATT and 59 AAT GGT TGT GGC ATT CAC CGC GAG AC. Login to comment
55 The His6-tagged L558A and L555/ 558A mutants were created by cloning the PstI-SacI site from pAcUW21-L/ ABCG2-L558A or L555/558A into the pAcUW21-L/His6-ABCG2. Login to comment
119 We have generated the Leu to Ala mutations L555A, L558A, and L555A/L558A in this motif. Login to comment
122 As shown in Fig. 2B, the L555A, L558A, and L555A/L558A mutants exhibited a well-measurable vanadate-sensitive ATPase activity. Login to comment
123 However, given the similar expression levels of the wild-type and the mutant proteins, we found that L555A and L555A/L558A had only about one-third the basal ATPase activity as compared with wtABCG2. Login to comment
124 Moreover, in the case of the L555A and L555A/L558A mutants, ATPase turnover in the presence of quercetin was also well below of that measured for the wild-type protein or the L558A mutant. Login to comment
133 In these experiments, we examined the effect of cholesterol on the [3 H]methotrexate ([3 H]MTX) and [3 H]estradiol-glucuronide ([3 H]ESG) transport activity of ABCG2 L555A, L558A, and L555A/L558A mutant variants expressed in Sf9 insect cells. Login to comment
136 We found that despite their comparable expression level to wtABCG2, the L555A and L555A/L558A mutants did not show any detectable vesicular transport activity for MTX in either control or cholesterol-rich membranes. Login to comment
137 Even in the case of the L558A mutant, which showed high ATPase activity, we could detect only very low MTX transport activity, similar to that observed in the R482 mutants (Supplemental Figs. Login to comment
139 When we analyzed [3 H]MTX transport by the L558A mutant in membranes loaded with cholesterol, we found only a nonsignificant increase in this transport activity (Supplemental Fig. 3A). Login to comment
147 To analyze the cholesterol sensing of the purified SBE mutants, we have generated N-terminally His6-tagged versions of the L558A and L555/558A variants. Login to comment
148 The His6-L558A and His6-L555A/ L558A ABCG2 mutants were successfully expressed in Sf9 cells, and we also found that tagging did not alter their functionality (data not shown). Login to comment
149 The membrane isolation as well as the purification and reconstitution of the L558A variant were successful. Login to comment
159 the expression level of the His6-L555A/L558A variant in the Sf9 cells was comparable to that of the other variants, the purification yielded a much lower amount of this mutant (data not shown). Login to comment
160 We analyzed the ATPase activity of the purified L558A variant, reconstituted in E. coli lipids in the absence and in the presence of cholesterol. Login to comment
204 (A) ATPase activity of purified wtABCG2, ABCG2-L558A, and L555/558A in proteoliposomes. Login to comment
240 Figure 6C shows that CA does not influence ATP hydrolysis of the L555A/L558A mutant-that is, both basal and substrate-stimulated activities remained unaltered. Login to comment
241 GC and TC also did not influence the activity of the L555A/L558A mutant (data not shown). Login to comment
243 In the case of the L558A mutant, a similar effect of bile acids was observed as in the case of the R482G variant: CA and TC inhibited both basal and substrate-stimulated ATPase activity (Supplemental Fig. 5), but the relative substrate activation was practically unchanged (Fig. 6D). Login to comment
244 GC had no effect on either baseline or substrate-stimulated activity of the L558A mutant (Supplemental Fig. 5). Login to comment
258 In their study, Velamakanni et al. (2008) found that the ABCG2-L555A/L558A mutant does not have an altered cholesterol sensing, but progesterone and estradiol binding as well as transport were abolished. Login to comment
259 In our present work, we expressed and analyzed in detail the SBE (or LxxL motif) mutants L555A, L558A, and L555/558A of human ABCG2. Login to comment
262 When examining the effect of cholesterol on their function, we found that although a slight increase in the baseline ATP hydrolysis of the L555A and L558A mutants occurred in cholesterol-enriched membranes (fold activation was 1.260.1 and 1.560.1, respectively), their relative substrate stimulation (ratio of ATP hydrolysis in the presence and absence of substrates) did not change (Fig. 2B and Supplemental Fig. 2). Login to comment
264 Moreover, the L555A/L558A mutant was absolutely insensitive to cholesterol loading in the ATPase-activity measurements (Fig. 2B). Login to comment
265 This apparent cholesterol independence of the L555A/L558A Fig. 5. Login to comment
271 mutant contradicted the results described by Velamakanni et al. (2008), which may be due to the fact that they investigated a triple mutant of ABCG2, which had R482G besides the L555A/L558A mutation, whereas we performed our experiments using the wild-type ABCG2 (482R) as a background. Login to comment
273 To solve this contradiction, we also generated the triple mutant R482G/L555A/L558A of ABCG2 and expressed this protein in insect cells. Login to comment
275 Because even very low levels of membrane sterols may affect ABCG2 function, we have purified and reconstituted the L558A and L555A/L558A mutants in cholesterol-free liposomes. Login to comment
276 Surprisingly, we found that the L558A mutant also needs cholesterol for its full activity. Login to comment