ABCMdb

PMID: 23800412

Saranko H, Tordai H, Telbisz A, Ozvegy-Laczka C, Erdos G, Sarkadi B, Hegedus T
Effects of the gout-causing Q141K polymorphism and a CFTR DeltaF508 mimicking mutation on the processing and stability of the ABCG2 protein.
Biochem Biophys Res Commun. 2013 Jul 19;437(1):140-5. doi: 10.1016/j.bbrc.2013.06.054. Epub 2013 Jun 22., [PubMed]

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0 ABCG2 p.Gln141Lys Effects of the gout-causing Q141K polymorphism and a CFTR DF508 mimicking mutation on the processing and stability of the ABCG2 protein Hajnalka Sarank&#f3; a,b,1 , Hedvig Tordai b,1 , &#c1;gnes Telbisz c , Csilla &#d6;zvegy-Laczka d , G&#e1;bor Erd} os a,b , Bal&#e1;zs Sarkadi b,c,d , Tam&#e1;s Heged} us a,b,d1; a MTA-SE Molecular Biophysics Research Group, Budapest, Hungary b Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary c Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary d National Blood Center, Budapest, Hungary a r t i c l e i n f o Article history: Received 12 June 2013 Available online 22 June 2013 Keywords: ABCG2 Polymorphism Gout Cystic fibrosis Stability Structure a b s t r a c t ABCG2 is an important multidrug transporter involved also in urate transport, thus its mutations can lead to the development of gout and may also alter general drug absorption, distribution and excretion. Login to comment 1 ABCG2 p.Gln141Lys The frequent ABCG2 polymorphism, Q141K, is associated with an elevated risk of gout and has been controversially reported to reduce the plasma membrane expression and/or the transport function of the protein. Login to comment 2 ABCG2 p.Gln141Lys In the present work we examined the stability and cellular processing of the Q141K ABCG2 variant, as well as that of the DF142 ABCG2, corresponding to the DF508 mutation in the CFTR (ABCC7) protein, causing cystic fibrosis. Login to comment 6 ABCG2 p.Gln141Lys We find that the Q141K variant has a mild processing defect which can be rescued by low temperature, a slightly reduced activity, and a mild folding defect, especially affecting the NBD. Login to comment 20 ABCG2 p.Gly188Glu ABCG2 p.Arg191Gln http://dx.doi.org/10.1016/j.bbrc.2013.06.054 Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator, ABCC7; HEK, human embryonic kidney 293 cells; NBD, nucleotide binding domain; MBP, maltose binding protein; TMD, transmembrane domain; PBA, 4-phenylbutyrate; Sf9, Spodoptera frugiperda cells; SNP, single nucleotide polymorphism; 3R, G188E, R191Q, and R193K rescue mutations in ABCG2-NBD. Login to comment 26 ABCG2 p.Gln141Lys A frequent SNP in the ABCG2 gene produces a Q141K variant [5] of the protein. Login to comment 28 ABCG2 p.Gln141Lys The Q141K variation has been reported to result in altered processing and a less efficient substrate transport by ABCG2 [6-8]. Login to comment 29 ABCG2 p.Gln141Lys Expression of ABCG2 variants in single gene copy systems suggested that the Q141K variant has a reduced plasma membrane expression level and increased intracellular concentration [9,10]. Login to comment 30 ABCG2 p.Gln141Lys In order to rescue the Q141K variant phenotype, which may be relevant in preventing gout and increasing protection against xenobiotics, the molecular details associated with this amino acid change have to be explored. Login to comment 31 ABCG2 p.Gln141Lys The Q141K change is located in the NBD of ABCG2, adjacent to F142, which has an analogous position to F508 of the CFTR (ABCC7) protein (Figs. Login to comment 34 ABCG2 p.Gln141Lys Therefore it has been suggested that the ABCG2 Q141K variant may cause similar changes to the DF508 mutation, disrupting the interface between the NBD and the TMD [2,11]. Login to comment 40 ABCG2 p.Gln141Lys In the present study we compared the biochemical properties of the wild-type ABCG2 to those of the Q141K variant and the DF142 mutation. Login to comment 43 ABCG2 p.Gln141Lys Our results indicate that the Q141K polymorphism reduces the efficient processing and membrane targeting, while has only a mild effect on the structure and function of ABCG2. Login to comment 80 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys The Q141K variant localizes to the plasma membrane, in contrast to the DF142 mutant In order to examine the effects of Q141K and DF142 alterations on the ABCG2 protein expression and localization, we transiently expressed these constructs in HEK cells. Login to comment 82 ABCG2 p.Gln141Lys As shown in Fig. 1A, the Q141K variant exhibited significant level of maturation, although the amount of the fully glycosylated form was significantly lower, compared to that of the wild type ABCG2. Login to comment 87 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Both the wild type and Q141K ABCG2 were found to be expressed mostly in the plasma membrane, although the Q141K variant exhibited a higher level of intracellular staining (Fig. 1B). Login to comment 90 ABCG2 p.Gln141Lys These experiments confirmed lower cell surface expression of the Q141K variant compared to the wild type, whereas cells expressing the DF142 mutant did not bind the 5D3 antibody. Login to comment 91 ABCG2 p.Gln141Lys Our results demonstrate that the effect of the Q141K variation causes a milder phenotype in ABCG2 than the DF142 mutation, while this latter mutation results in an expression deficiency similar to that of DF508 CFTR. Login to comment 93 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Phenylbutyrate treatment increases the level of mature WT and Q141K ABCG2, while do not rescue DF142 ABCG2 To evaluate the potential folding and trafficking deficiency of the ABCG2 Q141K and DF142 proteins, their expression was characterized under more permissive conditions. Login to comment 97 ABCG2 p.Gln141Lys As shown in Fig. 2, in transiently transfected HEK cells the level of the wild type and the Q141K proteins slightly decreased at low temperature (Fig. 2), indicating the adverse effect of low temperature on protein synthesis. Login to comment 100 ABCG2 p.Gln141Lys Expression of ABCG2 WT, Q141K, and DF142 constructs in HEK cells. Login to comment 113 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCG2 p.Arg193Lys ABCG2 p.Gly188Glu ABCG2 p.Arg191Gln Therefore all these three mutations were introduced into the corresponding regions of the ABCG2 DF142 construct (3R: G188E, R191Q, and R193K). Login to comment 120 ABCG2 p.Gln141Lys ABCG2 p.Gly188Glu Interestingly, one of the rescue mutations, G188E has been reported to promote Q141K maturation [11]. Login to comment 121 ABCG2 p.Gln141Lys Most likely, similarly to the homologous CFTR G550E [13,21], this mutation increases the thermostability of NBD in an extent that is sufficient to counteract the effect of the mild Q141K mutation, but not that of the DF142. Login to comment 122 ABCC7 p.Gly550Glu ABCG2 p.Gly188Glu While this G188E mutation does not increase the maturation of WT ABCG2 [11], the analogous G550E mutation promotes the maturation of WT CFTR [21] that again suggest fundamental differences between the NBDs of the two proteins. Login to comment 123 ABCC7 p.Gly550Glu While this G188E mutation does not increase the maturation of WT ABCG2 [11], the analogous G550E mutation promotes the maturation of WT CFTR [21] that again suggest fundamental differences between the NBDs of the two proteins. Login to comment 128 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys The Q141K NBD is more stable than the DF142 NBD, indicated by limited proteolysis experiments Since the quality control system in insect cells is less stringent than in mammalian cells, Sf9 cells were used to express the Q141K, DF142, and WT ABCG2 for functional and biochemical studies. Login to comment 129 ABCG2 p.Gln141Lys In Sf9 cells the expression levels of the wild type and the Q141K ABCG2 variant were similar, while the expression of the deletion mutant was significantly lower (Fig. 3A). Login to comment 130 ABCG2 p.Gln141Lys The vanadate sensitive ATPase activity of the Q141K variant in isolated Sf9 membrane was approximately 70% of that of the wild type protein ([8] and Fig. S6A). Login to comment 134 ABCG2 p.Gln141Lys Membranes expressing Q141K, DF142, and WT ABCG2 were Table 1 Cell surface expression of the ABCG2 constructs quantitated by flow cytometry. Login to comment 135 ABCG2 p.Gln141Lys Geometric mean values HEK parent EGFP only ABCG2 WT ABCG2 Q141K ABCG2 DF142 ABCG2 3R ABCG2DF142 3R Isotype control 7.5 7.6 7.3 7.2 7 8.4 7 Isotype control + Ko143 6.99 7.3 7.3 7.1 7 8.3 7.44 5D3 9.45 12 110 78 10 146 8.4 5D3 + Ko143 30 29.5 210 150 27 152 27.4 Transfection efficiency EGFP FL1 0% 85% 93% 89% 89% 83% 89% Fig. 2. Login to comment 150 ABCG2 p.Gln141Lys Although the rate of Q141K degradation was somewhat greater than that of the WT, it was clearly smaller than the rate of the DF142 fragment. Login to comment 153 ABCG2 p.Gln141Lys (A) WT and Q141K ABCG2 are expressed at similar levels in membranes isolated from Sf9 insect cells, as detected by Western blotting using the BXP-21 antibody at 60 kDa. Login to comment 162 ABCG2 p.Gln141Lys (A) Far UV CD spectra of WT, Q141K, and DF142 NBD fused to MBP, expressed in and isolated from E. coli. Login to comment 164 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys (B) Thermal unfolding of isolated MBP-fused NBD constructs was followed by measuring CD at 222 nm between 20 &#b0;C and 90 &#b0;C. Q141K variant may also be caused by a potential extra trypsin cleavage site introduced by the Q141K variation [11]. Login to comment 166 ABCG2 p.Gln141Lys The thermal stability of the isolated Q141K NBD is similar to the WT, in contrast to the DF142 To identify the effects of the amino acid changes on the NBD conformation and dynamics, isolated NBD constructs fused to MBP, were purified from bacteria (the MBP could not be removed because of a rapid precipitation of the isolated NBDs). Login to comment 172 ABCG2 p.Gln141Lys We found that the melting curve of the Q141K variant was similar to that of the wild type, while the melting profile of the DF142 differed significantly. Login to comment 173 ABCG2 p.Gln141Lys As a summary, we found that the ABCG2 Q141K variant is expressed in the plasma membrane at a lower level and with a somewhat decreased function, as compared to the wild type protein. Login to comment 175 ABCG2 p.Gln141Lys While the Q141K variation is located in a position adjacent to the F508 in CFTR, their structural roles and functions are significantly different based on our experiments and in silico analysis (Fig. S2). Login to comment 177 ABCG2 p.Gln141Lys Moreover, studies focusing on positions similar to the ABCG2 Q141K variant in other ABC transporters may help to understand alterations at the protein level in various diseases, and promote the development of successful treatments. Login to comment