ABCMdb

PMID: 26294421

Haider AJ, Cox MH, Jones N, Goode AJ, Bridge KS, Wong K, Briggs D, Kerr ID
Identification of residues in ABCG2 affecting protein trafficking and drug transport, using co-evolutionary analysis of ABCG sequences.
Biosci Rep. 2015 Jul 17;35(4). pii: e00241. doi: 10.1042/BSR20150150., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCG2 p.Ile573Ala One mutant (I573A) showed disrupted glycosylation and reduced trafficking kinetics. Login to comment 7 ABCG2 p.Ile573Ala In contrast with many ABC transporter folding mutations which appear to be 'rescued` by chemical chaperones or low temperature incubation, the I573A mutation was not enriched at the cell surface by either treatment, with the majority of the protein being retained in the endoplasmic reticulum (ER). Login to comment 8 ABCG2 p.Pro485Ala ABCG2 p.Met549Ala Two other mutations (P485A and M549A) showed distinct effects on transport of ABCG2 substrates reinforcing the role of TM helix 3 in drug recognition and transport and indicating the presence of intracellular coupling regions in ABCG2. Login to comment 32 ABCG2 p.Arg482Gly Residue 482 in transmembrane helix (TM) 3 of the TMD is an arginine in the wild-type (WT) sequence, but a drug selected cell line expressing a mutant version (where the arginine is replaced by glycine; R482G) shows a broader resistance profile including doxorubicin and rhodamine 123 in its substrates [16-18]. Login to comment 84 ABCG2 p.Ile573Ala For immunofluorescence determination of the localization of the I573A isoform, cells were fixed on cover slips with 4% PFA in PBS for 5 min at room temperature before treatment with 50 mM NH4Cl for 10 min to quench the free aldehyde groups of the fixative preventing autofluorescence [21]. Login to comment 109 ABCG2 p.Lys473Ala ABCG2 p.Pro485Ala ABCG2 p.Lys453Ala ABCG2 p.Ile573Ala ABCG2 p.Met549Ala ABCG2 p.Ser195Ala ABCG2 p.Met131Ala ABCG2 p.Trp564Ala We made individual substitutions of each amino acid to alanine: M131A, S195A, K453A, K473A, P485A, M549A, W564A and I573A. Login to comment 110 ABCG2 p.Asn596Gln ABCG2 p.Glu211Gln ABCG2 p.Lys86Ala A further three control mutations were analysed, namely a substitution of lysine to alanine in the Walker-A motif (K86A), a substitution of glutamate to glutamine in the Walker-B motif (E211Q) and a substitution of asparagine to glutamine at the site of glycosylation (N596Q). Login to comment 114 ABCG2 p.Ile573Ala Analysis of the expression level (given by the ratio 5D3-PE fluorescence labelling compared with IgG-PE fluorescence labelling) of all mutants suggested that, of the novel mutants examined in the present study, only I573A had reduced cell surface expression (Figures 2E and 2F). Login to comment 123 ABCG2 p.Ile573Ala variability in expression of I573A was not a reflection of the conformational sensitivity of the 5D3 antibody [31], we performed western blotting of whole cell lysates with the BXP-21 antibody which recognizes an intracellular epitope. Login to comment 124 ABCG2 p.Ile573Ala ABCG2 p.Ile573Ala This confirmed that I573A was expressed at reduced levels compared with other isoforms (Figure 2G; I573A expression is essentially undetectable in this exposure). Login to comment 125 ABCG2 p.Ile573Ala The failure of one of the isoforms (I573A) to effectively reach the cell surface piqued our interest as membrane trafficking is clearly of cellular and clinical relevance, both for ABCG2 and ...................................................................... .......................................................... .................................................................... ........................................................... ........................................................... ................................................ c 2015 Authors. Login to comment 127 ABCG2 p.Ile573Ala 5 Figure His12-I573A shows impaired glycosylation (A) Following transient transfection cell lysates (30 bc;g) were resolved on 10 % w/v acrylamide gels and blotted with BXP-21 antibody. Login to comment 128 ABCG2 p.Met131Ala Transfection reagent only (PEI) served as negative control, WT His12-ABCG2 and His12-M131A serve as positive controls. Login to comment 129 ABCG2 p.Asn596Gln His12-N596Q is a glycosylation defective form of ABCG2. Login to comment 130 ABCG2 p.Ile573Ala ABCG2 p.Ile573Ala His12-I573A is exclusively present at an intermediate molecular mass at 24 h and even at 48 h the majority of His12-I573A isoform is not the mature molecular mass. Login to comment 133 ABCG2 p.Asn596Gln His12-N596Q shows no change in molecular mass upon PNGaseF treatment. Login to comment 134 ABCG2 p.Ile573Ala By contrast, the lower molecular mass band following transfection with the His12-I573A isoform shows a small (estimated < 5 kDa) change in molecular mass upon PNGaseF treatment indicative of the removal of core glycosylation. Login to comment 135 ABCG2 p.Met131Ala WT and a representative other mutant (His12-M131A) are fully glycosylated at both 24 and 48 h post transfection. Login to comment 142 ABCG2 p.Ile573Ala To enable live cell imaging of this isoform and to rule out the possibility that these results are a consequence of a transient transfection system, we generated stable cell lines expressing WT and I573A isoforms of ABCG2 with an N-terminal sfGFP extension. Login to comment 145 ABCG2 p.Ile573Ala For sfGFP-I573A, the cellular fluorescence level (i.e. a measure of protein expression) is reduced compared with WT sfGFP-ABCG2 (compare intensity of Figure 4A with Figure 4B obtained using identical image acquisition and processing conditions), in agreement with data from transient expression of untagged isoforms (Figure 2). Login to comment 146 ABCG2 p.Ile573Ala Moreover, sfGFP-I573A is retained in perinuclear and reticular intracellular compartments with very little protein at the cell surface. Login to comment 147 ABCG2 p.Ile573Ala Co-localization of the sfGFP-I573A isoform was demonstrated with anti-calnexin antibodies confirming the ER retention (Figure 4C). Login to comment 149 ABCG2 p.Ile573Ala ABCG2 p.Ile573Ala ABCG2 p.Ile573Ala We investigated both of these possibilities with the sfGFP-I573A isoform and demonstrated that neither incubation at 30e6; C (Figure 4D, right hand panel), nor incubation with the chaperone 4-phenylbutyrate rescued the cell surface expression of I573A (Figure 4D, left hand panel), although the latter did result in a qualitatively higher expression of sfGFP-I573A (compare Figure 4B right hand and 4D left hand panel which are obtained under identical image capture and processing settings on the same day). Login to comment 153 ABCG2 p.Lys473Ala ABCG2 p.Lys453Ala ABCG2 p.Ser195Ala ABCG2 p.Met131Ala ABCG2 p.Met131Ala ABCG2 p.Trp564Ala Firstly, there was a group of five mutants which showed FTC-inhibited MX export comparable to WT protein (e.g. M131A, S195A, K453A, K473A, W564A; M131A data shown in Figure 3C). Login to comment 154 ABCG2 p.Pro485Ala ABCG2 p.Pro485Ala ABCG2 p.Met549Ala Secondly, there were a pair of mutant isoforms which showed little or no FTC-mediated inhibition of MX export (i.e. having high accumulation of MX even in the absence of FTC) but normal ABCG2 surface protein expression (P485A and M549A; P485A data shown in Figure 5D). Login to comment 155 ABCG2 p.Glu211Gln ABCG2 p.Lys86Ala These two mutants behaved in this assay in essentially the same way as the K86A and E211Q catalytically inactive mutants (result not shown). Login to comment 156 ABCG2 p.Pro485Ala ABCG2 p.Met549Ala To investigate further the two TMD mutations with impaired drug transport (M549A and P485A), we generated stable expressing cell lines expressing sfGFP-tagged isoforms. Login to comment 157 ABCG2 p.Ile573Ala ABCG2 p.Ile573Ala sfGFP- ................................................................... ............................................................ .................................................................. ............................................................. ........................................................ .................................................... 6 Figure sfGFP-I573A is retained in the endoplasmic reticulum (ER) and cell surface expression is not rescued by low temperature incubation To visualize trafficking of I573 in live cells, stable cell lines expressing WT and I573A isoforms of ABCG2 with an N-terminal sfGFP tag were generated and examined by confocal imaging. Login to comment 158 ABCG2 p.Ile573Ala The majority of WT ABCG2 is present at the cell surface (A, phase and merged image), whereas the majority of I573A is retained intracellularly (B). Login to comment 159 ABCG2 p.Ile573Ala ABCG2 p.Ile573Ala (C) Immunostaining of cells with anti-calnexin antibodies (red, left hand panel) shows significant co-localization of the sfGFP-I573A fluorescence signal (green, middle panel and yellow in the merged image, right hand panel) indicative of the retention of the majority of sfGFP-I573A in the ER. Login to comment 161 ABCG2 p.Ile573Ala ABCG2 p.Ile573Ala (D) Incubation of sfGFP-I573A expressing cells at 30e6; C had little effect on the localization of the I573A to the cell surface (right hand panel). Login to comment 165 ABCG2 p.Pro485Ala ABCG2 p.Met131Ala Data are representative of at least four independent transfections and reflect negative control data (empty vector, A), positive control data (WT ABCG2 expression, B), a mutant with normal MX transport function (M131A, C) and a mutant with abrogated function but normal surface expression (P485A, D). Login to comment 171 ABCG2 p.Pro485Ala sfGFP-P485A retained limited MX transport function with a 1.6-fold increase in accumulation in the presence of Ko143. Login to comment 172 ABCG2 p.Met549Ala ABCG2 p.Lys86Ala sfGFP-M549A and the catalytically inactive sfGFP-K86A isoform showed no Ko143-inhibited MX transport. Login to comment 173 ABCG2 p.Met549Ala ABCG2 p.Lys86Ala With PhA, neither sfGFP-K86A nor sfGFP-M549A showed Ko143-inhibitable transport. Login to comment 174 ABCG2 p.Pro485Ala In contrast, both sfGFP-ABCG2-WT and sfGFP-P485A were able to export PhA in a Ko143-inhibitable manner, with indistinguishable levels of Ko143 inhibition (Figure 6B), indicating positional specific effects on drug binding and transport. Login to comment 179 ABCG2 p.Pro485Ala ABCG2 p.Ile573Ala ABCG2 p.Met549Ala Of the eight residues we mutated, two resulted in altered drug export function (P485A and M549A) and a further residue (I573A) perturbs the trafficking and maturation of ABCG2 glycosylation. Login to comment 181 ABCG2 p.Pro485Ala Intriguingly, two of the residues we have analysed (I573 and P485A) are coupled to a residue Ser195 , which can be mutated to alanine without any apparent affect. Login to comment 188 ABCG2 p.Ile573Ala One of the eight residues we mutated in the present study (I573A) proved to have an effect on protein trafficking and maturation, an effect previously seen with a mutation of a residue located close to the intracellular side of TM1 [39]. Login to comment 190 ABCG2 p.Ile573Ala I573A is located within TM5 in the model of Mao and colleagues [30], although sequence based topological models place it within the extracellular loop between TM5 and TM6. Login to comment 191 ABCG2 p.Ile573Ala Irrespective of its exact location, the effect of mutating I573A on targeting and glycosylation is consistent with a critical role for the TM5 and 6 region in the integrity of ABCG2 [40,41]. Login to comment 192 ABCG2 p.Ile573Ala In contrast with some recent reports on the rescue of folding mutants by chemical 'correctors` or by low incubation temperatures [6,34,35], we could find not positive effect on I573A. Login to comment 193 ABCG2 p.Pro485Ala ABCG2 p.Met549Ala Two of the remaining residues we mutated (M549A and P485A) affected drug export and provide some further evidence for a role for TM3 in forming part of a drug-binding site on ABCG2. Login to comment 200 ABCG2 p.Pro485Ala A previous study [42] also showed P485A mutation resulted in altered substrate specificity (although the majority of MX transport was retained in that study [42]). Login to comment 206 ABCG2 p.Pro485Ala ABCG2 p.Met549Ala 9 to determine whether the lack of function of the P485A and M549A isoforms is due to impaired substrate binding (i.e. a direct effect on the drug-binding site) and/or impaired TMD-NBD communication. Login to comment