ABCMdb

PMID: 21763656

Suetani RJ, Sorrenson B, Tyndall JD, Williams MJ, McCormick SP
Homology modeling and functional testing of an ABCA1 mutation causing Tangier disease.
Atherosclerosis. 2011 Oct;218(2):404-10. Epub 2011 Jun 29., [PubMed]

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0 ABCA1 p.Arg1068His Atherosclerosis 218 (2011) 404-410 Contents lists available at ScienceDirect Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis Homology modeling and functional testing of an ABCA1 mutation causing Tangier disease Rachel J. Suetania , Brie Sorrensona , Joel D.A. Tyndallb , Michael J.A. Williamsc , Sally P.A. McCormicka,* a Department of Biochemistry, University of Otago, Dunedin, New Zealand b School of Pharmacy, University of Otago, Dunedin, New Zealand c Department of Medical and Surgical Sciences, University of Otago, Dunedin, New Zealand a r t i c l e i n f o Article history: Received 27 January 2011 Received in revised form 8 June 2011 Accepted 8 June 2011 Available online 29 June 2011 Keywords: ABCA1 HDL Cholesterol efflux Homology modeling Tangier disease a b s t r a c t Objective: To investigate the impact of the p.R1068H mutation on the structure and function of the ATP-binding cassette A1 (ABCA1) protein. Login to comment 2 ABCA1 p.Arg1068His ABCA1 p.Arg1068His Cholesterol efflux assays were performed on fibroblasts obtained from members of a Tangier disease (TD) family carrying the p.R1068H mutation and in HEK293 cells transfected with a p.R1068H mutant cDNA vector. Login to comment 3 ABCA1 p.Arg1068His Confocal microscopy was used to investigate the localisation of the wildtype and mutant p.R1068H protein in HEK293 cells. Login to comment 5 ABCA1 p.Arg1068His Cholesterol efflux studies showed the efflux from TD fibroblasts and HEK293 cells expressing the mutant p.R1068H protein to be markedly reduced compared to wildtype. Login to comment 6 ABCA1 p.Arg1068His Localisation of the mutant p.R1068H protein in HEK293 cells showed intracellular retention of the protein indicating a defect in trafficking to the plasma membrane. Login to comment 7 ABCA1 p.Arg1068His Conclusion: Homology modeling of the ABCA1 protein showed that the p.R1068H mutation would likely disrupt the conformation of NBD-1. Login to comment 8 ABCA1 p.Arg1068His Functional studies of p.R1068H showed a lack of cholesterol efflux function due to defective trafficking to the plasma membrane, most likely caused by impaired oligomerisation. Login to comment 26 ABCA1 p.Arg1068His We previously reported a missense mutation (c.3516G>A) in exon 22 of the ABCA1 gene, causing a p.R1068H amino acid substitution in a highly conserved region of the ABCA1 protein [11]. Login to comment 28 ABCA1 p.Arg1068His Heterozygotes with the p.R1068H mutation displayed a wide range of HDL levels (0.48-1.22 mmol L-1) [11]. Login to comment 31 ABCA1 p.Arg1068His doi:10.1016/j.atherosclerosis.2011.06.019 model for the human ABCA1 protein in order to better predict the effect of the p.R1068H mutation. Login to comment 32 ABCA1 p.Arg1068His This was followed with direct testing of the functional impact of the p.R1068H mutation on the ABCA1 protein via cholesterol efflux assays and analysis of sub-cellular localisation. Login to comment 34 ABCA1 p.Arg1068His Materials and methods 2.1. Computational modeling The functional impact of the p.R1068H substitution was predicted with the PANTHER [12] and SIFT [13] programmes, using the NCBI reference sequence NP005493.2. Login to comment 38 ABCA1 p.Arg1068His Study subjects We have previously described the p.R1068H proband and her extended pedigree [11]. Login to comment 50 ABCA1 p.Arg1068His Mutagenic primers were used to introduce the c.3516G>A nucleotide change in exon 22 of the ABCA1 sequence causing the p.R1068H mutation. Login to comment 51 ABCA1 p.Arg1068His Mutagenic primers were used to introduce the c.3516G>A nucleotide change in exon 22 of the ABCA1 sequence causing the p.R1068H mutation. Login to comment 78 ABCA1 p.Arg1068His ABCA1 p.Arg1068His Results 3.1. Computational analysis of p.R1068H Previous computational analysis of the p.R1068H mutation using PolyPhen suggested that the mutation was 'probably damaging` [11], however, further analysis of the mutation here using PANTHER and SIFT predicted the mutation to be 'damaging` and 'tolerated`, respectively. Login to comment 79 ABCA1 p.Arg1068His ABCA1 p.Arg1068His Results 3.1. Computational analysis of p.R1068H Previous computational analysis of the p.R1068H mutation using PolyPhen suggested that the mutation was 'probably damaging` [11], however, further analysis of the mutation here using PANTHER and SIFT predicted the mutation to be 'damaging` and 'tolerated`, respectively. Login to comment 109 ABCA1 p.Arg1068His Functional activity of the p.R1068H protein Cholesterol efflux assays in primary fibroblast cultures established from the TD subject showed a markedly reduced efflux compared to wildtype family members (0.9% versus 7.3%; Fig. 2A). Login to comment 110 ABCA1 p.Arg1068His ABCA1 p.Arg1068His Heterozygous carriers of the p.R1068H mutation displayed an intermediate level of cholesterol efflux (2.9%) which was significantly lower than wildtype. Login to comment 111 ABCA1 p.Arg1068His Heterozygous carriers of the p.R1068H mutation displayed an intermediate level of cholesterol efflux (2.9%) which was significantly lower than wildtype. Login to comment 113 ABCA1 p.Arg1068His ABCA1 protein levels in both unstimulated and stimulated cells appeared unaffected by the presence of the p.R1068H mutation (Fig. 2C and D). Login to comment 114 ABCA1 p.Arg1068His ABCA1 p.Arg1068His This was confirmed by quantification of triplicate Western blots that showed no significant difference in ABCA1 protein levels in p.R1068H heterozygotes versus wildtype fibroblasts under unstimulated (Fig. 2E) or stimulated (Fig. 2F) conditions. Login to comment 115 ABCA1 p.Arg1068His This was confirmed by quantification of triplicate Western blots that showed no significant difference in ABCA1 protein levels in p.R1068H heterozygotes versus wildtype fibroblasts under unstimulated (Fig. 2E) or stimulated (Fig. 2F) conditions. Login to comment 116 ABCA1 p.Arg1068His The presence of the p.R1068H mutation did not affect ABCA1 expression, as the levels of wildtype R1068 and the mutant H1068 protein were comparable (Fig. 3A). Login to comment 117 ABCA1 p.Arg1068His ABCA1 p.Arg1068His Cholesterol efflux assays showed that HEK293 cells expressing the p.R1068H vector had markedly reduced cholesterol efflux compared to cells Fig. 3. Login to comment 118 ABCA1 p.Arg1068His Cholesterol efflux assays showed that HEK293 cells expressing the p.R1068H vector had markedly reduced cholesterol efflux compared to cells Fig. 3. Login to comment 119 ABCA1 p.Arg1068His Cultured HEK293 cells were transiently transfected with a GFP-tagged ABCA1 cDNA expression vector for either wildtype or p.R1068H. Login to comment 120 ABCA1 p.Arg1068His Cultured HEK293 cells were transiently transfected with a GFP-tagged ABCA1 cDNA expression vector for either wildtype or p.R1068H. Login to comment 127 ABCA1 p.Arg1068His ABCA1 p.Arg1068His Indeed, there was no statistical difference in cholesterol efflux between nontransfected HEK293 cells and cells expressing the mutant p.R1068H protein (p = 0.163), again confirming that the p.R1068H mutation renders the ABCA1 protein completely dysfunctional. Login to comment 128 ABCA1 p.Arg1068His 3.3. Sub-cellular localisation of the ABCA1 protein in vitro Confocal microscopy was used to investigate the effect of the p.R1068H mutation on localisation of the GFP-tagged ABCA1 protein. Login to comment 130 ABCA1 p.Arg1068His Wildtype ABCA1 protein primarily localised to the extracellular membrane as expected while the p.R1068H ABCA1 protein localised to intracellular regions of the cells indicative of a defect in trafficking of the mutant protein (Fig. 4). Login to comment 136 ABCA1 p.Arg1068His The p.R1068H mutation studied here was deemed to be damaging by PolyPhen and PANTHER, yet tolerated by SIFT. Login to comment 137 ABCA1 p.Arg1068His ABCA1 p.Arg1068Cys ABCA1 p.Arg1068Trp Indeed, SIFT predicts any residue substitution at position 1068 to be tolerated except R1068W which does not align with the observation that p.R1068H and the p.R1068C mutation are associated with extremely reduced HDL levels [11,23]. Login to comment 139 ABCA1 p.Arg1068His For these reasons, we constructed a model of the dimeric NBDs of ABCA1 based on the crystal structure of the E. coli HlyB ABC transporter in order to visualise the p.R1068H mutation in a three-dimensional context. Login to comment 141 ABCA1 p.Arg1068His The three dimensional model indicates potential ionic interactions between R1068 and two adjacent carboxylic acid containing residues, D1092 and E1093, which would be disrupted by the p.R1068H mutation. Login to comment 143 ABCA1 p.Arg1068His The p.R1068H mutation does not appear to affect expression or stability of the ABCA1 protein as shown by Western blot analysis of both primary human fibroblasts and transfected HEK293 cells. Login to comment 148 ABCA1 p.Arg1068His Preliminary studies investigating the oligomerisation state of the p.R1068H mutant showed only limited formation of a tetramer band (>800 kDa) and no dimer (Supplementary Fig. 1). Login to comment 150 ABCA1 p.Arg1068His Furthermore, higher molecular weight species were apparent with p.R1068H suggesting a tendency to aggregate. Login to comment 151 ABCA1 p.Arg1068His This could potentially explain why a much lower amount of the p.R1068H protein was detected on native gels compared to wildtype, despite similar amounts of ABCA1 being detected under denaturing conditions (Supplementary Fig. 1). Login to comment 152 ABCA1 p.Arg1068His Based on our model (Fig. 1B), we propose that the R1068H mutation disrupts the structure of the ABCA1 monomer around the ATP-binding site altering the conformation of NBD-1 to disrupt dimerisation and subsequent tetramer formation. Login to comment 155 ABCA1 p.Arg1068His The virtual absence of HDL in the TD proband would indicate that the p.R1068H mutation was highly detrimental to ABCA1 function. Login to comment 156 ABCA1 p.Arg1068His ABCA1 p.Arg1068His Functional testing of the p.R1068H mutation in primary fibroblast cell lines established from p.R1068H family members confirmed this, with the mutant protein failing to mediate efflux to apoA-I even after stimulation. Login to comment 157 ABCA1 p.Arg1068His Cholesterol efflux assays were also performed in HEK293 cells with cDNA expression vectors for either wildtype or p.R1068H ABCA1. Login to comment 158 ABCA1 p.Arg1068His These assays also confirmed that the p.R1068H protein was completely dysfunctional. Login to comment 160 ABCA1 p.Arg1068His HEK293 cells were transiently transfected with GFP-tagged ABCA1 cDNA expression vectors for either wildtype or p.R1068H. Login to comment 163 ABCA1 p.Arg1068His To conclude, functional studies confirm that the p.R1068H mutation produces a dysfunctional ABCA1 protein due to defective trafficking. Login to comment 166 ABCA1 p.Arg1068His Acknowledgements We wish to thank the p.R1068H family members for their participation in this study. Login to comment