title: Homology modeling and functional testing of an ABCA1 mutation causing Tangier disease.
abstract: OBJECTIVE
  To investigate the impact of the p.R1068H mutation on the structure and function of the ATP-binding cassette A1 (ABCA1) protein.
  
  
  METHODS
  A homology model of the nucleotide binding domains of ABCA1 was constructed to identify the three-dimensional orientation of R1068. 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. Confocal microscopy was used to investigate the localisation of the wildtype and mutant p.R1068H protein in HEK293 cells.
  
  
  RESULTS
  Sequence alignments and modeling indicated residue R1068 to be located in an α-helix downstream of the Walker B motif in the first nucleotide binding domain (NBD-1), in a position to form ionic interactions with D1092 and E1093. 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. 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.
  
  
  CONCLUSIONS
  Homology modeling of the ABCA1 protein showed that the p.R1068H mutation would likely disrupt the conformation of NBD-1. 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.
authors.affiliation: Department of Biochemistry, University of Otago, Dunedin, New Zealand.
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Suetani
authors.forename: Brie
authors.initials: B
authors.lastname: Sorrenson
authors.forename: Joel D A
authors.initials: JD
authors.lastname: Tyndall
authors.forename: Michael J A
authors.initials: MJ
authors.lastname: Williams
authors.forename: Sally P A
authors.initials: SP
authors.lastname: McCormick
mesh_terms: D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D019295:Computational Biology / Q000379:methods; D005347:Fibroblasts / Q000378:metabolism; D057809:HEK293 Cells; D006579:Heterozygote; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013631:Tangier Disease / Q000235:genetics*
chemical_list: C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate