The central helices of ApoA-I can promote ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux. Amino acid residues 220-231 of the wild-type ApoA-I are required for lipid efflux in vitro and high density lipoprotein formation in vivo.
We have mapped the domains of lipid-free apoA-I that promote cAMP-dependent and cAMP-independent cholesterol and phospholipid efflux. The cAMP-dependent lipid efflux in J774 mouse macrophages was decreased by approximately 80-92% by apoA-I[delta(185-243)], only by 15% by apoA-I[delta(1-41)] or apoA-I[delta(1-59)], and was restored to 75-80% of the wild-type apoA-I control value by double deletion mutants apoA-I[delta(1-41)delta(185-243)] and apoA-I[delta(1-59)delta(185-243)]. Similar results were obtained in HEK293 cells transfected with an ATP-binding cassette transporter A1 (ABCA1) expression plasmid. The double deletion mutant of apoA-I had reduced thermal and chemical stability compared with wild-type apoA-I. Sequential carboxyl-terminal deletions showed that cAMP-dependent cholesterol efflux was diminished in all the mutants tested, except the apoA-I[delta(232-243)] which had normal cholesterol efflux. In cAMP-untreated or in mock-transfected cells, cholesterol efflux was not affected by the amino-terminal deletions, but decreased by 30-40% and 50-65% by the carboxyl-terminal and double deletions, respectively. After adenovirus-mediated gene transfer in apoA-I-deficient mice, wild-type apoA-I and apoA-I[delta(1-41)] formed spherical high density lipoprotein (HDL) particles, whereas apoA-I[delta(1-41)delta(185-243)] formed discoidal HDL. The findings suggest that although the central helices of apoA-I alone can promote ABCA1-mediated lipid efflux, residues 220-231 are necessary to allow functional interactions between the full-length apoA-I and ABCA1 that are required for lipid efflux and HDL biogenesis.
Chroni
Angeliki
A
Section of Molecular Genetics, Whitaker Cardiovascular Institute, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Liu
Tong
T
Gorshkova
Irina
I
Kan
Horng-Yuan
HY
Uehara
Yoshinari
Y
Von Eckardstein
Arnold
A
Zannis
Vassilis I
VI
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000378:metabolism*; D016367:Baculoviridae / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000378:metabolism; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008024:Ligands; D050356:Lipid Metabolism; D008099:Liver / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D009154:Mutation; D010743:Phospholipids / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D014280:Triglycerides / Q000378:metabolism
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D018076:DNA, Complementary; D008024:Ligands; D010743:Phospholipids; D020033:Protein Isoforms; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014280:Triglycerides; D000255:Adenosine Triphosphate; D002784:Cholesterol; D000242:Cyclic AMP; D019791:Guanidine