Structure-function studies of apoA-I variants: site-directed mutagenesis and natural mutations.
Five mutants of apolipoprotein A-I (apoA-I), apoA-I(Delta63-73), apoA-I(Delta140-150), apoA-I(63-73@140-150), apoA-I(R149V), and apoA-I(P143A) were compared with human plasma apoA-I for their ability to promote cholesterol and phospholipid efflux from HepG2 cells. A significantly lower capacity to promote cholesterol and phospholipid efflux was observed with lipid-free apoA-I(Delta63-73), while mutations apoA-I(Delta140-150) and apoA-I(P143A) affected phospholipid efflux only. When added as apoA-I/palmitoyloleoyl phosphatidylcholine (POPC) complex, mutations apoA-I(63-73@140-150) and apoA-I(Delta140-150) affected cholesterol efflux. None of the mutations affected alpha-helicity of the lipid-free mutants or their self-association. Five natural mutations of apoA-I, apoA-I(A95D), apoA-I (Y100H), apoA-I(E110K), apoA-I(V156E), and apoA-I (H162Q) were studied for their ability to bind lipids and promote cholesterol efflux. None of the mutations affected lipid-binding properties, cholesterol efflux, or alpha-helicity of lipid-free mutants. Two mutations affected self-association of apoA-I: apoA-I(A95D) was more prone to self-association, while apoA-I(E100H) did not self-associate. The following conclusions could be made from the combined data: i) regions 210-243 and 63-100 are the lipid-binding sites of apoA-I and are also required for the efflux of lipids to lipid-free apoA-I, suggesting that initial lipidation of apoA-I is rate limiting in efflux; ii) in addition to the lipid-binding regions, the central region is important for cholesterol efflux to lipidated apoA-I, suggesting its possible involvement in interaction with cells.
Sviridov
Dmitri
D
Baker Medical Research Institute, P.O. Box 6492, St. Kilda Road Central, Melbourne, Victoria, 8008, Australia. dimitri.sviridov@baker.edu.au
Hoang
Anh
A
Huang
Wei
W
Sasaki
Jun
J
D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002784:Cholesterol / Q000378:metabolism; D002942:Circular Dichroism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010743:Phospholipids / Q000378:metabolism; D013329:Structure-Activity Relationship
D016632:Apolipoprotein A-I; D010743:Phospholipids; D002784:Cholesterol