The N-terminal globular domain and the first class A amphipathic helix of apolipoprotein A-I are important for lecithin:cholesterol acyltransferase activation and the maturation of high density lipoprotein in vivo.
To investigate the role of the N terminus of apolipoprotein A-I (apoA-I) in the maturation of high density lipoproteins (HDL), two N-terminal mutants with deletions of residues 1-43 and 1-65 (referred to as Delta 1-43 and Delta 1-65 apoA-I) were studied. In vitro, these deletions had little effect on cellular cholesterol efflux from macrophages but LCAT activation was reduced by 50 and 70% for the Delta 1-43 and Delta 1-65 apoA-I mutants, respectively, relative to wild-type (Wt) apoA-I. To further define the role of the N terminus of apoA-I in HDL maturation, we constructed recombinant adenoviruses containing Wt apoA-I and two similar mutants with deletions of residues 7-43 and 7-65 (referred to as Delta 7-43 and Delta 7-65 apoA-I, respectively). Residues 1-6 were not removed in these mutants to allow proper cleavage of the pro-sequence in vivo. Following injection of these adenoviruses into apoA-I-deficient mice, plasma concentrations of both Delta 7-43 and Delta 7-65 apoA-I were reduced 4-fold relative to Wt apoA-I. The N-terminal deletion mutants, in particular Delta 7-65 apoA-I, were associated with greater proportions of pre beta-HDL and accumulated fewer HDL cholesteryl esters relative to Wt apoA-I. Wt and Delta 7-43 apoA-I formed predominantly alpha-migrating and spherical HDL, whereas Delta 7-65 apoA-I formed only pre beta-HDL of discoidal morphology. This demonstrates that deletion of the first class A amphipathic alpha-helix has a profound additive effect in vivo over the deletion of the globular domain alone (amino acids 1-43) indicating its important role in the production of mature alpha-migrating HDL. In summary, the combined in vitro and in vivo studies demonstrate a role for the N terminus of apoA-I in lecithin:cholesterol acyltransferase activation and the requirement of the first class A amphipathic alpha-helix for the maturation of HDL in vivo.
Scott
B R
BR
Lipoprotein and Atherosclerosis Research Group, University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4W7, Canada.
McManus
D C
DC
Franklin
V
V
McKenzie
A G
AG
Neville
T
T
Sparks
D L
DL
Marcel
Y L
YL
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D004789:Enzyme Activation; D018014:Gene Transfer Techniques; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism* / Q000648:ultrastructure; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D010743:Phospholipids; D002784:Cholesterol; D007862:Phosphatidylcholine-Sterol O-Acyltransferase