In vivo studies of HDL assembly and metabolism using adenovirus-mediated transfer of ApoA-I mutants in ApoA-I-deficient mice.
We have used adenovirus-mediated gene transfer in apoA-I-deficient (A-I-/-) mice to probe the in vivo assembly and metabolism of HDL using apoA-I variants, focusing primarily on the role of the C-terminal 32 amino acids (helices 9-10). Lipid, lipoprotein, and apoA-I analyses showed that plasma levels of apoA-I and HDL of the mutants were 40-88% lower than that of wild type (WT) human apoA-I despite comparable levels of expression in the liver. WT apoA-I and mutant 1 (P165A, E172A) formed spherical particles with the size and density of HDL2 and HDL3. Mutant 2 (E234A, E235A, K238A, K239A) generated spherical particles with density between HDL2 and HDL3. Mutant 3 (L211V, L214V, L218V, L219V) and mutant 4 (L222K, F225K, F229K), which have substitutions of hydrophobic residues in the C-terminus, generated discoidal HDL particles indicating a defect in their conversion to mature spherical HDL. Significant amounts of mutant 4 and mutant 5 (truncated at residue 219) were found in the lipid poor fractions after ultracentrifugation of the plasma (18 and 35%, respectively, of total apoA-I). These findings suggest that hydrophobic residues in and/or between helices 9 and 10 are important for the maturation of HDL in vivo.
Reardon
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Department of Pathology, University of Chicago, 5841 S. Maryland Avenue, Chicago, Illinois 60637, USA. reardon@midway.uchicago.edu.
Kan
H Y
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Cabana
V
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Blachowicz
L
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Lukens
J R
JR
Wu
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Liadaki
K
K
Getz
G S
GS
Zannis
V I
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D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D016632:Apolipoprotein A-I / Q000097:blood / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D008076:Cholesterol, HDL / Q000097:blood; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D017353:Gene Deletion; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008055:Lipids / Q000097:blood; D008075:Lipoproteins, HDL / Q000097:blood / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009154:Mutation; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000378:metabolism
D016632:Apolipoprotein A-I; D008076:Cholesterol, HDL; D008055:Lipids; D008075:Lipoproteins, HDL; D012333:RNA, Messenger; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D004134:Dimyristoylphosphatidylcholine