Abstract |
Apolipoprotein A-I is a key protein for the biogenesis of High Density Lipoproteins and
protects from atherosclerosis. Mutations in apoA-I gene are associated with low HDL
levels and predisposition to atherosclerosis. The purpose of the present thesis was to
explore the structure-function relationship in human apoA-I and the role of specific
amino acid residues in HDL biogenesis and maturation. The thesis is organized in two
parts.
Part I: Alteration of Negatively Charged Residues in the 89 to 99 Domain of
ApoA-I Affects Lipid Homeostasis and the Maturation of HDL (J Lipid Res. 2011
Jul;52(7):1363-72). Adenovirus-mediated gene transfer in apoA-I-/- mice showed that an
apoA-I[D89A/E91A/E92A] mutant increased plasma cholesterol and caused severe
hypertriglyceridemia. HDL levels were reduced and approximately 40% of the apoA-I
was distributed in VLDL/IDL. The HDL consisted of mostly spherical and few discoidal
particles and contained preβ1 and α4-HDL subpopulations. The mutant protein had
increased affinity for triglyceride-rich emulsions. The lipid, lipoprotein and HDL profiles
of the apoA-I[K94A/K96A] mutant were similar, but not identical, to those of wild type
apoA-I. Co-expression of apoA-I[D89A/E91A/E92A] and human lipoprotein lipase
abolished hypertriglyceridemia, restored in part the α1,2,3,4 HDL subpopulations,
redistributed apoA-I in the HDL2/HDL3 regions, but did not prevent the formation of
discoidal HDL particles. We conclude that residues D89, E91 and E92 of apoA-I are
important for plasma cholesterol and triglyceride homeostasis as well as for the
maturation of HDL. The present and two previous studies raise the possibility that
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mutations in apoA-I in the general population may alter the functions of apoA-I and HDL
and contribute to hypertriglyceridemia.
Part II: Contribution of the residues 218 to 226 of apoA-I in the biogenesis of
HDL. Adenovirus mediated gene transfer of an apoA-I [L218A/L219A/V221A/L222A]
mutant in apoA-I-/- mice resulted in decreased plasma cholesterol and apoA-I levels to
approximately 10 % as compared to WT control and generated preβ and α4-HDL
particles. The HDL cholesterol peak of the mutant protein was greatly diminished. When
expressed in double deficient mice for apoA-I and apoE the apoAI[
L218A/L219A/V221A/L222A] mutant failed to form HDL particles as determined by
2D gel electrophoresis and electron microscopy. The apoA-I[E223A/K226A] mutant had
similar plasma apoA-I levels and similar but not identical lipid and lipoprotein profiles
with WT apoA-I. Overall the findings suggest that crucial changes in the C-terminal 218-
222 hydrophobic residues of apoA-I impair seriously the functional interactions of apoAI
with ABCA1 and/or LCAT and inhibit biogenesis of HDL.
In the context of this thesis we also investigated, in collaboration with other
groups, the role of apoA-I C-terminus in endothelial transcytosis of HDL (J Biol Chem.
2011 Mar 11;286(10):7744-54), the bactericidal activity of apoA-I against Yersinia
enterocolitica serotypre O:3 (manuscript in preparation) and apoA-I mutations in
patients with increased risk of ischaemic heart disease and total mortality in the
population of Copenhagen (J Intern Med. 2011 Mar 28. doi: 10.1111 in press)
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