Hypertriglyceridemia is a common pathological condition in humans of mostly unknown etiology. Here we report induction of dyslipidemia characterized by severe hypertriglyceridemia as a result of point mutations in human apolipoprotein A-I (apoA-I). Adenovirus-mediated gene transfer in apoA-I-deficient (apoA-I(-)(/)(-)) mice showed that mice expressing an apoA-I[E110A/E111A] mutant had comparable hepatic mRNA levels with WT controls but greatly increased plasma triglyceride and elevated plasma cholesterol levels. In addition, they had decreased apoE and apoCII levels and increased apoB48 levels in very low-density lipoprotein (VLDL)/intermediate-density lipoprotein (IDL). Fast protein liquid chromatography (FPLC) analysis of plasma showed that most of cholesterol and approximately 15% of the mutant apoA-I were distributed in the VLDL and IDL regions and all the triglycerides in the VLDL region. Hypertriglyceridemia was corrected by coinfection of mice with recombinant adenoviruses expressing the mutant apoA-I and human lipoprotein lipase. Physicochemical studies indicated that the apoA-I mutation decreased the alpha-helical content, the stability, and the unfolding cooperativity of both lipid-free and lipid-bound apoA-I. In vitro functional analyses showed that reconstituted HDL (rHDL) particles containing the mutant apoA-I had 53% of scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux capacity and 37% capacity to activate lecithin:cholesterol acyltransferase (LCAT) as compared to the WT control. The mutant lipid-free apoA-I had normal capacity to promote ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. The findings indicate that subtle structural alterations in apoA-I may alter the stability and functions of apoA-I and high-density lipoprotein (HDL) and may cause hypertriglyceridemia.

To probe the secondary structure of the C-terminus (residues 165-243) of lipid-free human apolipoprotein A-I (apoA-I) and its role in protein stability, recombinant wild-type and seven site-specific mutants have been produced in C127 cells, purified, and studied by circular dichroism and fluorescence spectroscopy. A double substitution (G185P, G186P) increases the protein stability without altering the secondary structure, suggesting that G185 and G186 are located in a loop/disordered region. A triple substitution (L222K, F225K, F229K) leads to a small increase in the alpha-helical content and stability, indicating that L222, F225, and F229 are not involved in stabilizing hydrophobic core contacts. The C-terminal truncation Delta(209-243) does not change the alpha-helical content but reduces the protein stability. Truncation of a larger segment, Delta(185-243), does not affect the secondary structure or stability. In contrast, an intermediate truncation, Delta(198-243), leads to a significant reduction in the alpha-helical content, stability, and unfolding cooperativity. The internal 11-mer deletion Delta(187-197) has no significant effect on the conformation or stability, whereas another internal 11-mer deletion, Delta(165-175), dramatically disrupts and destabilizes the protein conformation, suggesting that the presence of residues 165-175 is crucial for proper apoA-I folding. Overall, the findings suggest the presence of stable helical structure in the C-terminal region 165-243 of lipid-free apoA-I and the involvement of segment 209-243 in stabilizing interactions in the molecule. The effect of the substitution (G185P, G186P) on the exposure of tryptophans located in the N-terminal half suggests an apoA-I tertiary conformation with the C-terminus located close to the N-terminus.

To probe the structure and stability of the central region of lipid-free apolipoprotein (apo) A-I (residues 123-165), we studied the effects of four mutations made in this region on the conformation, stability, dimyristoylphosphatidylcholine (DMPC) binding kinetics, and size of discoidal reconstituted high-density lipoprotein (rHDL) particles. The apoA-I deletion delta(144-165) leads to a red shift in the wavelength of maximum fluorescence and a reduction in the alpha-helical content, the stability, the initial rate of association with DMPC liposomes, and the size of the discoidal particles. The data are consistent with the helical structure of residues 144-165, and the deletion appears to perturb the tertiary organization of the N-terminal half of apoA-I. In contrast, the deletion of the adjacent region, delta(136-143), leads to stabilization without altering the number of residues in the helical conformation or the initial rate of association with DMPC liposomes. The quadruple substitution E125K/E128K/K133E/E139K leads to approximately 17 additional residues in the helical conformation and an increase in the stability, the initial rate of association with DMPC liposomes, and the size of the rHDL particles. The findings are consistent with the disordered structure of the segment of residues 123-142, which becomes helical as a result of the quadruple mutation or upon lipid binding. The naturally occurring mutation L141R (also associated with coronary heart disease) that is located in this segment does not change the protein conformation but leads to a reduced stability and a decreased rate of association with DMPC liposomes that may relate to the observed altered functions of this mutant.

To identify residues and segments in the central region of apolipoprotein A-I (apoA-I) that are important for the protein structure and stability, we studied the effects of four double charge ablations, D102A/D103A, E110A/E111A, R116V/K118A, and R160V/H162A, and two deletion mutations, Δ(61-78) and Δ(121-142), on the conformation and stability of apoA-I in the lipid-free state and in reconstituted discoidal phospholipid:cholesterol:apoA-I particles (rHDL). The findings suggest that D102/D103 and E110/E111 located in helix 4, and segment(s) between residues 61 and 78 are involved in maintenance of the conformation and stability of apoA-I in both the lipid-free state and in rHDL. R116/K118 located in helix 4 are essential for the conformation and stabilization of apoA-I in rHDL, but not vital for the lipid-free state of the protein. The R160V/H162A substitutions in helix 6 lead to a less compact tertiary structure of lipid-free apoA-I without notable effects on the lipid-free or lipid-bound secondary conformation suggesting involvement of R160/H162 in important inter-helical interactions. The results on the Δ(121-142) mutant, together with our earlier findings, suggest disordered structure of a major segment between residues 121 and 143, likely including residues 131-143, in lipid-free apoA-I. Our findings provide the first experimental evidence for stabilization of rHDL by electrostatic inter-helical interactions, in agreement with the double belt model. The effects of alterations in the conformation and stability of the apoA-I mutants on in vitro and in vivo functions of apoA-I and lipid homeostasis are discussed.Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoprotein (HDL) that plays a key role in the biogenesis and atheroprotective functions of HDL and in reverse cholesterol transport (1-3). Lipid-free apoA-I secreted by cells can interact functionally with ATP binding cassette transporter 1 (ABCA1) to promote efflux of cholesterol and phospholipids from cells. This process leads to the lipidation of apoA-I and formation of discoidal HDL (4,5). ApoA-I in discoidal HDL activates the enzyme lecithin:cholesterol acyltransferase (LCAT) converting cholesterol to cholesterol ester and thereby promoting the maturation of HDL particles from discoidal to spherical. ApoA-I bound to discoidal and spherical HDL also interacts functionally with scavenger receptor class B type I (SR-BI) (4, 6,7). On binding HDL, SR-BI mediates selective uptake of cholesterol esters and other lipids χ This work was supported by PO1HL 26335 and grant HL 48739 from the National Institute of Heath *To whom correspondence should be addressed at Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, W-322, Boston, MA 02118. Fax: (617) from HDL by cells and net efflux of excess cholesterol (4,6). In the course of apoA-I metabolism, the protein function is modulated by alterations in its structure and stability (5,(8)(9)(10)(11)(12)(13)(14)(15). Therefore, detail...

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