Substitutions of Glutamate 110 and 111 in the Middle Helix 4 of Human Apolipoprotein A-I (apoA-I) by Alanine Affect the Structure and in Vitro Functions of apoA-I and Induce Severe Hypertriglyceridemia in apoA-I-Deficient Mice

Abstract

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.

Structure and Stability of Apolipoprotein A-I in Solution and in Discoidal High-Density Lipoprotein Probed by Double Charge Ablation and Deletion Mutation

“…In agreement with previous observations (5,12,29,30), the physicochemical characteristics of WT apoA-I proteins expressed in different expression systems appeared to be slightly different (Tables 1-3). This difference may result from a difference in the propeptide at the N-terminus of the expressed proteins.…”

“…This agrees with data showing that the segment of residues 63-73, which overlaps with the 61-78 region, is essential for apoA-I function, such as phospholipid binding (10). Remarkably, of all the mutations studied in this work, the Δ(61-78) and E110A/ E111A lead to the least compact tertiary packing and to the most significant increase in disordered conformation in lipid-free apoA-I ( Figure 1B (12,32). Similarly in human plasma, one of the apoE isoforms, apoE4 that has the less organized and more flexible structure, is preferably associated with larger very low density lipoprotein, in contrast to apoE2 and apoE3 isoforms that are more structurally organized and preferably bound to smaller HDL particles (49).…”

“…However, even with this considerable amount of accumulated data, the structural details of the lipid-free and lipid-bound conformation are not fully defined and experimental results leading to definition of residues involved in stabilizing the protein lipid-free and lipid-bound conformations are not sufficient. So far, there have been no reported experimental data demonstrating that electrostatic interactions between specific basic and acidic residues located in two anti-parallel apoA-I molecules on the edge of rHDL are essential for the stabilization of the discs, as predicted by the double belt model (27).Studies of bioengineered mutant apoA-I forms remain efficient to obtain information that leads to detailed understanding of the conformation and structure-function relationships of apoA-I (5,(7)(8)(9)(10)(11)(12)(13)(14)(15)24,29,30). Our earlier study (29) using mutation to probe the conformation and stability of the C-terminus of apoA-I suggested the presence of stable helical structure in the region between residues 165 and 243 and a close proximity of the N-and C-termini in the lipid-free protein.…”

“…Studies of bioengineered mutant apoA-I forms remain efficient to obtain information that leads to detailed understanding of the conformation and structure-function relationships of apoA-I (5,(7)(8)(9)(10)(11)(12)(13)(14)(15)24,29,30). Our earlier study (29) using mutation to probe the conformation and stability of the C-terminus of apoA-I suggested the presence of stable helical structure in the region between residues 165 and 243 and a close proximity of the N-and C-termini in the lipid-free protein.…”

“…The D102A/ D103A and R160V/H162A mutations have been shown to affect functional interactions of rHDL and SR-B1 and thereby affect cholesterol efflux (7). The D102A/D103A, E110A/ E111A, R160V/H162A, and Δ(61-78) mutations affect the LCAT-activation ability of apoA-I (12,31,32). Moreover, the E110A/E111A and Δ(61-78) mutations have been recently shown to affect lipid homeostasis and induce severe hypertriglyceridemia when expressed in A-Ideficient mice (12,32).…”