Deletion of Single Amino Acid E235 Affects the Structure and Lipid Interaction of Human Apolipoprotein A-I C-Terminal Peptides

Abstract

Human apolipoprotein (apo) A-I, a 243 amino acid protein, is the major protein component of high-density lipoprotein (HDL). ApoA-I plays an important role in lipoprotein metabolism, reverse cholesterol transport pathway, and protection against the development of atherosclerosis.1) In reverse cholesterol transport pathway, lipid-free or lipid-poor apoA-I molecules remove cholesterol from peripheral cells and transport it in the form of HDL back to the liver for excretion.2) Lipid-free apoA-I is folded into two domains, comprising an N-terminal domain forming a four-helix bundle and a discrete C-terminal domain, which is predominantly involved in lipid-protein interactions. These interactions are critical for HDL generation, including initial lipid binding and cholesterol efflux from plasma membrane.3) ApoA-I Nichinan, a naturally occurring human apoA-I variant with a deletion of E235 located in the C-terminus, is associated with low HDL cholesterolemia probably due to the impaired lipid binding and cellular interactions. 4,5) Computational analysis of the amino acid sequence of human apoA-I reveals that residues 44-243, encoded by exon 4, is composed of 11-or 22-amino acid tandem repeats, which can form amphipathic a-helices mostly punctuated by proline residues. 6) Studies of synthetic peptides corresponding to each of 22-residue amphipathic segments of apoA-I have shown that the last repeated helix (residues 220-241) has greatest lipid affinity. 7,8) In addition, our previous studies using deletion mutants of apoA-I demonstrated that deletion in the last helix leads to significant decreases in lipid-binding affinity compared to intact apoA-I. 9,10) A lack of E235 in apoA-I Nichinan variant is considered to bring about at least two possible structural influences such as 1) a negative charge ablation and 2) disturbance of amino acid distribution on the helix cross-section (i.e., loss of amphipathicity) (Fig. 1). Previously, Panagotopulos et al. have revealed that the E235K mutation in full-length apoA-I does not affect lipid microsolubilization and cellular cholesterol efflux, 11) suggesting the possibility that amphipathic nature of the C-terminal region in apoA-I Nichinan is influenced by the disturbance of amino acid distribution caused by E235 deletion. To examine the influences of E235 deletion on the a-helical structure and lipid interaction of the C-terminal region in apoA-I, we used a series of variant peptides corresponding to residues 220-241 of the apoA-I molecule in addition of DE235: E235A in which E235 was substituted with nonpolar alanine residue to examine the influence of a negative charge ablation, and L230P in which L230 was substituted with proline residue to disrupt the putative a-helical structure. We previously demonstrated that proline insertion instead of leucine residue at position 230 disrupts the C-ter- The C-terminal domain of apolipoprotein (apo) A-I plays an important role in lipid binding. ApoA-I Nichinan, a naturally occurring human apoA-I variant with a deletion of E235 located i...

Disruption of the C-terminal helix by single amino acid deletion is directly responsible for impaired cholesterol efflux ability of apolipoprotein A-I Nichinan

“…The apoA-I preparations were at least 95% pure as assessed by SDS-PAGE. The C-terminal apoA-I 209-241 and 209-241/ ⌬ E235 peptides were synthesized using Fmoc chemistry as described ( 30,31 ). The N and C termini were capped with an acetyl group and an amide group, respectively.…”

“…It is thought lamellar vesicles (MLVs) and peptides (typically 34 g/ml DMPC and 2-20 g/ml peptides) were used to avoid the aggregation of DMPC/peptide complexes during experiments. The decrease in turbidity was monitored by the right-angle light scattering intensity using a Hitachi F-4500 spectrophotometer with both excitation and emission wavelengths set at 600 nm ( 31,39 ).…”

“…Indeed, comparison of the ␣ -helix contents between synthetic C-terminal peptides, apoA-I 209-241 and 209-241/ ⌬ E235 in the lipid-free or SUVbound states ( Table 3 ) clearly demonstrates that the E235 deletion markedly reduces the ability of the C-terminal region of apoA-I to form ␣ -helix both in the absence and presence of lipids. Because the helical propensity of apolipoproteins is critical for their lipid-binding affi nities ( 19,31,50,54 ), such a weak propensity to form ␣ -helix caused by the E235 deletion results in the less effective ability of the Nichinan protein and its C-terminal peptide to solubilize DMPC vesicles (Figs. 4 and 6).…”