No abstract

Although the N-terminal region in human apolipoprotein (apo) A-I is thought to stabilize the lipid-free structure of the protein, its role in lipid binding is unknown. Using synthetic fragment peptides, we examined the lipid-binding properties of the first 43 residues (1-43) of apoA-I in comparison with residues 44-65 and 220-241, which have strong lipid affinity in the molecule. Circular dichroism measurements demonstrated that peptides corresponding to each segment have potential propensity to form alpha-helical structure in trifluoroethanol. Spectroscopic and thermodynamic measurements revealed that apoA-I (1-43) peptide has the strong ability to bind to lipid vesicles and to form alpha-helical structure comparable to apoA-I (220-241) peptide. Substitution of Tyr-18 located at the center of the most hydrophobic region in residues 1-43 with a helix-breaking proline resulted in the impaired lipid binding, indicating that the alpha-helical structure in this region is required to trigger the lipid binding. In contrast, apoA-I (44-65) peptide exhibited a lower propensity to form alpha-helical structure upon binding to lipid, and apoA-I (44-65/S55P) peptide exhibited diminished, but not completely impaired, lipid binding, suggesting that the central region of residues 44-65 is not pivotally involved in the formation of the alpha-helical structure and lipid binding. These results indicate that the most N-terminal region of apoA-I molecule, residues 1-43, contributes to the lipid interaction of apoA-I through the hydrophobic helical residues.

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...