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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:210.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

A Single Amino Acid Deletion in the Carboxy Terminal of Apolipoprotein A-I Impairs Lipid Binding and Cellular Interaction

Wei Huang; Jun Sasaki; Akira Matsunaga; Hua Han; Wei Li; Takafumi Koga; Mari Kugi; Setsuko Ando; Kikuo Arakawa

From the Department of Internal Medicine (W.H., J.S., A.M., H.H., W.L., T.K., M.K., K.A.), School of Medicine; and the Department of Chemistry (S.A.), Fukuoka University, Fukuoka, Japan.

Correspondence to Jun Sasaki, MD, Department of Internal Medicine, School of Medicine, Fukuoka University, 45-1, 7-chome Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. E-mail mm034515{at}msat.fukuoka-u.ac.jp

Abstract—The carboxy-terminal region of apolipoprotein (apo) A-I has been shown by mutagenesis or synthetic peptides to play an important role in lipid binding. However, the precise functional domain of the C-terminal remains to be defined. In this study, apoA-I Nichinan, a naturally occurring human apoA-I variant with a deletion of glutamic acid 235, was expressed in Escherichia coli to examine the effect of this mutation on the functional domain of apoA-I for lipid binding and related consequences. A dimyristoyl phosphatidylcholine binding study with recombinant (r-) proapoA-I Nichinan showed a significantly slow initial rate of lipid binding. On preincubation with human plasma lipoprotein fractions (d<1.225 g/mL) at 37°C for 1 hour, ¹²⁵I-labeled normal r-proapoA-I was chromatographed as a single peak at the high density lipoprotein (HDL) fraction, whereas ¹²⁵I-labeled r-proapoA-I Nichinan was chromatographed into the HDL fraction as well as the free r-proapoA-I fraction (23% of radioactivity). Circular dichroism measurements showed that the -helix content of lipid-bound r-proapoA-I Nichinan was reduced, being 62% (versus 73%) of normal r-proapoA-I. Nondenaturing gradient gel electrophoresis of reconstituted HDL particles assembled with r-proapoA-I Nichinan and normal r-proapoA-I showed similar particle size. To study cholesterol efflux, human skin fibroblasts were labeled with [³H]cholesterol, followed by incubation with either lipid-free r-proapoA-I or DMPC/r-proapoA-I complex. Fractional cholesterol efflux from [³H]cholesterol-labeled fibroblasts to lipid-free r-proapoA-I Nichinan or DMPC/r-proapoA-I Nichinan complexes was significantly reduced relative to that of normal r-proapoA-I or DMPC/r-proapoA-I during the 6-hour incubation. Binding assays of human skin fibroblasts by lipid-free r-proapoA-I showed that r-proapoA-I Nichinan was 32% less bound to fibroblasts than was normal r-proapoA-I. Our data demonstrate that the deletion of glutamic acid 235 at the C-terminus substantially reduces the lipid-binding properties of r-proapoA-I Nichinan, which may cause a reduction in its capacity to interact with plasma membranes as well as to promote cholesterol efflux from cultured fibroblasts.

Key Words: apo A-I variants • lipid binding • cellular binding • secondary structure

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