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Journal of Lipid Research, Vol. 50, 1409-1419, July 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Structural and functional consequences of the Milano mutation (R173C) in human apolipoprotein A-I

Eric T. Alexander^*, Masafumi Tanaka, Momoe Kono, Hiroyuki Saito, Daniel J. Rader and Michael C. Phillips^1,*

^* Gastroenterology/Nutrition/Hepatology Division, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4318
Children's Hospital of Philadelphia, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4318
Biophysical Chemistry Department, Kobe Pharmaceutical University, Kobe 658-8558, Japan

This work was supported by American Heart Association Grant 0625372U, National Institutes of Health Grant HL-22633, the Takeda Science Foundation, and the Suzuken Memorial Foundation.

Published, JLR Papers in Press, March 24, 2009.

¹ To whom correspondence should be addressed. e-mail: phillipsmi{at}email.chop.edu

Carriers of the apolipoprotein A-I_Milano (apoA-I_M) variant, R173C, have reduced levels of plasma HDL but no increase in cardiovascular disease. Despite intensive study, it is not clear whether the removal of the arginine or the introduction of the cysteine is responsible for this altered functionality. We investigated this question using two engineered variations of the apoA-I_M mutation: R173S apoA-I, similar to apoA-I_M but incapable of forming a disulfide bond, and R173K apoA-I, a conservative mutation. Characterization of the lipid-free proteins showed that the order of stability was wild typeR173K>R173S>R173C. Compared with wild-type apoA-I, apoA-I_M had a lower affinity for lipids, while R173S apoA-I displayed intermediate affinity. The in vivo effects of the apoA-I variants were measured by injecting apoA-I-expressing adeno-associated virus into apoA-I-null mice. Mice that expressed the R173S variant again showed an intermediate phenotype. Thus, both the loss of the arginine and its replacement by a cysteine contribute to the altered properties of apoA-I_M. The arginine is potentially involved in an intrahelical salt bridge with E169 that is disrupted by the loss of the positively charged arginine and repelled by the cysteine, destabilizing the helix bundle domain in the apoA-I molecule and modifying its lipid binding characteristics.

Supplementary key words high density lipoprotein • cholesterol • lipoprotein • protein structure

Abbreviations: AAV, adeno-associated virus; apo, apolipoprotein; ANS, 8-anilino-1-naphthalensulfonic acid; CD, circular dichroism; DMPC, dimyristoylphosphatidylcholine; GdnHCl, guanidine hydrochloride; ITC, isothermal titration calorimetry; PC, phosphatidylcholine; SUV, small unilamellar vesicles; Trx, thioredoxin; WT, wild type

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