title: Structural and functional consequences of the Milano mutation (R173C) in human apolipoprotein A-I.
abstract: 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 type approximately R173K>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.
authors.affiliation: Gastroenterology/Nutrition/Hepatology Division, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4318, USA.
authors.forename: Eric T
authors.initials: ET
authors.lastname: Alexander
authors.forename: Masafumi
authors.initials: M
authors.lastname: Tanaka
authors.forename: Momoe
authors.initials: M
authors.lastname: Kono
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Saito
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Rader
authors.forename: Michael C
authors.initials: MC
authors.lastname: Phillips