Structure And Function Of Plasma Lipoproteins And Apolip

Heart, Lung, And Blood Institute

Investigators

Abstract

ApoA-I: To evaluate the physiological sites of degradation of apoA-I and apoA-II in vivo, non-degradable 125I Tyramine Cellobiose (TC) was covalently attached to isolated apoA-I and apoA-II and the individual labeled apolipoproteins were reassociated with mouse HDL followed by injection into the saphenous vein of either control C57BL mice, hypoalphalipoproteinemic LCAT-ko, or hyperalphalipoproteinemic ABCA1-transgenic mice. The catabolism of 125I-TC-apoA-I-HDL and 125I-TC-apoA-II-HDL was compared to that of apoA-I-HDL and apoA-II-HDL labeled with 131I by the traditional iodine monochloride method. In C57BL controls, the plasma decay curves were similar for 125I-TC-apo-A-I and 131I-apoA-I, as well as 125I-TC-apoA-II and 131I-apoA-II. More than 85% of 125I-TC-apoA-I and 125I-TC-apoA-II cleared from the plasma compartment was recovered either in the liver or kidneys. The relative hepatic and renal accumulations of 125I-TC-apoA-I were 60.4+/-2.7% and 37.9+/-2.6%, and 125I-TC-apoA-II were 57.8+/-2.7% and 40.8+/-0.8%, respectively. The FCR of apoA-I and apoA-II were markedly increased in the LCAT-ko when compared to controls. In contrast to controls, in LCAT-ko mice 53.8+/-0.8% and 64.9+/-0.6% of 125I-TC-apoA-I and those of 125I-TC-apoA-II were cleared by the kidney with 46+/-0.8% and 35+/-0.6% removed by the liver. Conversely, the catabolism of apoA-I and apoA-II was delayed in ABCA1-Tg mice, however, the relative hepatic and renal recovery of 125I-TC-apoA-I and 125-TC-apoA-II were similar (58.9+/-0.4% and 32.2+/-1.6%) to controls. These findings demonstrate that the catabolism of 125I-TC-apoAI/apoAII HDL is similar to that of 131I-apoAI/apoAII HDL and that the use of individually TC labeled apoA-I and apoA-II permits the specific sites and percentage of degradation to be ascertained for the kidney and liver. The proportion of renal degradation of both apoA-I and apoA-II is increased in hypoalphalipoproteinemias due to LCAT deficiency. ABCA1: Study of Cholesterol & Phospholipid Acceptors ABCA1, the defective lipid transport protein in Tangier disease, mediates the efflux of lipids to apolipoprotein acceptors. In order to characterize the protein structural requirement of lipid acceptors, synthetic peptides were tested for lipid efflux on ABCA1 transfected HELA cells (ABCA1 cells) and on control cells lacking ABCA1. The 37pA peptide (40 ug/ml), which contains two class A amphipathic helices (DWLKAFYDKVAEKLKEAF) linked by proline, but not the 18A peptide, which contains a single helix, caused a 650% increase in cholesterol efflux from the ABCA1 cell line compared to the BSA blank control. The 37pA peptide, unlike apoA-I, also stimulated significant cholesterol efflux above the blank for the control cells (37pA:265%; apoA-I:20%). A stereoisomer of the 37pA peptide synthesized with D-amino acids was as effective as the L-amino acid form in effluxing cholesterol and phospholipid from both cell lines. Glyburide (60 uM), an ABCA1 inhibitor, blocked by 60% cholesterol efflux by apoA-I from ABCA1 cells but only partially (30%) blocked efflux by the 37pA peptide on ABCA1 cells and had no effect on 37pA mediated efflux from the control cells. Fixation of ABCA1 cells with paraformaldehyde completely blocked cholesterol efflux by apoA-I, but only partially inhibited (25%) efflux by the 37pA efflux. Fixation of the control cells had a minimal effect on 37pA peptide. Unlike apoA-I, the 37pA peptides were also capable of stimulating increased (300%) cholesterol efflux above the blank from Tangier disease fibroblasts. By cross-competition experiments, the 37pA peptides bound to the same binding site on ABCA1 cells as apoA-I and apoA-II, but also bound to additional sites not competed by apoA-I and apoA-II. In summary: (1) synthetic peptides were shown to be capable of mediating lipid efflux by both an ABCA1 dependent and independent pathway (2) lipid efflux by the peptides was not based on a stereoselective interaction of the peptides with ABCA1, but was dependent upon the presence of amphipathic helices, which may promote lipid efflux by acting as protein detergents (3) these results provide new insights into the mechanism of action of ABCA1 and may be valuable in the design of therapeutic peptides for promoting reverse cholesterol transport.