Tracey A-M. Neville

EFFECT OF THE SURFACE LIPID COMPOSITION OF RECONSTITUTED LPA-I ON APOLIPOPROTEIN A-I STRUCTURE AND LECITHIN:CHOLESTEROL ACYLTRANSFERASE ACTIVITY

Characterization of the factors that regulate plasma cholesterol esterification shows that the increased activity of lecithin:cholesterol acyltransferase (LCAT) in the plasma of hyperlipidemic subjects is due to enhanced interactions with a preferred substrate. The details of how the physical properties of high density lipoproteins (HDL) may affect their ability to stimulate cholesterol esterification by LCAT have been investigated in homogeneous reconstituted HDL particles containing two molecules of apolipoprotein (apo) A-I (Lp2A-I) and palmitoyl-oleoyl phosphatidylcholine (POPC). Increasing the POPC or sphingomyelin (SPH) content in an Lp2A-I complex increases particle size and stability but decreases the negative surface charge of apoA-I. Increasing Lp2A-I POPC or SPH content also significantly inhibits cholesterol esterification by LCAT. Increase in the maximum rate of CE production (Vmax) by LCAT is directly related to an increased negative charge on the different Lp2A-I particles and to a reduced amount and stability of amphipathic alpha-helices in apoA-I. In contrast, increasing the Lp2A-I complex negative charge directly by addition of a charged lipid, phosphatidylinositol (PI), has minimal effect on apoA-I conformation and LCAT activation. While variations in Lp2A-I PI content have little effect on the interfacial binding of LCAT, increasing POPC content appears to directly increase the binding affinity of LCAT for the different Lp2A-I particles. These results show that LCAT is stimulated by an apoA-I conformation-dependent increase in negative charge but is less sensitive to electrostatic changes in the lipid interface of discoidal Lp2A-I. The activation of LCAT appears to be dependent on the exposure of both central (residues 98-132) and N-terminal (residues 2-8) domains in apoA-I. A strong relationship between the immunoreactivity of two specific mAbs, 4H1 and A11, and LCAT reactivity suggests that the N-terminus of apoA-I may interact with a central domain in a manner that may regulate the accessibility of LCAT to the edge of the disc. This indicates that the conformation and charge of apoA-I are sensitive to the surface-lipid composition of HDL particles and play a central role in regulating LCAT activation. Since alterations in the surface lipid composition of HDL particles from hyperlipidemic subjects also modify the charge and structure of these particles, this may stimulate the rates of cholesterol esterification by making these lipoproteins preferred LCAT substrates.

Apolipoprotein AI tertiary structures determine stability and phospholipid‐binding activity of discoidal high‐density lipoprotein particles of different sizes

Human high‐density lipoprotein (HDL) plays a key role in the reverse cholesterol transport pathway that delivers excess cholesterol back to the liver for clearance. In vivo, HDL particles vary in size, shape and biological function. The discoidal HDL is a 140–240 kDa, disk‐shaped intermediate of mature HDL. During mature spherical HDL formation, discoidal HDLs play a key role in loading cholesterol ester onto the HDL particles by activating the enzyme, lecithin:cholesterol acyltransferase (LCAT). One of the major problems for high‐resolution structural studies of discoidal HDL is the difficulty in obtaining pure and, foremost, homogenous sample. We demonstrate here that the commonly used cholate dialysis method for discoidal HDL preparation usually contains 5–10% lipid‐poor apoAI that significantly interferes with the high‐resolution structural analysis of discoidal HDL using biophysical methods. Using an ultracentrifugation method, we quickly removed lipid‐poor apoAI. We also purified discoidal reconstituted HDL (rHDL) into two pure discoidal HDL species of different sizes that are amendable for high‐resolution structural studies. A small rHDL has a diameter of 7.6 nm, and a large rHDL has a diameter of 9.8 nm. We show that these two different sizes of discoidal HDL particles display different stability and phospholipid‐binding activity. Interestingly, these property/functional differences are independent from the apoAI α‐helical secondary structure, but are determined by the tertiary structural difference of apoAI on different discoidal rHDL particles, as evidenced by two‐dimensional NMR and negative stain electron microscopy data. Our result further provides the first high‐resolution NMR data, demonstrating a promise of structural determination of discoidal HDL at atomic resolution using a combination of NMR and other biophysical techniques.

Apolipoprotein A-I Regulates Lipid Hydrolysis by Hepatic Lipase

Association of hepatic lipase (HL) with pure heparan sulfate proteoglycans (HSPG) has little effect on hydrolysis of high density lipoprotein (HDL) particles, but significantly inhibits (>80%) the hydrolysis of low (LDL) and very low density lipoproteins (VLDL). Lipolytic inhibition is associated with a differential ability of the lipoproteins to remove HL from the HSPG. LDL and VLDL are unable to displace HL, whereas HDL readily displaces HL from the HSPG. These data show that HSPG-bound HL is inactive. Purified apolipoprotein (apo) A-I is more efficient than HDL at liberating HL from HSPG, and HL displacement is associated with the direct binding of apoA-I to HSPG. However, displacement of HL by apoA-I does not enhance hydrolysis of VLDL particles. This appears due to the direct inhibition of HL by apoA-I. Both apoA-I and HDL are able to inhibit VLDL lipid hydrolysis by up to 60%. Inhibition of VLDL hydrolysis is associated with the binding of apoA-I to the surface of the VLDL particle and a concomitant decreased affinity for HL. These data show that apoA-I can regulate lipid hydrolysis by HL by liberating/activating the enzyme from cell surface proteoglycans and by directly modulating lipoprotein binding and hydrolysis.

The lipid composition of high-density lipoprotein affects its re-absorption in the kidney by proximal tubule epithelial cells.

The kidney is believed to play a major role in the clearance of apoA-I (apolipoprotein A-I) and HDL (high-density lipoprotein) particles from the bloodstream. Proximal tubule epithelial cells of the kidney appear to prevent the loss of these proteins in the urine by re-absorbing them from the urinary filtrate. Experiments were undertaken to investigate the factors that regulate the renal re-absorption of apoA-I and small HDL in a transformed human proximal tubule epithelial (HKC-8) cell line. Fluorescent microscopic studies show that HKC-8 cells can readily bind and take up HDL particles. Intracellular localization of fluorescently labelled native HDL shows its accumulation in endocytotic vesicles, in a perinuclear region after 1 h. Binding studies reveal a saturable cell association of (125)I-HDL with the HKC-8 cell surface after 2 h. HKC-8 cells do not degrade apoA-I or other HDL-apoproteins. The specific cell association of lipid-free apoA-I is approx. 2-fold less than that observed for native HDL. Similarly, reconstituted HDL prepared from HDL-apoproteins and pure phospholipids also exhibits a low cell association with the HKC-8 cells. In contrast, reconstituted HDL prepared with the extracted lipids of HDL and pure apoA-I exhibits an even higher cell association than that observed with the native lipoprotein. A detailed characterization of the major lipid classes in reconstituted HDL shows that only cholesteryl ester increases the cell association of the recombinant particles. These results show that the cholesteryl ester content of HDL may play an important role in the re-absorptive salvage of HDL by the proximal tubule cells of the kidney.