Time-course studies by synchrotron X-ray solution scattering of the structure of human low-density lipoprotein during Cu2+-induced oxidation in relation to changes in lipid composition

Abstract

Low-density lipoproteins (LDLs) in plasma are constructed from a single molecule of apolipoprotein B-100 (apoB) (M(r) 512,000) in association with lipid [approximate M(r) (2-3) x 10(6)]. LDL oxidation is an important process in the development of atherosclerosis, and can be imitated by the addition of Cu2+ ions. Synchrotron X-ray scattering of LDL yields curves without radiation damage effects at concentrations close to physiological. The radius of gyration RG for preparations of LDL from different donors ranged between 12.1 and 16.0 nm, with a mean of 13.9 nm. At 4 degrees C, the distance distribution curve P(r) indicated a maximum dimension of 25-27 nm for LDL, a peak at 19.5 nm which corresponds to a surface shell of protein and phospholipid head groups in LDL, and submaxima between 1.7 and 13.5 nm, which correspond to an ordered lipid core in LDL. LDL from different donors exhibited distinct P(r) curves. For oxidation studies of LDL by X-rays, data are best obtained at 4 degrees C at a concentration of > or = 2 mg of LDL protein/ml together with controls based on non-oxidized LDL. LDL oxidation (2 mg of apoB/ml) was studied at 37 degrees C in the presence of 6.4, 25.6 and 51.2 mu of Cu2+/g of apoB. Large changes in P(r) were reproducibly observed in the inter-particle distance range between 13 and 16 nm shortly after initiation of oxidation. This corresponds to the phospholipid hydrocarbon in LDL, which has either increased in electron density during oxidation or become increasingly disordered. After 25 h, the structural changes subsequently spread to regions of the P(r) curves assigned to surface apoB and the central core of cholesteryl esters and triacyl-glycerols. Lipid analyses were carried out under the same solution conditions. The alpha-tocopherol and beta-carotene antioxidant contents of LDL were consumed within 1-2 h. Analyses of the formation of thiobarbituric acid-reactive substances and lipid hydroperoxides indicated that arachidonic acid was preferentially oxidized before the maximal formation of lipid hydroperoxides at 8-12 h after initiation of oxidation. High-performance TLC showed that phosphatidylcholine was continuously converted into lysophosphatidylcholine during oxidation, which is consistent with the early changes in the X-ray P(r) curves. The neutral core lipids became modified only after 12-15 h of oxidation. The combination of X-ray scattering structural analyses with biochemical analyses shows that the oxidation of LDL first affects the outer shell of surface phospholipid, then it spreads towards damage of apoB and the internal neutral lipid core of LDL.

Untangling the role of amyloid in atherosclerosis

“…Furthermore, analysis of oxidized LDL by x-ray powder diffraction shows a diffuse powder diffraction ring with an atomic spacing of %9.8 Å that is consistent with the presence of cross-b structure, a defining characteristic of fibrillar amyloid [8 ]. Significant protein conformational changes associated with LDL oxidation have also been documented by infrared spectroscopy, circular dichroism, and synchrotron x-ray scattering [9][10][11]. These modifications are thought to result from conformational constraints placed on the b-stranded domain of apoB-100 as a consequence of lipid loss from the oxidized LDL particle.…”

Comparison of the Inhibitory Effects of ApoB100 and Tissue Factor Pathway Inhibitor on Tissue Factor and the Influence of Lipoprotein Oxidation

“…However, mild oxidation of LDL does not result in a significant increase in the electrophoretic mobility of LDL. 37 This domain is within a region of ApoB100 capable of local structural alterations that changes even at low levels of LDL oxidation. 20 Moreover, the oxidation of lipids alone (Table 2), or the addition of pure lipid peroxides, did not enhance the tissue factor activity, but rather inhibited it slightly, concurring with our previous report that the enhancement of tissue factor activity by oxidized LDL arises from the modification of ApoB100.…”

The mechanism of kinetic inhibition of Cu(II)-induced oxidation of low density lipoprotein by lanthanide ions

“…X-ray and spin-labeling studies have demonstrated that during the course of Cu(II)-induced LDL oxidation, LDL oxidation first affects the outer shell of surface phospholipids, then it spreads toward damage of the apoB protein and the internal neutral lipid core of LDL [53,54]. The present work showed that treatment with Cu(II) (24 h, at 25°C) caused significant changes of the secondary structure of LDL apoB.…”