Thermal stability of human plasma electronegative low-density lipoprotein: A paradoxical behavior of low-density lipoprotein aggregation

Abstract

Low-density lipoprotein (LDL) aggregation is central in triggering atherogenesis. A minor fraction of electronegative plasma LDL, termed LDL(-), plays a special role in atherogenesis. To better understand this role, we analyzed the kinetics of aggregation, fusion and disintegration of human LDL and its fractions, LDL(+) and LDL(-). Thermal denaturation of LDL was monitored by spectroscopy and electron microscopy. Initially, LDL(-) aggregated and fused faster than LDL(+), but later the order reversed. Most LDL(+) disintegrated and precipitated upon prolonged heating. In contrast, LDL(-) partially retained lipoprotein morphology and formed soluble aggregates. Biochemical analysis of all fractions showed no significant degradation of major lipids, mild phospholipid oxidation, and an increase in non-esterified fatty acid (NEFA) upon thermal denaturation. The main baseline difference between LDL subfractions was higher content of NEFA in LDL(-). Since NEFA promote lipoprotein fusion, increased NEFA content can explain rapid initial aggregation and fusion of LDL(-) but not its resistance to extensive disintegration. Partial hydrolysis of apoB upon heating was similar in LDL subfractions, suggesting that minor proteins importantly modulate LDL disintegration. Unlike LDL(+), LDL(-) contains small amounts of apoA-I and apoJ. Addition of exogenous apoA-I to LDL(+) hampered lipoprotein aggregation, fusion and precipitation, while depletion of endogenous apoJ had an opposite effect. Therefore, the initial rapid aggregation of LDL(-) is apparently counterbalanced by the stabilizing effects of minor proteins such as apoA-I and apoJ. These results help identify key determinants for LDL aggregation, fusion and coalescence into lipid droplets in vivo.

Keywords: Apolipoprotein A-I; Apolipoprotein J; Atherogenesis; Electronegative LDL; Lipoprotein aggregation, fusion and droplet formation; Thermal denaturation.

Fig. 1

Secondary structure and thermal stability of LDL fractions monitored by circular dichroism spectroscopy. Far-UV CD spectra of intact LDL(total), LDL(+), and LDL(−) (A). The melting data recorded of LDL(+) and LDL(−) at 320 nm by turbidity to monitor changes in the particle size (B) and by near-UV CD to monitor lipid re-packing upon lipoprotein disintegration and release of core lipids (C). The data were recorded using LDL samples under standard conditions (0.35 mg protein/mL, 20 mM Na phosphate buffer, pH 7.0). The melting data in panels B and C were recorded simultaneously during sample heating and cooling at a constant rate of 11 °C/h. Steep decline in CD and turbidity amplitude observed upon heating of LDL(+) above 88 °C is due to sample precipitation (D). The melting data of LDL(total) (not shown) were similar to those of LDL(+) within the error of their experimental determination.

Fig. 2

Kinetics of the heat-induced aggregation, fusion and rupture of LDL(+) and LDL(−). All data were recorded from LDL samples under standard conditions in a temperature jump from 25 °C to 82 °C. (A) The time course of the transition was monitored at 320 nm by turbidity for increase in the particle size. (B) Simultaneously, near-UV CD at 320 nm signal was recorded to monitor lipid re-packing upon LDL rupture and coalescence into lipid droplets. For LDL(+), the signal loss due to sample precipitation was observed after ∼60 min. (C) NGGE of LDL subfractions at a baseline and after 90 min of thermal denaturation. (D) Repetitive kinetic data recorded from LDL(+). The sample was subjected to a T-jump from 25 °C to 82 °C (light-grey). An identical sample was subjected to similar T-jump for 15 min, followed by rapid sample cooling on ice, equilibration at 25 °C, and the second consecutive T-jump to 82 °C (grey, *).

Fig. 3

Electron micrographs of negatively stained LDL(total), LDL(+) and LDL(−). Intact particles (left) were incubated at 82 °C in T-jump experiments as described in Fig. 2; sample aliquots were taken after 15 min (middle) or 30 min (right) of incubation. TEM was performed as described in the Experimental Section. White arrows indicate fused LDL particles. Inserts: zoomed-out views illustrate LDL aggregation. The bar size is 50 nm.

Fig. 4

Heat-induced changes in biochemical composition of LDL subfractions. LDL(total), LDL(+) and LDL(−) were analyzed prior to heating (baseline) or after incubation for 15, 30 or 90 min at 82 °C. Relative changes (compared to the baseline) of the major protein, apoB, and major lipids are shown for (A) LDL(total), (B) LDL(+), and (C) LDL(−). (D) Quantification of cholesterol and triglycerides (Chol + TG), phosphatidylcholine (PC), sphingomyelin (SM) and other phospholipids in LDL subfractions; relative content as a fraction of total lipids is shown (see Supplemental Fig.S6 for details). (E) Phospholipid oxidation characterized by the absorbance ratio at 205 nm to 234 nm of the PC peak. (F) NEFA content in LDL subfractions. Data in A, B, C and Fare the mean ± SD of 8 independent samples. Data in D and E are the mean ± SD of 4 independent samples. See text for details of the biochemical analyses.*indicates P <0.05 vs. baseline values. ^# indicates P < 0.05 vs. LDL(total) or LDL(+).

Fig. 5

Protein degradation upon thermal denaturation of LDL. (A) SDS-PAGE (10%, Coomassie blue staining) was used to assess changes to apoB after 15, 30 and 90 min of incubation at 82 °C. Gels were run at 100 V for 1.5 h. (B, C) Following SDS-PAGE, Western blot analysis was used to detect apoB (B), apoJ (C) and apoA-I (D).

Fig. 6

Effects of apoA-I and apoJ on thermal stability and solubility of LDL. (A) Effects of exogenous apoA-I on LDL stability. Lipid-free human apoA-I (0.2 mg/mL) was added to LDL(+) under standard conditions. The data were recorded from LDL(+) alone (grey) or from LDL(+) and apoA-I (black) during a T-jump from 25 to 82 °C by turbidity at 320 nm. (B) Effects of endogenous apoJ on LDL solubility. ApoJ-depleted (LDL/J-) and apoJ-containing LDL (LDL/J+) were obtained from LDL(total) by affinity chromatography as described in Methods. LDL subfractions were incubated at 82 °C for 90 min, were centrifuged at 14,000 rpm for 10 min at 4 °C, and cholesterol was measured in the supernatant. The results are expressed as the proportion of soluble LDL cholesterol.