Thermal Transitions in Human Very-Low-Density Lipoprotein:  Fusion, Rupture, and Dissociation of HDL-like Particles

Abstract

Very low-density lipoproteins (VLDL) are metabolic precursors of low-density lipoproteins (LDL) and a risk factor for atherosclerosis. Human VLDL are heterogeneous complexes containing triacylglyceride-rich apolar lipid core and polar surface comprised of phospholipids, a nonexchangeable apolipoprotein B, and exchangeable apolipoproteins E and Cs. We report the first stability study of VLDL. Circular dichroism and turbidity data reveal an irreversible heat-induced VLDL transition that involves formation of larger particles and repacking of apolar lipids but no global protein unfolding. Heating rate effect on the melting temperature indicates a kinetically controlled reaction with high activation energy, E a . Arrhenius analysis of the turbidity data reveals two kinetic phases with E a =53±7 kcal/mol that correspond to distinct morphological transitions observed by electron microscopy. One transition involves VLDL fusion, partial rupture and dissociation of small spherical particles (d=7-15 nm), and another involves complete lipoprotein disintegration and lipid coalescence into droplets accompanied by dissociation of apolipoprotein B. The small particles, which are unique to VLDL denaturation, are comparable in size and density to high-density lipoproteins (HDL); they have apolar lipid core and polar surface comprised of exchangeable apolipoproteins (E and possibly Cs) and phospholipids. We conclude that, similar to HDL and LDL, VLDL are stabilized by kinetic barriers that prevent particle fusion and rupture and decelerate spontaneous inter-conversion among lipoprotein classes and subclasses. In addition to fusion, VLDL disruption involves transient formation of HDL-like particles that may mimic protein exchange among VLDL and HDL pools in plasma.Plasma lipoproteins, including high-, low-, intermediate-, and very-low density lipoproteins (HDL, LDL, IDL and VLDL), are macromolecular complexes of lipids and proteins (termed apolipoproteins) that mediate lipid transport and metabolism and are central in the development of coronary artery disease. HDL are anti-atherogenic, LDL are pro-atherogenic, and VLDL are not only direct metabolic precursors of LDL but also an independent risk factor for atherosclerosis (1)(2)(3)(4)(5)(6)(7)(8). VLDL are the major carriers of triacylglycerides (TG) in plasma. Human VLDL form heterogeneous population of spherical particles that contain apolar core comprised mainly of TG and cholesterol esters (CE) and polar surface comprised of cholesterol-containing phospholipid monolayer and proteins. The proteins include one copy of non-exchangeable apolipoprotein B (apoB, 550 kD) and multiple copies of exchangeable apolipoproteins, mainly apoE (34 kD) and apoCs (6-9 kD), that comprise over 50% of the total VLDL protein content. Metabolic remodeling by lipolytic enzymes converts VLDL into LDL that contain apoB as their sole protein, while the dissociated apoE and apoCs enter the HDL pool (2,4). Structural Our recent thermal and chemical denaturation analyses have shown that HDL and LDL...

Kinetic analysis of thermal stability of human low density lipoproteins: a model for LDL fusion in atherogenesis

“…The uncertainly in this value incorporates the fi tting errors and the deviations among the data sets recorded from different batches of LDL. Notably, this value of E a is nearly twice as high as the maximal values obtained from heat denaturation studies of human HDL ( 35 ) or VLDL ( 36 ). High activation energy of LDL denaturation refl ects a steep temperature dependence of its rate constant, k(T), and explains why LDL heat denaturation could be monitored in a relatively narrow temperature range.…”

“…I). Thermal denaturation studies revealed that stability of LDL and other lipoproteins is determined by kinetic barriers and suggested that similar barriers modulate lipoprotein remodeling and fusion in vivo ( 30,(33)(34)(35)(36). In fact, the products of the heatinduced LDL fusion are similar in size and morphology to LDL-derived extracellular deposits in early atherosclerotic lesions ( 37 ).…”

“…In fact, the products of the heatinduced LDL fusion are similar in size and morphology to LDL-derived extracellular deposits in early atherosclerotic lesions ( 37 ). To quantify LDL stability, we used a kinetic approach applied in the previous thermal stability studies of high-and very low-density lipoproteins, HDL and VLDL (34)(35)(36), and HDL subclasses ( 38 ). The application of this approach to LDL has been complicated by the relatively narrow range of experimental conditions allowing kinetic data collection.…”

“…1A ), and was monitored by turbidity (dynode voltage, V) as described ( 33,36 ). LDL heat denaturation showed sigmoidal time course that contrasts with the exponential decay time course observed in other lipoproteins (33)(34)(35)(36). At each temperature, the turbidity data, V(t), in Fig.…”

“…In literature, the term "aggregation" is often used to describe increase in lipoprotein size that may result from particle aggregation, fusion, and/or rupture. To differentiate among these transitions, we use electron microscopy to visualize particle size and morphology, together with near-UV circular dichroism to monitor rupture as previously described ( 30,36 ). …”