Novel Phospholipolytic Activities Associated with Electronegative Low-Density Lipoprotein Are Involved in Increased Self-Aggregation

Abstract

Electronegative low-density lipoprotein (LDL(-)) is a minor LDL subfraction present in plasma with increased platelet-activating factor acetylhydrolase (PAF-AH) activity. This activity could be involved in the proinflammatory effects of LDL(-). Our aim was to study the presence of additional phospholipolytic activities in LDL(-). Total LDL was fractionated into electropositive (LDL(+)) and LDL(-) by anion-exchange chromatography, and phospholipolytic activities were measured by fluorometric methods. Phospholipolytic activity was absent in LDL(+) whereas LDL(-) presented activity against lysophosphatidylcholine (LPC, 82.4 +/- 34.9 milliunits/mg of apoB), sphingomyelin (SM, 53.3 +/- 22.5 milliunits/mg of apoB), and phosphatidylcholine (PC, 25.7 +/- 4.3 milliunits/mg of apoB). LDL(-), but not LDL(+), presented spontaneous self-aggregation at 37 degrees C in parallel to phospholipid degradation. This was observed in the absence of lipid peroxidation and suggests the involvement of phospholipolytic activity in self-aggregation of LDL(-). Phospholipolytic activity was not due to PAF-AH, apoE, or apoC-III and was not increased in LDL(+) modified by Cu (2+) oxidation, acetylation, or secretory phospholipase A 2 (PLA 2). However, LDL(-) efficiently degraded phospholipids of lipoproteins enriched in LPC, such as oxidized LDL or PLA 2-LDL, but not native or acetylated LDL. This finding supports that LPC is the best substrate for LDL(-)-associated phospholipolytic activity. These results reveal novel properties of LDL(-) that could play a significant role in its atherogenic properties.

2D-NMR reveals different populations of exposed lysine residues in the apoB-100 protein of electronegative and electropositive fractions of LDL particles

“…Composition results and TNBS reactivity agreed with previously reported data ( 7,14 ). LDL(-) contained more triglyceride, more NEFA, and less apoB-100 proportion.…”

“…It has been reported that active Lys are clustered in basic microenvironments, some of them being involved in LDLr recognition (11)(12)(13). These conformational differences between LDL(+) and LDL(-) agree with the observation of misfolded apoB-100 in LDL(-) ( 9 ), a property that increases its overall amyloidogenic propensity ( 10 ) and could also be involved in its higher susceptibility to particle aggregation ( 7,8 ).…”

Immunochemical Analysis of the Electronegative LDL Subfraction Shows That Abnormal N-terminal Apolipoprotein B Conformation Is Involved in Increased Binding to Proteoglycans

“…Phosphatidylcholine and sphingomyelin were quantified by normal phase HPLC in a Gold System chromatograph (Beckman) after lipid extraction as described (22). The oxidative level of LDL was estimated by measuring the absorbance at 234 nm of the phosphatidylcholine peak (which corresponds to oxidized phosphatidylcholine) and expressed as the 205/234 nm ratio (24). LDL aggregation level was determined by measuring the absorbance at 450 nm and by nondenaturing acrylamide gradient gel electrophoresis as described (5).…”

High binding affinity of electronegative LDL to human aortic proteoglycans depends on its aggregation level

“…Phospholipolytic activities [lysophospholipase C (lysoPLC) and SMase activities] were measured using 15 mg of LDL by a commercial fluorimetric method (Amplex Red, Molecular Probes) using lysophosphatidylcholine or sphingomyelin as substrates (30). SMase activity was also analyzed by incubation of LDL (25 mg of LDL protein) with a fluo-rescently labeled substrate (borondipyrromethene (BODIPY)-FL-C12-SM, Molecular Probes) for 3 h at 137°C followed by lipid extraction and separation by TLC (30,31). Aggregation studies.…”