Sphingomyelinase Induces Aggregation and Fusion, but Phospholipase A2 Only Aggregation, of Low Density Lipoprotein (LDL) Particles: TWO DISTINCT MECHANISMS LEADING TO INCREASED BINDING STRENGTH OF LDL TO HUMAN AORTIC PROTEOGLYCANS

Katariina Öörni,Jukka K Hakala,Arto Annila,Mika Ala-Korpela,Petri T Kovanen

doi:10.1074/jbc.273.44.29127

Katariina Öörni, Jukka K Hakala + Show 3 more

Open Access

https://doi.org/10.1074/jbc.273.44.29127

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Abstract

During atherogenesis, low density lipoprotein (LDL) particles bind to extracellular matrix proteoglycans in the arterial wall, become modified, and appear as aggregated and fused particles. Sphingomyelinase (SMase) and phospholipase A2 (PLA2) have been found in the arterial wall, and, moreover, lesional LDL shows signs of hydrolysis of both sphingomyelin and phosphatidylcholine. We have now studied the effects of these two lipolytic modifications on the aggregation and fusion of LDL particles by hydrolyzing the particles with Bacillus cereus SMase or bee venom PLA2. In addition, the binding strengths of the modified LDL to human aortic proteoglycans (PG) were analyzed on an affinity column. We found that SMase induced aggregation and fusion of LDL, but PLA2 induced only aggregation of the particles. In addition, the SMase-induced aggregation and fusion of LDL was promoted by pretreatment of LDL with PLA2. Determination of the binding strengths of the hydrolyzed LDL revealed that mere lipolysis of LDL without aggregation or fusion, either by SMase or PLA2, did not affect the binding of the particles to PG. Aggregation and fusion of lipolyzed LDL particles, however, increased their strength of binding to PG. Active lysine residues in apolipoprotein B-100 (apoB-100) appear to be involved in the binding of LDL to PG, and, in fact, quantitative 13C NMR analysis revealed that, in the fused LDL particles, the number of active lysine residues per apoB-100 moiety was increased. Moreover, aggregation and fusion of LDL increased the number of apoB-100 copies and, consequently, the number of active lysine residues per aggregate or fused particle. Our present findings therefore (i) show that treatment of LDL with SMase and PLA2 generates modified LDL particles, which then bind to human aortic PG with increased strength, and (ii) suggest that SMase- and PLA2-induced aggregation and fusion of LDL are potential mechanisms leading to focal retention of extracellular lipid in the arterial wall.

Highlights

During atherogenesis, low density lipoprotein (LDL) particles bind to extracellular matrix proteoglycans in the arterial wall, become modified, and appear as aggregated and fused particles
We have examined the effects of hydrolysis of SM with Bacillus cereus SMase and hydrolysis of PC with bee venom phospholipase A2 (PLA2) on the morphology of LDL particles and on the interaction between LDL particles and human aortic proteoglycans
To study the effect of SM and PC hydrolysis of LDL particles, 1 mg/ml 3H-LDL was first treated with 95 ng/ml (50 milliunits/ ml) SMase for 1 h at 37 °C or with 50 ng/ml (60 milliunits/ml) PLA2 in the presence of 2% bovine serum albumin (BSA) for 1 h at 37 °C

Summary

EXPERIMENTAL PROCEDURES

Materials—Essentially fatty acid-free bovine serum albumin (BSA), ⑀-aminocaproic acid, PLA2 (from bee venom), immobilized PLA2 (from bee venom), and sphingomyelinase (from B. cereus) were from Sigma. t-Butoxycarbonyl-L-[3H]methionine N-hydroxysuccinimidyl ester (the 3H-labeling reagent) was from Amersham Pharmacia Biotech. Treatment of LDL with Phospholipase A2 and Sphingomyelinase— LDL (1 mg/ml) was incubated with 95 ng/ml (50 milliunits/ml) SMase in 5 mM HEPES, 2 mM CaCl2, 5 mM MgCl2, and 140 mM NaCl, pH 7.4, or with 50 ng/ml (60 milliunits/ml) PLA2 or 16 ␮g/ml agarose beads containing 60 units/ml immobilized PLA2 in the absence or presence of 2% (w/v) fatty acid-free BSA in 5 mM HEPES, 5 mM CaCl2, 2 mM MgCl2, and 140 mM NaCl, pH 7.4. Individual phospholipid subclasses were visualized by dipping the TLC plate into a solution containing 3% copper acetate (w/v) and 8% phosphoric acid (w/v) and heating the plate at 150 °C for 20 min. Isolation of Aggregated/Fused LDL—The degree of aggregation and/or fusion of lipolyzed 3H-LDL was determined by gel filtration chromatography. Glycosaminoglycans were determined by the method of Bartold and Page [31], and the amounts of the proteoglycans are expressed in terms of their glycosaminoglycan content

Affinity Chromatography of Lipolyzed LDL on a Proteoglycan Affinity

RESULTS

DISCUSSION

Active lysine residues nm LDL