Abstract
Lipoprotein lipase (LPL) efficiently mediates the binding of lipoprotein particles to lipoprotein receptors and to proteoglycans at cell surfaces and in the extracellular matrix. It has been proposed that LPL increases the retention of atherogenic lipoproteins in the vessel wall and mediates the uptake of lipoproteins in cells, thereby promoting lipid accumulation and plaque formation. We investigated the interaction between LPL and low density lipoproteins (LDLs) with special reference to the protein-protein interaction between LPL and apolipoprotein B (apoB). Chemical modification of lysines and arginines in apoB or mutation of its main proteoglycan binding site did not abolish the interaction of LDL with LPL as shown by surface plasmon resonance (SPR) and by experiments with THP-I macrophages. Recombinant LDL with either apoB100 or apoB48 bound with similar affinity. In contrast, partial delipidation of LDL markedly decreased binding to LPL. In cell culture experiments, phosphatidylcholine-containing liposomes competed efficiently with LDL for binding to LPL. Each LDL particle bound several (up to 15) LPL dimers as determined by SPR and by experiments with THP-I macrophages. A recombinant NH(2)-terminal fragment of apoB (apoB17) bound with low affinity to LPL as shown by SPR, but this interaction was completely abolished by partial delipidation of apoB17. We conclude that the LPL-apoB interaction is not significant in bridging LDL to cell surfaces and matrix components; the main interaction is between LPL and the LDL lipids.
Highlights
Lipoprotein lipase (EC 3.1.1.34; LPL)1 is a key enzyme regulating the disposal of lipid fuels in the body [1,2,3]
We investigated the interaction between LPL and low density lipoproteins (LDLs) with special reference to the protein-protein interaction between LPL and apolipoprotein B
LPL has been proposed to have a role in atherosclerosis, functioning as a bridge that links atherogenic LDL with the subendothelial matrix of the arterial wall
Summary
Materials—LPL was isolated from bovine milk as previously described [24]. THP-I monocytes were purchased from the American Type Culture Collection (Manassas, VA). Immunoaffinity-purified apoB17 (2 ml) was mixed with 4 ml of buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 3% sodium deoxycholate) and protease inhibitors (aprotinin (final concentration, 100 kallikrein inhibitor units/ ml), leupeptin (0.1 mM), and phenylmethylsulfonyl fluoride (1 mM)). The macrophages were washed twice with Dulbecco’s phosphate-buffered saline and dissolved in 0.2 M NaOH for measurement of the 125I-LDL remaining associated with cells (heparin-resistant LDL) and cellular protein content [22]. Determination of Mass/Response Relationship for LDL and LPL— Iodinated LDL and LPL were immobilized on the dextran matrix of sensor chips with the N-ethyl-NЈ-[(diethylamino)propyl]carbodiimide/ N-hydroxysuccinimide method [36]. The mass of bound LPL and LDL was calculated from their respective specific radioactivities
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