Abstract
An initial event in atherosclerosis is the retention of lipoproteins within the intima of the vessel wall. Previously we identified Site B (residues 3359-3369) in apolipoprotein (apo) B100 as the proteoglycan binding sequence in low density lipoproteins (LDLs) and showed that the atherogenicity of apoB-containing lipoproteins is linked to their affinity for artery wall proteoglycans. However, both apoB100- and apoB48-containing lipoproteins are equally atherogenic even though Site B lies in the carboxyl-terminal half of apoB100 and is absent in apoB48. If binding to proteoglycans is a key step in atherogenesis, apoB48-containing lipoproteins must bind to proteoglycans via other proteoglycan binding sites in the amino-terminal 48% of apoB. In vitro studies have identified five clusters of basic amino acids in delipidated apoB48 that bind negatively charged glycosaminoglycans. To determine which of these sites is functional on LDL particles, we analyzed the proteoglycan binding activity of recombinant human LDLs from transgenic mice or rat hepatoma cells. Substitution of neutral amino acids for the basic amino acids in Site B-Ib (residues 84-94) abolished the proteoglycan binding activity of recombinant apoB53. Carboxyl-truncated apoB80 bound biglycan with higher affinity than apoB100 and apoB48. ApoB80 in which Site B was mutated had the same affinity for proteoglycans as apoB48. These data support the hypothesis that the carboxyl terminus of apoB100 "masks" Site B-Ib, the amino-terminal proteoglycan binding site, and that this site is exposed in carboxyl-truncated forms of apoB. The presence of a proteoglycan binding site in the amino-terminal region of apoB may explain why apoB48- and apoB100-containing lipoproteins are equally atherogenic.
Highlights
Elevated levels of low density lipoproteins (LDLs)1 and other lipoproteins containing apolipoprotein B cause increased
If binding to artery wall proteoglycans is a key step in atherogenesis, these apoB48-containing lipoproteins must bind to proteoglycans at other proteoglycan binding sites in the amino-terminal 48% of the apoB molecule
To verify that the binding of apoB48 LDLs to proteoglycans was mediated by apoB48 and not by the lipids of the lipoprotein, we treated the lipoproteins with CHD or acetic anhydride to modify the arginines and lysines, respectively
Summary
Materials—The QuikChange mutagenesis kit was from Stratagene (La Jolla, CA), and primers were from Oligos Etc. (Wilsonville, OR). A 4.2-kb fragment containing the RK3359 –3369SA mutation [3] was excised from the P1 plasmid RK3359 –3369SA apoB100 [3] using oligomers EcoRI-35763 and EcoRI-39948 as described previously [3] and inserted into the pZErO 5.7-kb plasmid containing a stop codon at residue 3620 (i.e. apoB80) [3]. Mutagenesis of the Heparin Binding Sites in the Amino Terminus of ApoB—A 2.45-kb EcoRI-HindIII fragment was excised from pB100L-L and inserted into the pZErO-1 vector (Invitrogen) to generate a subclone designated pZErO-2.45kb as described previously [15]. 100 l of a horseradish peroxidase-conjugated polyclonal antibody against human apoB (The Binding Site; diluted 1:750 in HBS buffer with 0.1% bovine serum albumin and 0.02% Tween 20) was added and incubated at room temperature for 1.5 h. The reactions were stopped with an equal volume of 2 M H2SO4 and measured at 450 nm in a spectrophotometer
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