LDL protein nitration: Implication for LDL protein unfolding

Oxiclatively- or enzymatically-modified low-density lipoprotein (LDL) is intimately involved in the initiation and progression of atherosclerosis. The in vivo modified LDL is electro-negative (LDL-) and consists of peroxidized lipid and unfolded apoB-100 protein. This Study was aimed at establishing specific protein modifications and conformational changes in LDL- assessed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and circular dichroism analyses, respectively. The functional significance of these chemical modifications and Structural changes were validated with binding and uptake experiments to- and by bovine aortic endothelial cells (BAEC). The plasma LDL- fraction showed increased nitrotyrosine and lipid peroxide content as well as a greater cysteine oxidation as compared with native- and total-LDL. LC/MS/MS analyses of LDL- revealed specific modifications in the apoB-100 moiety, largely involving nitration of tyrosines in the alpha-helical structures and beta(2) sheet as well as cysteine oxidation to cysteic acid in beta(1) sheet. Circular dichroism analyses showed that the of-helical content of LDL- was substantially lower (similar to 25%) that) that of native LDL (similar to 90%): conversely, LDL- showed greater content of beta-sheet and random coil structure, in agreement With unfolding of the protein. These results were mimicked by treatment of LDL subfractions with peroxynitrite (ONOO-) or SIN-1: similar amino acid modifications as well as conformational changes (loss of of-helical structure and gain in beta-sheet Structure) were observed. Both LDL- and ONOO--treated LDL,showed a statistically significant increase in binding and uptake to- and by BAEC compared to native LDL. We further found that most binding and Uptake in control-LDL was through LDL-R with minimal oxLDL-R-dependent uptake. ONOO center dot-treated LDL was significantly bound and endocytosed by LOX-1, CD36, and SR-A with minimal contribution from LDL-R. It is suggested that lipid peroxidation and protein nitration may account for the mechanisms leading to apoB- 100 protein unfolding and consequential increase in modified LDL binding and Uptake to and by endothelial cells that is dependent on oxLDL scavenger receptors. (c) 2008 Elsevier Inc. All rights reserved.