LDL protein nitration: Implication for LDL protein unfolding

• Published in: Archives of biochemistry and biophysics Vol. 479; no. 1; pp. 1 - 14
• Main Authors: Hamilton, Ryan T., Asatryan, Liana, Nilsen, Jon T., Isas, Jose M., Gallaher, Timothy K., Sawamura, Tatsuya, Hsiai, Tzung K.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-11-2008
• Subjects: Animals; Aorta - cytology; Apolipoprotein B-100 - chemistry; Apolipoprotein B-100 - metabolism; Cattle; Cells, Cultured; Cysteine - metabolism; Dose-Response Relationship, Drug; Endothelial cells; Endothelial Cells - metabolism; Endothelium, Vascular - cytology; Endothelium, Vascular - metabolism; Humans; LDL; Lipid Peroxidation - drug effects; Lipid Peroxides - analysis; Lipoproteins, LDL - chemistry; Lipoproteins, LDL - isolation & purification; Lipoproteins, LDL - metabolism; Models, Chemical; Molsidomine - analogs & derivatives; Molsidomine - pharmacology; Nitration; Oxidation; Oxidation-Reduction; Peroxynitrous Acid - pharmacology; Protein Conformation - drug effects; Protein Denaturation; Protein Processing, Post-Translational; Protein Structure, Secondary; Proteins - chemistry; Proteins - metabolism; Reproducibility of Results; Tyrosine - analogs & derivatives; Tyrosine - metabolism; Nitration; Oxidation; LDL; Endothelial cells
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2008.07.026

Oxidatively- 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 α-helical structures and β
2 sheet as well as cysteine oxidation to cysteic acid in β
1 sheet. Circular dichroism analyses showed that the α-helical content of LDL
− was substantially lower (∼25%) than that of native LDL (∼90%); conversely, LDL
− showed greater content of β-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 α-helical structure and gain in β-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
−-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.