Effects of Post-Translational Modifications on the Structure and Stability of Human LDL

Abstract

LDL remodeling in vivo (by hydrolysis, oxidation, glycosylation, lipid transfer, drugs, etc.) may affect LDL entrapment in the arterial wall, which causes inflammation and promotes atherosclerosis. The molecular basis underlying the pro- or anti-atherogenic effects of modified LDL is unclear. To test whether LDL modifications lead to changes in LDL structure and stability, we used (i) myeloperoxidase and Cu2+ to produce LDL oxidized to various stages, (ii) phospholipase A2 (PLA2) to hydrolyze LDL phospholipids, (iii) beta-glucose to glycosylate apoB in LDL. Earlier we showed that heat denaturation of LDL is a kinetically controlled reaction that involves partial unfolding of the beta-sheet structure in apoB, protein dissociation, and changes in LDL morphology such as fusion and rupture. Here we test the effects of LDL modifications on these structural transitions.Our results show that LDL oxidation leads to a gradual unfolding of the secondary structure in apoB (observed by far-UV circular dichroism, CD) and inhibits heat-induced LDL fusion (observed by turbidity, near-UV CD and electron microscopy). We propose that fusion inhibition results from modifications that increase surface-to-core ratio (e.g., transfer of polar lipids to LDL or lipolysis of apolar lipids), and/or from protein cross-linking upon advanced oxidation.To assess the effect of PC hydrolysis, we hydrolyzed LDL phospholipids by PLA2, removed free fatty acids by albumin, and analyzed the structure and stability of modified LDL. CD spectroscopy showed no significant changes in the apoB secondary structure. Turbidity and electron microscopy showed that PC hydrolysis promotes LDL fusion, an effect that is reversed by albumin treatment. Consequently, free fatty acids promote lipoprotein fusion. Interestingly, glycosylation of apoB and LDL treatment with niacin also promote lipoprotein fusion. These results help understand molecular basis for LDL fusion in vivo and in vitro.