Apolipoprotein E (apoE) is an anti-atherogenic protein that plays a critical role in maintaining plasma cholesterol and triglyceride homeostasis by virtue of its ability to act as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins. In this study, we characterized the biochemical and biophysical features of recombinant rat apoE, in comparison with those of human apoE3. Rat apoE was overexpressed in Escherichia coli using a codon optimized system and purified by affinity chromatography. SDS-PAGE and RP-HPLC of rat apoE confirmed the purity, while immunoblot verified the identity and cross-reactivity with the LDLr-binding region of apoE3. The α-helical content was calculated to be ~45% by circular dichroism spectroscopy. The protein exists in a predominantly tetrameric form in lipid-free state. Chemical denaturation studies reveal that the unfolding pattern is biphasic with mid points of denaturation corresponding to 0.8 and 2.2 M guanidine hydrochloride, suggesting the presence of two domains. Rat apoE converts DMPC vesicles to smaller DMPC/apoE complexes with a first order rate constant of 0.12 min−1. It has the ability to bind the LDLr and to heparin. Our studies indicate that although its sequence resembles apoE4, an isoform of apoE3, rat apoE displays the biophysical behavior of apoE3.

The objective of our study was to assess if second hand smoke exposure predisposes subjects towards developing heart disease. We monitored the effect of reactive aldehydes in tobacco smoke on the status of plasma apolipoprotein E (apoE). ApoE plays a critical role in regulating plasma cholesterol homeostasis, since it serves as a ligand for the low density lipoprotein family of receptors. ELISA indicated that rats exposed to low doses of tobacco smoke had lower plasma apoE levels compared to the control group. Immunoprecipitation analysis revealed that the smoke‐exposed group also had higher levels of modified apoE in their lipid‐free fractions. There were no differences between the two groups in lipid peroxidation products levels or in the susceptibility of the lipoproteins to lipid peroxidation. In vitro analysis using recombinant apoE provided further support for modification of essential lysines. Our results suggest that the occurrence of oxidatively modified apoE in a lipid‐free state can result in decreased clearance of plasma lipoproteins. We propose that sustained second hand smoke exposure can potentially lead to a proatherogenic profile. Source of research support: Tobacco Related Disease Research Program, American Heart Association, NIH R25 HL096365‐01, CSULB funding and SCAC award.