A growing amount of evidence implicates the involvement of apolipoprotein E (apoE) in the development of late-onset and sporadic forms of Alzheimer's disease (AD). It is now generally believed that the epsilon4 allele is associated with AD and the oxidative stress is more pronounced in AD. However, only limited data are available on apoE isoform-specificity and its relationship to both the oxidative susceptibility and conformational stability of apoE. In this article, we use site-directed mutagenesis to investigate the structural role of amino acid residue 112, which is the only differing residue between apoE3 and E4. We examine the structural variation manipulating the oxidative susceptibility and conformational stability of apolipoprotein E isoforms. Arg112 in apoE4 was changed to Ala and Glu. Previous research has reported that apoE4 is more susceptible to free radicals than apoE3. In protein oxidation experiments, apoE4-R112A becomes more resistant to free radicals to the same extent as apoE3. In contrast, apoE4-R112E becomes the most susceptible protein to free radicals among all the apoE proteins. We also examine the conformational stability and the quaternary structural change by fluorescence spectroscopy and analytical ultracentrifugation, respectively. ApoE3 and E4 show apparent three- and two-state unfolding patterns, respectively. ApoE4-R112A, similar to apoE3, demonstrates a biphasic denaturation with an intermediate that appears. The denaturation curve for apoE4-R112E, however, also displays a biphasic profile but with a slight shoulder at approximately 1.5M GdmCl, implying that an unstable intermediate existed in the denaturation equilibrium. The size distribution of apoE isoforms display similar patterns. ApoE4-R112E, however, has a greater tendency to dissociate from high-molecular-weight species to tetramers. These experimental data suggest that the amino acid residue 112 governs the differences in salt-bridges between these two isoforms and thus has a significant impact on the free radical susceptibility and structural variation of the apoE isoforms.
2007 Wiley-Liss, Inc.