Biophysical Analysis of Progressive C-Terminal Truncations of Human Apolipoprotein E4: Insights into Secondary Structure and Unfolding Properties

Abstract

Apolipoprotein E4 (apoE4) is a risk factor for Alzheimer's disease and has been associated with a variety of neuropathological processes. ApoE4 C-terminally truncated forms have been found in brains of Alzheimer's disease patients. Structural rearrangements in apoE4 are known to be key to its physiological functions. To understand the effect of C-terminal truncations on apoE4 lipid-free structure, we produced a series of recombinant apoE4 forms with progressive C-terminal deletions between residues 166 and 299. Circular dichroism measurements show a dramatic loss in helicity upon removal of the last 40 C-terminal residues, whereas further truncations of residues 203-259 lead to recovery of helical content. Further deletion of residues 186-202 leads to a small increase in helical content. Thermal denaturation indicated that removal of residues 260-299 leads to an increase in melting temperature but truncations down to residue 186 did not further affect the melting temperature. The progressive C-terminal truncations, however, gradually increased the cooperativity of thermal unfolding. Chemical denaturation of the apoE4 forms revealed a two-step process with a clear intermediate stage that is progressively lost as the C-terminus is truncated down to residue 230. Hydrophobic fluorescent probe binding suggested that regions 260-299 and 186-202 contain hydrophobic sites, the former being solvent accessible in the wild-type molecule and the latter being accessible only upon truncation. Taken together, our results show an important but complex role of apoE4 C-terminal segments in secondary structure stability and unfolding and suggest that interactions mediated by the C-terminal segments are important for the structural integrity and conformational changes of apoE4. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 June 8. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptApolipoprotein E (apoE) 1 is an important protein of the lipid transport system that has an indisputable role in atherosclerosis, dyslipidemia, and Alzheimer's disease (AD) (1,2). ApoE, expressed in liver, brain, and other tissues, has three common isoforms (apoE2, apoE3, apoE4) in the general population, each differing in the amino acid positions 112 and 158 (3,4). ApoE3, the most common form, contains cysteine and arginine, respectively, whereas apoE2 has two cysteine residues and apoE4 has two arginine residues at these positions. ApoE4 has been associated with a variety of neuropathological processes, including AD (2). ApoE4 is a major genetic risk factor for AD since 40% of all patients have at least one ε4 allele (5). Being homozygous for the ε4 allele increases the risk of AD 4-fold and lowers the age of onset of late-onset AD (5). Recent studies have shown that apoE4 is also associated with carotid atherosclerosis and is a significant risk factor for coronary heart disease (6,7).ApoE contains 299 residues and in the lipid-free state is folded into two independent structu...

ApoA-IV promotes the biogenesis of apoA-IV-containing HDL particles with the participation of ABCA1 and LCAT

“…II A,B ). Chemical denaturation of apoA-IV revealed single-step transition with limited cooperativity that lacked the intermediate described for the thermal denaturation of apoE ( 49 ). Chemical denaturation of rHDL-A-IV showed a highly noncooperative transition (supplementary Fig.…”

“…Circular dichroism measurements revealed a significant helical content of 41.4%, albeit reduced compared with apoA-I and apoE ( 48,49 ). Upon mixing with egg yolk phosphatidyl-choline, recombinant apoA-IV readily formed HDL-like particles with increased helical content of 46.7% (supplementary Fig.…”

Complement Factor H Binds to Human Serum Apolipoprotein E and Mediates Complement Regulation on High Density Lipoprotein Particles

“…ApoE was purified from the culture medium of adenovirus-infected HTB-13 cells using dextran sulfate-Sepharose column fractionation (23).…”

The Adipophilin C Terminus Is a Self-folding Membrane-binding Domain That Is Important for Milk Lipid Secretion

“…The molar ellipticity ([]) in degrees cm 2 dmol Ϫ1 was calculated from the equation, [] ϭ ⅐MRW/10lc as described (26), where MRW represents mean residue weight, l is path length in cm, and c is concentration in mg/ml. The ␣-helical content of ADPH(172-425) was calculated from the equation, % ␣-helix ϭ (Ϫ[] 222 ϩ 3000)/39,000 (27).…”