Effect of carboxyl-terminal truncation on structure and lipid interaction of human apolipoprotein E4.
Apolipoprotein (apo) E4 has been identified as a major risk factor for Alzheimer's disease. Recently, apoE4 was found to undergo proteolytic cleavage in Alzheimer's disease brains, resulting in neurotoxic C-terminal-truncated fragments. In this study, we examined the effect of progressive truncation of the C-terminal domain in apoE4 on its lipid-free structure and lipid binding properties. Circular dichroism measurements demonstrated that deletion of residues 273-299 or 261-299 significantly decreased the number of helical residues, suggesting that the C-terminal residues 261-299 have alpha-helical structure. Although the progressive deletions in the C-terminal domain appear to somewhat increase thermal stability, apoE4 (delta273-299) and apoE4 (delta261-299) showed stability similar to that of the apoE4 22-kDa fragment (residues 1-191) when denatured with guanidine-HCl, indicating that residues 192-272 have a negligible effect on the stability of the C-terminal-truncated apoE4. Comparison of Trp-264 fluorescence in single Trp mutants of full-length and C-terminal-truncated apoE4 (delta273-299) indicated that the C-terminal domain structure in the latter is both less organized and cooperative. In addition, comparison of the binding of the C-terminal-truncated mutants to a hydrophobic fluorescent dye and to lipid emulsions revealed that residues 261-272 create a hydrophobic site which is critical for lipid binding. These results suggest that removal of a hydrophobic C-terminal alpha-helical segment (residues 273-299) to create C-terminal-truncated apoE4 forms found in brain leads to less organized C-terminal structure while still retaining a second alpha-helical lipid-binding region (residues 261-272) that is available for interaction with cell membranes and other proteins such as amyloid beta peptide.
Department of Biophysical Chemistry, Kobe Pharmaceutical University, Kobe 658-8558, Japan.
Masafumi
M
Tanaka
Charulatha
C
Vedhachalam
Takaaki
T
Sakamoto
Padmaja
P
Dhanasekaran
Michael C
MC
Phillips
Sissel
S
Lund-Katz
Hiroyuki
H
Saito
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D053327:Apolipoprotein E4; D001057:Apolipoproteins E / Q000737:chemistry* / Q000235:genetics; D002850:Chromatography, Gel; D004655:Emulsions; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics
D000817:Anilino Naphthalenesulfonates; D053327:Apolipoprotein E4; D001057:Apolipoproteins E; D004655:Emulsions; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D019791:Guanidine