Contributions of the carboxyl-terminal helical segment to the self-association and lipoprotein preferences of human apolipoprotein E3 and E4 isoforms

Abstract

To understand the molecular basis for the different self-association and lipoprotein preferences of apolipoprotein (apo) E isoforms, we compared the effects of progressive truncation of the C-terminal domain in human apoE3 and apoE4 on their lipid-free structure and lipid binding properties. A VLDL/HDL distribution assay demonstrated that apoE3 binds much better than apoE4 to HDL 3, whereas both isoforms bind similarly to VLDL. Removal of the C-terminal helical regions spanning residues 273-299 weakened the ability of both isoforms to bind to lipoproteins; this led to the elimination of the isoform lipoprotein preference, indicating that the C-terminal helices mediate the lipoprotein selectivity of apoE3 and apoE4 isoforms. Gel filtration chromatography experiments demonstrated that the monomer-tetramer distribution is different for the two isoforms with apoE4 being more monomeric than apoE3 and that removal of the C-terminal helices favors the monomeric state in both isoforms. Consistent with this, fluorescence measurements of Trp-264 in single-Trp mutants revealed that the C-terminal domain in apoE4 is less organized and more exposed to the aqueous environment than in apoE3. In addition, the solubilization of dimyristoylphosphatidylcholine multilamellar vesicles is more rapid with apoE4 than with apoE3; removal of the C-terminal helices significantly affected solubilization rates with both isoforms. Taken together, these results indicate that the C-terminal domain is organized differently in apoE3 and apoE4 so that apoE4 self-associates less and binds less than apoE3 to HDL surfaces; these alterations may lead to the pathological sequelae for cardiovascular and neurodegenerative diseases.

Fig. 1. Effects of C-terminal truncation of apoE3 and apoE4 on the relative distributions between VLDL and HDL₃

The apoE variants were incubated at 4 °C for 30 min with a mixture of human HDL₃ and VLDL. For ease of comparison, the ratios for the initial mixtures are normalized to one in each case. (A) apoE3/apoE4 (B) apoE3 (1–272)/apoE4 (1–272) (C) apoE3 (1–260)/apoE4 (1–260) (D) apoE3 (1–250)/apoE4 (1–250) (E) apoE3 (1–191)/apoE4 (1–191). The results from 2–5 independent experiments, each done in triplicate, are reported as mean ± SD.

Fig. 2. Effects of C-terminal truncations on the ability of apoE3 and apoE4 to bind to VLDL and HDL₃

(A) apoE3 C-terminal truncation mutants/apoE3. (B) apoE4 C-terminal truncation mutants/apoE4. The results from 2–5 independent experiments, each done in triplicate, are reported as mean ± SD.

Fig. 3. Far-UV CD spectra of apoE3 C-terminal-truncated mutants

apoE3 (a), apoE3 (1–272) (b), apoE3 (1–260) (c), and apoE3 22-kDa (d). The protein concentration was 50 µg/ml.

Fig. 4. GdnHCl denaturation of full-length apoE3, 22-kDa and 10-kDa fragments of apoE3, and C-terminal-truncated apoE3 monitored by Trp fluorescence (A) and CD (B)

Full-length apoE3 (▲), apoE3 (1–272) (●), apoE3 (1–260) (△), apoE3 (1–250) (▽), apoE3 22-kDa (□), and apoE 10-kDa (○).

Fig. 5. ANS fluorescence for apoE3 C-terminal-truncated mutants

(A) Emission spectra of ANS in the presence of apoE3 (a), apoE3 (1–272) (b), apoE3 (1–260) (c), apoE3 22-kDa (d), and buffer (e). (B) ANS fluorescence intensity ratio of the C-terminal-truncated mutants to full-length apoE. The results from at least 2 independent experiments, each done in triplicate, are shown.

Fig. 6. Elution profiles upon gel filtration of apoE3 and apoE4 variants on a Superdex 200 column

Proteins at a concentration of 5 µg/ml (Panel A and B) or 50 µg/ml (Panel C) were subjected to gel filtration. (A) Full-length apoE3 (●), full-length apoE4 (□); (B) apoE3 (1–272) (▲), apoE4 (1–272) (×); (C) apoE4(E255A) (◆). Representative profiles from at least 3 independent experiments using different batches of protein are shown.

Fig. 7. Fluorescence spectroscopy of apoE3 and apoE4 single Trp mutants

Solutions of the proteins (100 µg/ml) were excited at 295 nm to obtain the emission fluorescence from W264. (A) Fluorescence spectra for apoE3 W@264 (a) and apoE4 W@264 (b). (B) Stern-Volmer plot comparing quenching by KI of W264 fluorescence from apoE3 (○) and apoE4 (●). The data for N-acetyltryptophanamide are also shown for comparison (△). The inset shows a modified Stern-Volmer plot. The results from 2–4 independent experiments, each done in triplicate, are shown.

Fig. 8. GdnHCl denaturation of single Trp mutants of apoE3 and apoE4 monitored by Trp fluorescence

Normalized fluorescence intensity at 335 nm was plotted as a function of GdnHCl concentration. (A) ApoE3 W@264 (○), ApoE4 W@264 (●), (B) apoE3 (1–272) W@264 (△), and apoE4 (1–272) W@264 (▲). The results from 2–3 independent experiments, each done in duplicate, are shown.

Fig. 9. Solubilization of DMPC multilamellar vesicles by apoE C-terminal-truncated mutants

A: Time courses for turbidity clearance. DMPC alone (a), apoE3 (b), and apoE4 (c). The DMPC and protein concentrations were 0.25 and 0.1 mg/ml, respectively. B: Effect of protein concentration on fraction cleared in 10 min. ApoE3 (●), apoE4 (△), and apo E4(E255A) (○). C: Comparison of DMPC solubilization efficiency. Protein concentration was 0.1 mg/ml. The results from 3–5 independent experiments using different batches of protein are shown.