Human Apolipoprotein E4 Domain Interaction

Abstract

Human apolipoprotein (apo) E contains an amino-and a carboxyl-terminal domain, which are connected by a hinge region (approximately residues 165 to 215). The interaction of the two domains has been suggested to be responsible for the apoE4-binding preference for very low density lipoproteins (VLDL). In the absence of this interaction in apoE3, the preference is for high density lipoproteins (HDL). To exclude the possibility that the interaction of apoE with other apolipoproteins on the native particles may contribute to the isoform-specific preferences, VLDL-like emulsion particles were incubated with apoE, and the lipid-bound apoE was separated from free apoE on a Superose 6 column. The apoE4 bound more effectively to these particles than did apoE3, indicating that the apoE4 preference for VLDL is due not to interactions with other apolipoproteins but to an intrinsic property of apoE4, likely related to domain interaction. Previously, arginine 61 was shown to be critical for the isoform preferences, suggesting that it interacted with an acidic residue(s) in the carboxyl terminus. Substitution of arginine 61 with lysine did not alter the preference of apoE4 for VLDL, demonstrating that a positive charge rather than a specific requirement for arginine is critical for domain interaction. To identify the acidic residue(s) in the carboxyl terminus interacting with arginine 61, the six acidic residues (244, 245, 255, 266, 270, and 271) in a region known to be important for both lipoprotein association and isoformspecific preferences were substituted individually with alanine in apoE4. Only substitution of glutamic acid 255 altered the preference of apoE4 from VLDL to HDL, indicating that this was the sole residue in the carboxyl terminus that interacts with arginine 61. The participation of the hinge region in domain interaction was examined with internal deletion mutants. Deletion of the residues 186 -202 or 186 -223, representing major portions of the hinge region, had no effect on the apoE4 preference for VLDL. This suggests that the hinge region may act as a spacer that connects the two domains. Further deletion into the carboxyl-terminal domain (to residue 244) results in a loss of apoE4 VLDL binding. These studies establish that interaction of arginine 61 and glutamic acid 255 mediates apoE4 domain interaction. Apolipoprotein (apo)1 E (299 amino acids) plays an important role in lipoprotein metabolism through its interaction with the low density lipoprotein (LDL) receptor (1, 2). The three common isoforms of apoE (apoE2, apoE3, and apoE4) are distinguished from each other by their cysteine/arginine content at two polymorphic sites. Apolipoprotein E3 contains cysteine and arginine at positions 112 and 158, respectively, whereas apoE2 contains cysteine and apoE4 contains arginine at both sites (3).Apolipoprotein E4 is associated with higher plasma cholesterol and LDL concentrations than apoE3 and apoE2 and, as a result, is a risk factor for cardiovascular disease (4 -8). One of the distinct metabolic characterist...

Apolipoprotein E4 Domain Interaction Occurs in Living Neuronal Cells as Determined by Fluorescence Resonance Energy Transfer

“…5B), suggesting that the N and C termini of these two apoE4 mutants are separated by more than 100 Å. Interestingly, the ratios of YFP to CFP fluorescence in cells expressing the mutant proteins were similar to those of cells expressing YFP-apoE3-CFP. Thus, the two apoE4 mutants appear to be structurally similar to apoE3, as reported (35,36). These data strongly suggest that the FRET in YFP-apoE4-CFP cells is caused by apoE4 domain interaction.…”

“…This exception results in fundamental structural and biophysical differences (30 -34). For example, apoE4 displays domain interaction mediated by a salt bridge between Arg-61 in the N terminus and Glu-255 in the C terminus, leading to a compact structure (35,36). Consequently, apoE4 binds preferentially to triglyceride-rich very low density lipoproteins (35,36).…”

“…For example, apoE4 displays domain interaction mediated by a salt bridge between Arg-61 in the N terminus and Glu-255 in the C terminus, leading to a compact structure (35,36). Consequently, apoE4 binds preferentially to triglyceride-rich very low density lipoproteins (35,36). ApoE3, which lacks the domain interaction, is predicted to have a more open structure, and it binds preferentially to phospholipid-rich high density lipoproteins (35,36).…”

“…ApoE3, which lacks the domain interaction, is predicted to have a more open structure, and it binds preferentially to phospholipid-rich high density lipoproteins (35,36). ApoE4 domain interaction occurs on lipoprotein particles in vitro in human plasma (35,36) and in vivo in Arg-61 knock-in mice (37), in which domain interaction was introduced into mouse apoE by mutating Thr-61 to Arg.Although it was initially believed that apoE was primarily synthesized by astrocytes in the brain but not by neurons (38), numerous subsequent studies (39 -54) have demonstrated that central nervous system neurons also express apoE, albeit at lower levels than astrocytes, under diverse physiological and pathological conditions. Notably, transgenic mice expressing apoE4 specifically in central nervous system neurons are more susceptible to age-induced and excitotoxin-induced neurodegeneration (55-57) and behavioral deficits (55, 58) than transgenic mice with similar expression of apoE3.…”

“…ApoE3, which lacks the domain interaction, is predicted to have a more open structure, and it binds preferentially to phospholipid-rich high density lipoproteins (35,36). ApoE4 domain interaction occurs on lipoprotein particles in vitro in human plasma (35,36) and in vivo in Arg-61 knock-in mice (37), in which domain interaction was introduced into mouse apoE by mutating Thr-61 to Arg.…”