Characterization of the Heparin Binding Sites in Human Apolipoprotein E

Abstract

Apolipoprotein (apo) E mediates lipoprotein remnant clearance via interaction with cell-surface heparan sulfate proteoglycans. Both the 22-kDa N-terminal domain and 10-kDa C-terminal domain of apoE contain a heparin binding site; the N-terminal site overlaps with the low density lipoprotein receptor binding region and the Cterminal site is undefined. To understand the molecular details of the apoE-heparin interaction, we defined the microenvironments of all 12 lysine residues in intact apoE3 and examined their relative contributions to heparin binding. Nuclear magnetic resonance measurements showed that, in apoE3-dimyristoyl phosphatidylcholine discs, Lys-143 and -146 in the N-terminal domain and Lys-233 in the C-terminal domain have unusually low pK a values, indicating high positive electrostatic potential around these residues. Binding experiments using heparin-Sepharose gel demonstrated that the lipid-free 10-kDa fragment interacted strongly with heparin and a point mutation K233Q largely abolished the binding, indicating that Lys-233 is involved in heparin binding and that an unusually basic lysine microenvironment is critical for the interaction with heparin. With lipidated apoE3, it is confirmed that the Lys-233 site is completely masked and the N-terminal site mediates heparin binding. In addition, mutations of the two heparin binding sites in intact apoE3 demonstrated the dominant role of the N-terminal site in the heparin binding of apoE even in the lipid-free state. These results suggest that apoE interacts predominately with cell-surface heparan sulfate proteoglycans through the N-terminal binding site. However, Lys-233 may be involved in the binding of apoE to certain cell-surface sites, such as the protein core of biglycan. Apolipoprotein E (apoE)1 is a critical ligand for several hepatic lipoprotein receptors, including the low density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP), and for cell-surface heparan sulfate proteoglycans (HSPG) (1-3). Through its interaction with these receptors and with the HSPG-LRP pathway, apoE mediates the catabolism of remnant lipoproteins (4, 5). In the HSPG-LRP pathway, apoE is postulated to interact initially with cell-surface HSPG and then to transfer to the LRP for internalization (6, 7). Therefore, the interaction of apoE with HSPG is an initial step in the clearance of apoE-containing lipoproteins from the plasma. The apoE-HSPG interaction is also involved in the differential effects of the apoE isoforms on neurite outgrowth (8, 9), the existence of a pool of newly secreted apoE on the cell surface (10 -12), and the inhibition of platelet-derived growth factorstimulated smooth muscle cell proliferation (13). In addition, several studies suggest that binding of apoE to HSPG may be involved in Alzheimer's disease (5,14,15).ApoE, a 299-amino acid, single chain protein, contains two independently folded functional domains, a 22-kDa N-terminal domain (residues 1-191) and a 10-kDa C-terminal domain (residues 222-299) (2, 16). The N-terminal...

Role of the N- and C-Terminal Domains in Binding of Apolipoprotein E Isoforms to Heparan Sulfate and Dermatan Sulfate: A Surface Plasmon Resonance Study

“…As shown in Fig. 4A, large decreases in binding to HS and DS were observed with the K146E mutant whereas the K233E mutant exhibited responses similar to the wild-type, consistent with the previous finding that only the N-terminal site is available for the interaction with heparin even in the lipid-free state (27,28). Comparison of the free energy of binding for each step among lysine mutants (Fig.…”

“…4) (27,28). Rather, the C-terminal domain appears to contribute to the highaffinity binding of apoE to GAG through an oligomerization effect (27). Using C-terminally truncated mutants of apoE, we showed previously that the C-terminal helical residues, 261-299 or 272-299 modulate the tetramerization of apoE in solution (37,47).…”

“…The mutations in apoE to create K146E and K233E, and the truncated forms (Δ251-299, Δ261-299 and Δ273-299) were made using PCR methods as described (27,37). The apoE preparations were at least 95% pure as assessed by SDS-PAGE.…”

“…To confirm the importance of an ionic interaction in the apoE-GAG interaction, we examined the effects of substitutions of lysine residues 146 and 233 in apoE on the GAG binding; these residues are located in the N-and C-terminal heparin binding sites, respectively, and contribute to an ionic interaction with heparin (26,27). As shown in Fig.…”

N-methyl-d-aspartate receptor inhibition by an apolipoprotein E-derived peptide relies on low-density lipoprotein receptor-associated protein

“…6C) heparinase-I treatment. Thus, HSPG does not cofunction with LRP in this case, even though apoE [133][134][135][136][137][138][139][140][141][142][143][144][145][146][147][148][149] can interact with HSPG (Ji et al, 1993;Saito et al, 2003).…”