Swapping the N- and C-terminal domains of human apolipoprotein E3 and AI reveals insights into their structure/activity relationship.

Mark T Lek,Paul M M Weers,Vasanthy Narayanaswami,John K Bielicki,Siobanth Cruz,Wendy H J Beck,Nnejiuwa U Ibe,Ying-Mei Feng

doi:10.1371/journal.pone.0178346

Mark T Lek, Paul M M Weers + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0178346

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Abstract

Apolipoprotein (apo) E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma lipoprotein metabolism. ApoE3 (299 residues) is composed of an N-terminal (NT) domain bearing a 4-helix bundle and a C-terminal (CT) domain bearing a series of amphipathic α-helices. ApoAI (243 residues) also comprises a highly helical NT domain and a less structured CT tail. The objective of this study was to understand their structural and functional role by generating domain swapped chimeras: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. The bacterially overexpressed chimeras were purified by affinity chromatography and their identity confirmed by immunoblotting and mass spectrometry. Their α-helical content was comparable to that of the parent proteins. ApoE3-NT/apoAI-CT retained the denaturation profile of apoE3 NT domain, with apoAI CT tail eliciting a relatively unstructured state; its lipid binding ability improved dramatically compared to apoE3 indicative of a significant role of apoAI CT tail in lipid binding interaction. The LDL receptor interaction and ability to promote ABCA1-mediated cholesterol efflux of apoE3-NT/apoAI-CT was comparable to that of apoE3. In contrast, apoAI-NT/apoE-CT elicited an unfolding pattern and lipid binding ability that were similar to that of apoAI. As expected, DMPC/apoAI-NT/apoE-CT discoidal particles did not elicit LDLr binding ability, and promoted SR-B1 mediated cellular uptake of lipids to a limited extent. However, apoAI-NT/apoE-CT displayed an enhanced ability to promote cholesterol efflux compared to apoAI, indicative of a significant role for apoE CT domain in mediating this function. Together, these results indicate that the functional attributes of apoAI and apoE3 can be conferred on each other and that NT-CT domain interactions significantly modulate their structure and function.

Highlights

Apolipoprotein E3 and apolipoprotein AI are exchangeable apolipoproteins that are key players in cardiovascular disease predominantly due to their abilities to maintain and regulate plasma triglyceride and cholesterol homeostasis [1,2,3,4]
To determine the structural basis of the functional differences between the NT and CT domains of Apolipoprotein E3 (apoE3) and apolipoprotein AI (apoAI), chimeric apolipoproteins were generated by swapping the CT domain of each other, Fig 1
Our findings further showed that the nature of the CT tail is irrelevant for the low density lipoprotein receptor (LDLr) binding capability of apoE3

Summary

Introduction

Apolipoprotein E3 (apoE3) and apolipoprotein AI (apoAI) are exchangeable apolipoproteins that are key players in cardiovascular disease predominantly due to their abilities to maintain and regulate plasma triglyceride and cholesterol homeostasis [1,2,3,4]. Both show broadly similar functional characteristics with an ability to bind and transport lipids as large lipoprotein complexes, which serve as a vehicle for other amphipathic and hydrophobic proteins, lipid and nucleic acid moieties in an aqueous environment such as the plasma. ApoE resides predominantly on VLDL, chylomicron remnants and a sub species of HDL; in the brain, it is one of the major apolipoproteins that has been identified to play a crucial role in brain cholesterol metabolism, and is located on HDL-like particles [8]

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