Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Apr 3;112(7):1100-1109.e5.
doi: 10.1016/j.neuron.2023.12.018. Epub 2024 Jan 23.

Apolipoprotein E secreted by astrocytes forms antiparallel dimers in discoidal lipoproteins

Michael R Strickland  1 , Michael J Rau  2 , Brock Summers  2 , Katherine Basore  2 , John Wulf 2nd  2 , Hong Jiang  1 , Yun Chen  3 , Jason D Ulrich  4 , Gwendalyn J Randolph  5 , Rui Zhang  6 , James A J Fitzpatrick  2 , Anil G Cashikar  7 , David M Holtzman  8
Affiliations

Apolipoprotein E secreted by astrocytes forms antiparallel dimers in discoidal lipoproteins

Michael R Strickland et al. Neuron. .

Abstract

The Apolipoprotein E gene (APOE) is of great interest due to its role as a risk factor for late-onset Alzheimer's disease. ApoE is secreted by astrocytes in the central nervous system in high-density lipoprotein (HDL)-like lipoproteins. Structural models of lipidated ApoE of high resolution could aid in a mechanistic understanding of how ApoE functions in health and disease. Using monoclonal Fab and F(ab')2 fragments, we characterize the structure of lipidated ApoE on astrocyte-secreted lipoproteins. Our results provide support for the "double-belt" model of ApoE in nascent discoidal HDL-like lipoproteins, where two ApoE proteins wrap around the nanodisc in an antiparallel conformation. We further show that lipidated, recombinant ApoE accurately models astrocyte-secreted ApoE lipoproteins. Cryogenic electron microscopy of recombinant lipidated ApoE further supports ApoE adopting antiparallel dimers in nascent discoidal lipoproteins.

Keywords: Alzheimer’s disease; ApoE; HDL; apolipoprotein E; astrocytes; cryo-EM; glia; lipoprotein.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests D.M.H. is an inventor on a patent licensed by Washington University to NextCure on the therapeutic use of anti-ApoE antibodies. D.M.H. co-founded and is on the scientific advisory board of C2N Diagnostics. D.M.H. is on the scientific advisory board of Denali, Genentech, and Cajal Neuroscience; consults for Asteroid; and is on the advisory board for Cell.

Figures

Figure 1:
Figure 1:. ApoE Forms Antiparallel Dimers in Discoidal Astrocyte Secreted ApoE Lipoproteins
A. Negative stain TEM lowpass filtered images and 2D class averages of anti-ApoE HJ15.10 Fab fragment bound to astrocyte-secreted ApoE lipoprotein. 2D classes show the presence of two ApoE antibody Fab fragments bound to discoidal ApoE lipoprotein. B. Negative stain TEM lowpass filtered images and 2D class averages of anti-ApoE HJ15.30 F(ab’)2 fragment bound to astrocyte-secreted ApoE lipoprotein. 2D classes show the presence of two ApoE bound to discoidal ApoE lipoprotein. Binding of the F(ab’)2 fragment suggests that ApoE adopts an antiparallel orientation on astrocyte ApoE lipoprotein.
Figure 2:
Figure 2:. Lipidated Recombinant ApoE Forms Antiparallel Dimers in Discoidal Lipoproteins
A. 2D classes of rApoE4-DMPC lipoprotein bound by anti-ApoE HJ15.10 Fab fragment. Micrographs represent cryoEM images taken on Titan Krios operating at 300kV at 59,000x magnification with volta phase plate (1.081Å/pixel). Side views show lipid bilayer consistent with ApoE forming discoidal nanodiscs. Side views also show monoclonal Fab fragment binding to the “top” and “bottom” of the nanodisc. B. 2D classes of rApoE4-DMPC lipoprotein bound by anti-ApoE HJ15.30 F(ab’)2 fragment. Micrographs represent cryoEM images taken on Titan Krios operating at 300kV at 75,000x magnification with volta phase plate (0.928Å/pixel). Binding of the F(ab’)2 fragment suggests that ApoE adopts an antiparallel orientation consistent with the “double belt” model. C. 2D classes of gradient fixed rApoE4-DMPC lipoprotein bound by anti-ApoE HJ15.30 F(ab’)2 fragment. Micrographs represent cryoEM images taken on Glacios operating at 200kV at 150,000x magnification (0.928Å/pixel). D. Representative 2D classes from negative stain TEM imaging of rApoE2-DMPC, rApoE3-DMPC, rApoE4-DMPC bound by anti-ApoE HJ15.30 F(ab’)2 fragment. These data show that ApoE adopts an antiparallel conformation on discoidal HDL-like lipoprotein regardless of ApoE isoform. E. 2D classes from negative stain TEM micrographs of rApoE4-DMPC bound simultaneously by anti-ApoE antibody HJI 5.10 monoclonal Fab fragment and anti-ApoE antibody HJ15.30 monoclonal F(ab’)2 fragment.
Figure 3:
Figure 3:. CryoEM Density Maps of Lipidated ApoE
A. 3D cryoEM density map of lipidated rApoE4-DMPC bound by anti-ApoE HJ15.30 F(ab’)2 fragment. CryoEM density map shows binding of the F(ab’)2 fragment to the “top” and “bottom” of the nanodisc. Strong electron density is observed on the side of the nanodisc consistent with two α-helical chains wrapping around the nanodisc (cyan arrows). Strong electron density is also observed opposite of the F(ab’)2 binding site where the lipoprotein is slightly “pointed” (fuchsia arrow). Micrographs used were collected on a Titan Krios operating at 300kV at 75,000x magnification (0.928Å/pixel). B. 3D cryoEM density map of lipidated rApoE4-DMPC bound by anti-ApoE antibody HJ15.30 derived monoclonal F(ab’)2 fragment. Higher resolution of the cryoEM density map allows for better resolution of the electron density of two α-helical chains wrapping around the nanodisc (cyan arrows). Consistent with the lower resolution model, strong electron density is also observed opposite of the F(ab’)2 binding site where the lipoprotein is slightly “pointed” (fuchsia arrow). Micrographs used were collected on a Glacios operating at 200kV at 150,000x magnification (0.928Å/pixel). C. Cartoon model showing two ApoE wrapping around a nanodisc showing the “double belt” model of lipidated ApoE. Helical domains are represented by cylinders. Epitope of anti-ApoE antibody HJ15.30 F(ab’)2 fragment is represented as a red cylinder. Created with BioRender.com
See this image and copyright information in PMC

References

    1. Mahley RW, Weisgraber KH, and Huang Y (2009). Apolipoprotein E: structure determines function, from atherosclerosis to Alzheimer’s disease to AIDS. J. Lipid Res 50, S183–S188. 10.1194/jlr.R800069-JLR200. - DOI - PMC - PubMed
    1. Huang Y, and Mahley RW (2014). Apolipoprotein E: Structure and function in lipid metabolism, neurobiology, and Alzheimer’s diseases. Neurobiol. Dis 72, 3–12. 10.1016/j.nbd.2014.08.025. - DOI - PMC - PubMed
    1. Chen Y, Strickland MR, Soranno A, and Holtzman DM (2021). Apolipoprotein E: Structural Insights and Links to Alzheimer Disease Pathogenesis. Neuron 109, 205–221. 10.1016/j.neuron.2020.10.008. - DOI - PMC - PubMed
    1. Boyles JK, Pitas RE, Wilson E, Mahley RW, and Taylor JM (1985). Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system. J. Clin. Invest 76, 1501–1513. 10.1172/JCI112130. - DOI - PMC - PubMed
    1. Pitas RE, Boyles JK, Lee SH, Hui D, and Weisgraber KH (1987). Lipoproteins and their receptors in the central nervous system. Characterization of the lipoproteins in cerebrospinal fluid and identification of apolipoprotein B,E(LDL) receptors in the brain. J. Biol. Chem 262, 14352–14360. 10.1016/s0021-9258(18)47945-8. - DOI - PubMed

MeSH terms

LinkOut - more resources