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Insights into the C-terminal domain of apolipoprotein E from chimera studies with apolipophorin III

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Horn, James V. C., Kakutani, Leesa M., Narayanaswami, Vasanthy, Weers, Paul M. M.·2023·Molecular and Cellular Biochemistry
DOI 10.1007/s11010-022-04497-yPMC PMC11479662
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Abstract

Apolipoprotein E3 (apoE) is a critical cholesterol transport protein in humans and is composed of two domains: a well characterized N-terminal (NT) domain that harbors the low-density lipoprotein LDL receptor, and a less understood C-terminal (CT) domain that is the site of protein oligomerization and initiation of lipid binding. To better understand the domain structure of apoE, the CT domain was fused to apolipophorin III (apoLp-III), a single-domain, monomeric apolipoprotein of insect origin, to yield a chimeric protein, apoLp-III/CT-apoE. Recombinant apoLp-III/CT-apoE maintained an overall helical content similar to that of the parent proteins, while chemical induced unfolding studies indicated that its structural integrity was not compromised. Analysis using 1-anilinonaphthalene-8-sulfonic acid (ANS), a sensitive fluorescent indicator of exposed hydrophobic sites and protein folding, demonstrated that whereas apoLp-III provided few ANS binding sites, apoLp-III/CT-apoE harbored an abundance of ANS binding sites. Thus, this indicated tertiary structure formation in CT-apoE when part of the chimera. Size-exclusion chromatography and chemical crosslinking analysis demonstrated that while apoLp-III is monomeric, the chimeric protein formed large oligomeric complexes, similar to native apoE3. Compared to apoLp-III, the chimera showed a two-fold enhancement in phospholipid vesicle solubilization rates and a significantly improved ability to bind to lipolyzed low-density lipoprotein, preventing the onset of lipoprotein aggregation at concentrations comparable to that of parent CT-apoE. These results confirm that high lipid binding and self-association sites are located in the CT domain of apoE, and that these properties can be transferred to an unrelated apolipoprotein, demonstrating that these properties operate independently from the NT domain.

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Sorted by citations

Authors
Horn, J. V. C., Kakutani, L. M., Narayanaswami, V., & Weers, P. M. M.
Affiliations
  • California State University, Long Beach (US)
  • California State University System (US)
Corresponding author
Weers, Paul M. M.
ORCID
Venue
Molecular and Cellular Biochemistry
Publisher
Springer Science and Business Media LLC
Published
2022-06-28
Volume
478
Issue
1
Pages
173-183
ISSN
  • 0300-8177 (ISSN-L)
  • 1573-4919
DOI
10.1007/s11010-022-04497-y
PMC
PMC11479662
OpenAlex
W4283645110
Cited by
3
References
7
Citations by year
2025: 3
FWCI
0.24
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular and Cellular Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
National Institute of General Medical Sciences (GM089564, GM105561, 5UL1GM118979, 5TL4GM118980, 5RL5GM118978)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Health Sciences › Medicine › Surgery › Cholesterol and Lipid Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Lipid Membrane Structure and Behavior
Keywords
apolipoprotein E, C-terminal domain, apolipophorin III, fusion protein, protein oligomerization, lipid binding, low-density lipoprotein, cholesterol transport, protein structure, ANS binding, lipoprotein aggregation
MeSH terms
Apolipoproteins, Apolipoproteins E, Humans, Lipoproteins, LDL, Protein Binding, Recombinant Fusion Proteins, Recombinant Proteins

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