Differences in stability among the human apolipoprotein E isoforms determined by the amino-terminal domain
Abstract
Denaturation by guanidine-HCl, urea, or heating was performed on the common isoforms of human apolipoprotein (apo) E (apoE2, apoE3, and apoE4) and their 22-kDa and 10-kDa fragments in order to investigate the effects of the cysteine/arginine interchanges at residues 112 and 158. Previous physical characterization of apoE3 established that apoE contains two domains, the 10-kDa carboxyl-terminal and 22-kDa amino-terminal domains, which unfold independently and exhibit large differences in stability. However, the physical properties of apoE2, apoE3, and apoE4 have not been compared before. Analysis by circular dichroism showed that the different isoforms have identical alpha-helical contents and guanidine-HCl denaturation confirmed that the two domains unfold independently in all three isoforms. However, guanidine-HCl, urea, and thermal denaturation showed differences in stability among the 22-kDa amino-terminal fragments of the apoE isoforms (apoE4 < apoE3 < apoE2). Furthermore, guanidine-HCl denaturation monitored by circular dichroism and fluorescence suggested the presence of a folding intermediate in apoE, most prominently in apoE4. Thus, these studies reveal that the major isoforms of apoE, which are associated with different pathological consequences, exhibit significant differences in stability.
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The lower cortical APOE abundance in APOE3/4 mice likely reflects reduced stability of the APOE4 protein.
“The lower cortical APOE abundance in APOE3/4 mice likely re fl ects the reduced stability of the APOE4 protein, as previously reported 11 .”
From APOE2 attenuates APOE4-associated amyloid pathology and related toxicity in a mouse model of Alzheimer’s disease (2026) · cites this paper as [11]
ApoE undergoes a two-phase unfolding transition when treated with GndHCl, with an unfolding intermediate appearing between 1 and 2 M.
“ApoE has been shown to undergo a two-phase unfolding transition when treated with GndHCl, with an unfolding intermediate appearing between 1 and 2 M (36).”
From Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy (2018) · cites this paper as [36]
ApoE2, which harbors the R158C substitution, has similar α-helical content as apoE3 and appears to be more thermodynamically stable compared with apoE3.
“ApoE2, which harbors the R158C substitution, has similar -helical content as apoE3 and appears to be more thermodynamically stable compared with apoE3 (36).”
From Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy (2018) · cites this paper as [36]
The polymorphism at position 112 brings about alterations in the thermodynamic properties of the molecule that may underlie functional changes linked with disease predisposition.
“This polymorphic variation brings about alterations in the thermodynamic properties of the molecule that may underlie functional changes linked with disease predisposition [61], [65], [71], [72], [73], [74].”
From Biophysical Analysis of Apolipoprotein E3 Variants Linked with Development of Type III Hyperlipoproteinemia (2011) · cites this paper as [65]
Functional differences between apoE4 and apoE3 have been attributed to altered inter-domain interactions brought about by the polymorphism at position 112 of the N-terminal domain.
“Specifically, functional differences between apoE4 and apoE3 have been attributed to altered inter-domain interactions brought about by the polymorphism at position 112 of the N-terminal domain.”
From Biophysical Analysis of Apolipoprotein E3 Variants Linked with Development of Type III Hyperlipoproteinemia (2011) · cites this paper as [65]
Differences in function and disease predisposition are established between apoE3 and apoE2, although these two variants differ only at a single amino acid at position 158.
“Similarly, differences in function and disease predisposition are established between apoE3 and apoE2 although these two variants differ only at a single amino acid at position 158 [65], [72], [74].”
From Biophysical Analysis of Apolipoprotein E3 Variants Linked with Development of Type III Hyperlipoproteinemia (2011) · cites this paper as [65]
The second unfolding transition of apoE3 variant corresponds to the unfolding of the N-terminal domain where the K146E mutation is located.
“of the second unfolding transition of apoE3 variant (Table 3) that corresponds to the unfolding of the N-terminal domain where the K146E mutation is located [13], [44], [65].”
From Biophysical Analysis of Apolipoprotein E3 Variants Linked with Development of Type III Hyperlipoproteinemia (2011) · cites this paper as [65]
ApoE4 exhibits reduced thermodynamic stability compared to ApoE3 in prior studies using purified recombinant protein.
“ApoE4 exhibits reduced thermodynamic stability compared to ApoE3, but prior characterisation has relied on purified recombinant protein”
From Utilizing a cell culture based novel cellular thermal shift assay to understand the isoform-dependent thermal stability of ApoE variants (2026) · cites this paper as [32]
The delta of the stability difference between ApoE3 and ApoE4 in BiTSA is approximately 2-3°C.
“The delta of the stability difference between ApoE3 and ApoE4 (~2-3°C) was consistent with previous reports using purified recombinant protein 28,32”
From Utilizing a cell culture based novel cellular thermal shift assay to understand the isoform-dependent thermal stability of ApoE variants (2026) · cites this paper as [32]
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- Authors
- Morrow, J. A., Segall, M. L., Lund-Katz, S., Phillips, M. C., Knapp, M., Rupp, B., & Weisgraber, K. H.
- Affiliations
- Lawrence Livermore National Laboratory (US)
- Gladstone Institute of Neurological Disease, Cardiovascular Research Institute, Department of Pathology, University of California, San Francisco, California 94110, Department of Biochemistry, MCP Hahnemann University, Philadelphia, Pennsylvania 19129, and Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94550
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2000-08-26
- Volume
- 39
- Issue
- 38
- Pages
- 11657-11666
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2090688839
- Cited by
- 315
- References
- 6
- Citations by year
- 2026: 3 · 2025: 13 · 2024: 6 · 2023: 15 · 2022: 12 · 2021: 10 · 2020: 7 · 2019: 18 · 2018: 13 · 2017: 13 · 2016: 23 · 2015: 24 · 2014: 23 · 2013: 17 · 2012: 16
- FWCI
- 2.05
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Heart, Lung, and Blood Institute (HL-41633, P01HL041633, R01HL056083, HL-56083)
- National Institute of Neurological Disorders and Stroke (NS35939, R01NS035939)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Keywords
- apolipoprotein E, apolipoprotein E isoforms, protein stability, N-terminal domain, C-terminal domain, folding intermediates, guanidine hydrochloride denaturation, urea denaturation, thermal denaturation, alpha-helical content, protein folding
- MeSH terms
- Apolipoproteins E, Catalysis, Hot Temperature, Humans, Hydrolysis, Molecular Weight, Peptide Fragments, Protein Denaturation, Thermodynamics, Thrombin, Urea, Protein Structure, Secondary, Protein Structure, Tertiary, Protein Folding, Guanidine, Protein Isoforms
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