Apolipoprotein E secreted by astrocytes forms antiparallel dimers in discoidal lipoproteins.

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.

Structural analyses of ß2-glycoprotein I: is there a circular conformation?

BACKGROUND: Antiphospholipid antibodies targeting ß2-glycoprotein I (ß2GPI) cause thrombosis and pregnancy morbidity in antiphospholipid syndrome (APS) patients. How these antibodies recognize ß2GPI remains controversial. OBJECTIVES: This study aimed to elucidate the structure of ß2GPI and evaluate how pathogenic anti-domain I (DI) antibodies recognize it in human plasma. METHODS: ß2GPI was made recombinant and purified from human plasma using different protocols. Structural and functional analyses were conducted using orthogonal techniques, namely, electron microscopy, size-exclusion chromatography, single-molecule Förster resonance energy transfer, and microfluidic diffusional sizing. RESULTS: Electron microscopy and size-exclusion chromatography showed that the structure of ß2GPI produced recombinantly and purified from plasma is elongated, even when subjected to conditions previously reported to favor circularization. Single-molecule Förster resonance energy transfer analyses of ß2GPI labeled at positions 88 in DII and 278 in DV showed that these residues are located >90 Å apart, consistent with an elongated form. They also documented that the distance between these 2 residues did not change when the protein was reconstituted in human plasma. Microfluidic diffusional sizing documented that ß2GPI binds with moderate affinity to a prototypical anti-DI antibody targeting the epitope G40-R43 despite being elongated. CONCLUSION: Circulating ß2GPI is elongated and, therefore, fully capable of binding to anti-DI antibodies. Binding of ß2GPI to negatively charged phospholipids drives autoantibody recognition by increasing the local concentration of the antigen and not by dramatically changing its conformation. These findings clarify the structural properties of ß2GPI, which have important implications for understanding APS pathogenesis and the development of APS diagnostics and therapeutics.