Abstract 116: Apolipoprotein E Registry In Discoidal Lipoproteins Influences Uptake

Abstract

Introduction: Apolipoprotein E (apoE) is of great interest due to its role as a risk factor for Alzheimer's disease and atherosclerosis. ApoE is secreted by astrocytes in the central nervous system as HDL-like lipoparticles. Currently there is no high-resolution structural model of lipidated apoE, resulting in a limited mechanistic understanding of how apoE functions in health and disease. Research Questions: The goal of this research was to utilize cryoEM to characterize the structure of apoE in discoidal lipoproteins. Methods: Astrocyte secreted apoE was immunopurified from culture media and characterized by TEM and cryoEM. Recombinant apoE (rapoE) was lipidated with DMPC to model astrocyte secreted lipoproteins. Results: TEM and cryoEM imaging of astrocyte apoE and rapoE found that apoE forms a dimer on discoidal lipoproteins. These results provide support for the “double belt” model of two apoE in an anti-parallel conformation. This conformation was independent of apoE isoform, suggesting it is a general feature of apoE in discoidal lipoproteins. ApoE isoforms differ by the addition or loss of a Cys residue. ApoE2 has 2 Cys residues (Cys112, Cys158), apoE3 has 1 Cys residue (Cys112), and apoE4 has no Cys residues. Accordingly, apoE4 cannot form disulfide bonds, apoE3 can form intermolecular disulfide bonds, and apoE2 can form both intramolecular and intermolecular disulfide bonds. Due to the presence of two apoE on discoidal lipoproteins, apoE3 can form a disulfide bridge between the two apoE protein. Disulfide bridge formation and experimental reduction of these disulfide bridges revealed that disulfide-linked apoE3 was uptaken significantly more by Neuro2a cells (P ≤ 0.0001) than nondisulfide-linked apoE3. Conclusion: All isoforms of apoE adopt an antiparallel dimer in discoidal lipoprotein. Different registries, regulated through the formation of disulfide bonds between the anti-parallel apoE polypeptides, leads to differential uptake of apoE by Neuro2a cells. Since the different isoforms of apoE have inherently different capacity to generate disulfide bridges, we conclude that this property affecting receptor uptake may strongly impact differential biological properties of the different isoforms.