Structural Basis for the Recognition and Cross-linking of Amyloid Fibrils by Human Apolipoprotein E

Menachem J Gunzburg,Matthew A Perugini,Geoffrey J Howlett

doi:10.1074/jbc.m706425200

Menachem J Gunzburg, Matthew A Perugini + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m706425200

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Abstract

Apolipoprotein (apo) E is a well characterized lipid-binding protein in plasma that also exists as a common nonfibrillar component of both cerebral and systemic amyloid deposits. A genetic link between a common isoform of apoE, apoE4, and the incidence of late onset Alzheimer disease has drawn considerable attention to the potential roles of apoE in amyloid-related disease. We examined the interactions of apoE with amyloid fibrils composed of apoC-II and the amyloid-beta (Abeta) peptide. Aggregates of apoE with Abeta and apoC-II are found in Alzheimer and atherosclerotic plaques, respectively. Sedimentation velocity and fibril size distribution analysis showed that apoE3 and E4 isoforms bind and noncovalently cross-link apoC-II fibrils in a similar manner. This ability to cross-link apoC-II fibrils was abolished by the dissociation of the apoE tetramer to monomers or by thrombin cleavage to yield separate N- and C-terminal domains. Preparative ultracentrifuge binding studies indicated that apoE and the isolated N- and C-terminal domains of apoE bind with submicromolar affinities to both apoC-II and Abeta fibrils. Fluorescence quenching and resonance energy transfer experiments confirmed that both domains of apoE interact with apoC-II fibrils and demonstrated that the binding of the isolated N-terminal domain of apoE to apoC-II or Abeta fibrils is accompanied by a significant conformational change with helix three of the domain moving relative to helix one. We propose a model involving the interaction of apoE with patterns of aligned residues that could explain the general ability of apoE to bind to a diverse range of amyloid fibrils.

Highlights

Form and the occurrence of Alzheimer disease, with APOE4 conveying a higher risk and earlier age of onset of Alzheimer disease than APOE3 [5]
Recent studies suggest that apoE may form amyloid fibrils directly [7], the observation that apoE co-localizes in all amyloid deposits [2, 4] and interacts with amyloid fibrils in vitro, including fibrils composed of A␤-(1– 40), A␤-(1– 42), ␤2-microglobulin, gelsolin, and apoC-II (8 –11), suggest that apoE plays a general role as a nonfibrillar amyloid component
These results suggest that nonfibrillar components, such as serum amyloid P (SAP) and apoE, may act as mediators of amyloid fibril deposition and stability

Summary

EXPERIMENTAL PROCEDURES

Materials—The pET32a expression vectors for apoE3, apoE4, apoE3 N-terminal domain, apoE C-terminal domain, apoE4S53C, apoE4S76C, apoE4A241C, apoE4S263C, and apoE4T289C were kindly provided by Karl Weisgraber and Danny Hatters (Gladstone Institute, San Francisco) [22]. The concentration of unbound ligand was calculated and corrected for the presence of a small amount of free monomeric apoC-II or A␤-(1– 40) measured for control samples of apoC-II or A␤-(1– 40) fibrils in the absence of apoE. Acrylamide Quenching—AEDANS-labeled apoE derivatives in the presence and absence of preformed apoC-II fibrils were incubated in the presence of varying concentrations of acrylamide (0 – 0.5 M) in TBS. Emission scans of these samples were taken. Fluorescence Resonance Energy Transfer—The emission spectra of AEDANS-labeled and unlabeled apoE3 N-terminal domain was measured in the presence and absence of preformed apoC-II or A␤-(1– 40) fibrils in TBS. Images were generated using PyMOL [46]

RESULTS

KDa n

Relative change in emission intensityc

FRET distanceb