Abstract
Accumulation of amyloid β-protein (Aβ) into brain parenchymal plaques and the cerebral vasculature is a pathological feature of Alzheimer disease and related disorders. Aβ peptides readily form β-sheet-containing oligomers and fibrils. Previously, we reported a strong interaction between myelin basic protein (MBP) and Aβ peptides that resulted in potent inhibition of fibril assembly (Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2007) J. Biol. Chem. 282, 9952-9961; Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2009) Biochemistry 48, 4720-4727). MBP is recognized as a highly post-translationally modified protein. In the present study, we demonstrate that human MBP purified from either brain or a bacterial recombinant expression system comparably bound to Aβ and inhibited Aβ fibril assembly indicating that post-translational modifications are not required for this activity. We also show that purified mouse brain MBP and recombinantly expressed mouse MBP similarly inhibited Aβ fibril formation. Through a combination of biochemical and ultrastructural techniques, we demonstrate that the binding site for Aβ is located in the N-terminal 64 amino acids of MBP and that a stable peptide (MBP1) comprising these residues was sufficient to inhibit Aβ fibrillogenesis. Under conditions comparable with those used for Aβ, the fibrillar assembly of amylin, another amyloidogenic peptide, was not inhibited by MBP1, although MBP1 still bound to it. This observation suggests that the potent inhibitory effect of MBP on fibril formation is not general to amyloidogenic peptides. Finally, MBP1 could prevent the cytotoxic effects of Aβ in primary cortical neurons. Our findings suggest that inhibition of Aβ fibril assembly by MBP, mediated through its N-terminal domain, could play a role in influencing amyloid formation in Alzheimer disease brain and corresponding mouse models.
Highlights
The histopathology of Alzheimer disease (AD)2 is characterized by the prominent presence of plaques of amyloid -protein
We demonstrate that human myelin basic protein (MBP) purified from either brain or a bacterial recombinant expression system comparably bound to A and inhibited A fibril assembly indicating that post-translational modifications are not required for this activity
Human MBP Purified from Brain or Bacterial Recombinant Expression Binds to and Inhibits A Fibril Formation— We first compared the ability of purified MBP proteins from human brain white matter and bacterial recombinant expression to bind to A and inhibit its fibrillar assembly
Summary
Reagents and Chemicals—A peptides were synthesized on an ABI 430A solid phase peptide synthesizer (Applied Biosystems, Foster City, CA) using t-butoxycarbonyl chemistry, purified, and structurally characterized as described previously [25]. The His-MBP-containing fractions were dialyzed against 6 M urea, 80 mM glycine, pH 10.5; passed over a CM52 column; washed; and eluted with 80 mM glycine buffer, pH 10.5 containing 200 mM NaCl. Protein refolding was achieved by removal of denaturant and salt by slow dialysis into distilled water. For studies with IAPP, the peptide was resuspended to 2.5 mM in DMSO and diluted to 3.13 M in PBS containing 5 M thioflavin T either alone or with 0.391 M MBP1. Biotinylated A was immobilized to a Biacore CM4 sensor chip coated with streptavidin at 10 l/min to achieve an average Rmax of ϳ400 resonance units for each analyte leaving flow cell 1 as a reference This chip preparation procedure was found to result in a surface that minimized mass transfer effects for kinetic interaction studies. The assay was quantified by measuring the absorbance at 570 nm using a SpectraMax spectrofluorometer (Molecular Devices)
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