Cyclization of Peptides Enhances the Inhibitory Activity against Ganglioside-Induced Aβ Fibril Formation

Abstract

Alzheimer’s disease is a progressive neurodegenerative disease and is the most common cause of dementia. It has been reported that the assembly of amyloid β-protein (Aβ) on the cell membrane is induced by the interaction of the Aβ monomer with gangliosides such as GM1. The ganglioside-bound Aβ (GAβ) complex acts as a seed to promote the toxic assembly of the Aβ fibrils. In a previous study, we found that a GM1 cluster-binding peptide (GCBP) specifically recognizes Aβ-sensitive ganglioside nanoclusters and inhibits the assembly of Aβ on a GM1-containing lipid membrane. In this study, cysteine-substituted double mutants of GCBP were designed and cyclized by intramolecular disulfide bond formation. Affinity assays indicated that one of the cyclic peptides had a higher affinity to a GM1-containing membrane compared to that of GCBP. Furthermore, surface topography analysis indicated that this peptide recognizes GM1 nanoclusters on the lipid membrane. An evaluation of the inhibitory kinetics indicated that the cyclic peptide could inhibit the formation of Aβ fibrils with an IC50 value of 1.2 fM, which is 10,000-fold higher than that of GCBP. The cyclic peptide was also shown to have a clearance effect on Aβ fibrils deposited on the lipid membrane and suppressed the formation of toxic Aβ assemblies. Our results indicate that the cyclic peptide that binds to the Aβ-sensitive ganglioside nanocluster is a potential novel inhibitor of ganglioside-induced Aβ assembly.

Prevention of amyloid β fibril deposition on the synaptic membrane in the precuneus by ganglioside nanocluster-targeting inhibitors

“…Similar results were obtained by AFM in the case of the clearance of Aβ fibrils on the 10% GM1-containing membrane. 42,43 The control peptide cpVIII did not show any binding or release of Aβ.…”

“…Disruption of the GAβ complex by the binding of peptide inhibitors to ganglioside nanoclusters is a promising mechanism to reduce Aβ deposition, as reported previously. 42,43…”

“…44 In previous studies, GCBP and cy5A have been shown to inhibit GM1-induced Aβ aggregation with IC 50 values of 12 pM and 1.2 fM, respectively. 42,43 Alanine scanning has revealed that arginines (Arg3 and Arg12) and hydrophobic amino acids (Trp3 and Phe9) of GCBP and cy5A interact with sialic acid and the galactose ring of gangliosides. 17 Although several kinds of gangliosides in SPM lipids likely contribute to Aβ fibril formation, the design of inhibitors, based on our peptides, against ganglioside-induced Aβ assembly, is a promising strategy.…”

“…5(A) ). 43 To determine the binding of GCBP and cy5A, a monolayer of SPM PC and CC lipids was loaded on plastic discs. The amount of GCBP and cy5A (10 μM) bound to the membrane with PC lipids was 26.3% and 11.6% higher than that for CC lipids, respectively ( Fig.…”

“…Synthetic linear (GCBP and cpVIII) and cyclic (cy5A) peptide amides (purity >95%) carrying a biotinyl group at the C-terminus with biotinylated lysine are listed in Table S1 (ESI†). 42,43 Peptide stock solutions (0.1–1 mM) were prepared in PBS (pH 7.4) and stored at −20 °C until use.…”