Abstract
The Aβ peptide forms extracellular plaques associated with Alzheimer's disease. In addition to protein fibrils, amyloid plaques also contain non-proteinaceous components, including glycosaminoglycans (GAGs). We have shown previously that the GAG low-molecular-weight heparin (LMWH) binds to Aβ40 fibrils with a three-fold-symmetric (3Q) morphology with higher affinity than Aβ40 fibrils in alternative structures, Aβ42 fibrils, or amyloid fibrils formed from other sequences. Solid-state NMR analysis of the GAG–3Q fibril complex revealed an interaction site at the corners of the 3Q fibril structure, but the origin of the binding specificity remained obscure. Here, using a library of short heparin polysaccharides modified at specific sites, we show that the N-sulfate or 6-O-sulfate of glucosamine, but not the 2-O-sulfate of iduronate within heparin is required for 3Q binding, indicating selectivity in the interactions of the GAG with the fibril that extends beyond general electrostatic complementarity. By creating 3Q fibrils containing point substitutions in the amino acid sequence, we also show that charged residues at the fibril three-fold apices provide the majority of the binding free energy, while charged residues elsewhere are less critical for binding. The results indicate, therefore, that LMWH binding to 3Q fibrils requires a precise molecular complementarity of the sulfate moieties on the GAG and charged residues displayed on the fibril surface. Differences in GAG binding to fibrils with distinct sequence and/or structure may thus contribute to the diverse etiology and progression of amyloid diseases.
Highlights
Aggregation of proteins and peptides into amyloid fibrils is responsible for more than 50 human diseases [1]
The results showed clear evidence for seeded fibril growth in which an immediate and rapid increase in thioflavin T (ThT) fluorescence occurs upon the addition of seed under the conditions employed (Fig. 1a)
The results described above provide a rationale for the ubiquitous presence of GAGs in amyloid deposits and reveal that the interaction between low-molecular-weight heparin (LMWH) and 3Q fibrils displays a specificity that extends beyond a simple electrostatic model, involving precise interactions between specific sulfates on the GAG and positively charged residues on the fibril surface
Summary
Aggregation of proteins and peptides into amyloid fibrils is responsible for more than 50 human diseases [1]. One of the most well-documented amyloid pathologies is Alzheimer's disease (AD), which results from the extracellular deposition of fibrils formed from the Aβ peptide, which ranges from 38 to 43 amino acids in length, along with intracellular deposits of the protein tau [2,3]. Heparin and its shorter derivative, low-molecular-weight heparin (LMWH), have been shown to interact with a variety of amyloid proteins in vitro including amylin [10,11], α-synuclein [12], transthyretin [13,14], β2-microglobulin [15,16], gelsolin [17,18], tau [19,20], and Aβ40/42 [21,22]. There is increasing evidence that heparin and other GAGs may be active participants in the formation of amyloid fibrils. Heparin has been shown to accelerate fibril formation [21,23], enhance fibril stability [24,25], and decrease amyloid toxicity [26,27]
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