Nature and Significance of the Interactions between Amyloid Fibrils and Biological Polyelectrolytes ² Martino Calamai, ‡ Janet R. Kumita, ‡ John Mifsud, ‡ Claudia Parrini, § Matteo Ramazzotti, § Giampietro Ramponi, § Niccolo ´ Taddei, § Fabrizio Chiti, § and Christopher M. Dobson* ,‡ Department of Chemistry, UniVersity of Cambridge, Lensfield Road, Cambridge, CB2 1EW, U.K., and Dipartimento di Scienze Biochimiche, UniVersita ` degli Studi di Firenze, Viale Morgagni 50, 50134, Firenze, Italy ReceiVed May 29, 2006; ReVised Manuscript ReceiVed August 16, 2006 ABSTRACT: Charged polyelectrolytes such as glycosaminoglycans and nucleic acids have frequently been found associated with the proteinaceous deposits in the tissues of patients with amyloid diseases. We have investigated the nature and generality of this phenomenon by studying the ability of different polyanions, including DNA, ATP, heparin, and heparan sulfate, to promote the aggregation of amyloidogenic proteins and to bind to the resulting aggregates. Preformed amyloid fibrils of human muscle acylphosphatase and human lysozyme, proteins with a net positive charge at physiological pH values, were found to bind tightly to the negatively charged DNA or ATP. The effects of the polyelectrolytes on the kinetics of aggregation were studied for acylphosphatase, and the presence of ATP, DNA, or heparin was found to increase its aggregation rate dramatically, with a degree dependent on the net charge and size of the polyanion. Magnesium or calcium ions were found to attenuate, and ultimately to suppress, these interactions, suggesting that they are electrostatic in nature. Moreover, heparin was found to stabilize the aggregated state of acylphosphatase through compensation of electrostatic repulsion. Noteworthy, differences in affinity between native and aggregated acylphosphatase with heparin suggest that amyloid fibrils can themselves behave as polyelectrolytes, interacting very strongly with other polyelectrolytes bearing the opposite charge. Within an in vivo context, the strengthening of the electrostatic interactions with other biological polyelectrolytes, as a consequence of protein misfolding and aggregation, could therefore result in depletion of essential molecular components and contribute to the known cytotoxicity of amyloid fibrils and their precursors. Amyloid diseases are characterized by the deposition in a variety of tissues of specific proteins as aggregated species that share a distinctive fibrillar ultrastructure (1-3). Although amyloid deposits are predominantly proteinaceous, as dem- onstrated by Friedreich and Kekule in 1859 (4), careful examination of diseased tissues has revealed the presence of a significant quantity of polysaccharide species associated with the deposits. These polysaccharides belong to the glycosaminoglycan family (GAGs 1 ) and are long unbranched chains of repeating disaccharide units. Among these species, heparan sulfate is the most common, being found in a variety of amyloid disorders including Alzheimer’s disease, type II diabetes, light chain amyloidosis, and the prion related diseases (5-7). While direct binding of heparan sulfate or heparin, the hypersulfated form of heparan sulfate, to soluble amyloid precursor proteins has been established in many cases, the ability of these polymers to promote fibrillogenesis has been investigated for only a few peptides or proteins, such as the A peptides (8, 9), tau (10, 11), R-synuclein (12), and 2-microglobulin (13). GAGs are not, however, the only polyanions found to be associated with amyloid fibrils. In the brain tissue from victims of Alzheimer’s disease, for example, nucleic acids have been detected in neurofibrillary tangles, intracellular inclusions primarily composed of the tau protein, as well as in senile plaques composed of the A peptides (14). Moreover, it has been shown that RNA is able to stimulate the aggregation of tau (11, 15) and to induce the conversion of PrP C to PrP Sc (16), while DNA can promote fibril formation by R-synuclein (17). Interestingly, DNA can bind strongly after cell lysis to the amyloid-like Curli fibrils of Escherichia coli (18), and ATP has been found to promote the formation of fibrils of A peptide and amylin (19, 20). Despite the ubiquitous presence of polyanions in amyloid deposits, the degree of specificity and the nature of the interactions involved are controversial. While attempts to identify a heparin binding consensus sequence within amy- loidogenic precursors have been challenging (7), several studies have demonstrated the importance of electrostatic interactions in the binding of polyanions to amyloid fibrils (21-24). In particular, it has been shown that removal of ² This work was supported by grants from the Wellcome and Leverhulme Trust, from the European Commission (Research Director- ates, Project HPRN-CT-2002-00241), and from the Italian MIUR (PRIN 2003025755•003 and FIRB RBAVO15B47). * To whom correspondence should be addressed. E-mail: cmd44@ cam.ac.uk. Tel: +44(0)1223763070. Fax: +44(0)1223336362. ‡ University of Cambridge. § Universita ` degli Studi di Firenze. 1 Abbreviations: AcP, human muscle acylphosphatase; ATR-FTIR, attenuated total reflection Fourier transform infrared; bzP, benzoyl phosphate; GAGs, glycosaminoglycans; HL, human lysozyme; ssDNA and dsDNA, single and double stranded DNA, respectively; TFE, 2,2,2- trifluoroethanol; ThT, thioflavin T. 12806 Biochemistry 2006, 45, 12806-12815 10.1021/bi0610653 CCC: $33.50 © 2006 American Chemical Society Published on Web 09/19/2006