Heparin-binding Properties of the Amyloidogenic Peptides Aβ and Amylin

Abstract

Aggregation and deposition of the 40–42-residue amyloid β-protein (Aβ) are early and necessary neuropathological events in Alzheimer's disease. An understanding of the molecular interactions that trigger these events is important for therapeutic strategies aimed at blocking Aβ plaque formation at the earliest stages. Heparan sulfate proteoglycans may play a fundamental role since they are invariably associated with Aβ and other amyloid deposits at all stages. However, the nature of the Aβ-heparan sulfate proteoglycan binding has been difficult to elucidate because of the strong tendency of Aβ to self-aggregate. Affinity co-electrophoresis can measure the binding of proteoglycans or glycosaminoglycans to proteins without altering the physical state of the protein during the assay. We used affinity co-electrophoresis to study the interaction between Aβ and the glycosaminoglycan heparin and found that the aggregation state of Aβ governs its heparin-binding properties: heparin binds to fibrillar but not nonfibrillar Aβ. The amyloid binding dye, Congo red, inhibited the interaction in a specific and dose-dependent manner. The “Dutch” mutant AβE22Q peptide formed fibrils more readily than wild type Aβ and it also attained a heparin-binding state more readily, but, once formed, mutant and wild type fibrils bound heparin with similar affinities. The heparin-binding ability of aggregated AβE22Q was reversible with incubation in a solvent that promotes α-helical conformation, further suggesting that conformation of the peptide is important. Studies with another human amyloidogenic protein, amylin, suggested that its heparin-binding properties were also dependent on aggregation state. These results demonstrate the dependence of the Aβ-heparin interaction on the conformation and aggregation state of Aβ rather than primary sequence alone, and suggest methods of interfering with this association. Aggregation and deposition of the 40–42-residue amyloid β-protein (Aβ) are early and necessary neuropathological events in Alzheimer's disease. An understanding of the molecular interactions that trigger these events is important for therapeutic strategies aimed at blocking Aβ plaque formation at the earliest stages. Heparan sulfate proteoglycans may play a fundamental role since they are invariably associated with Aβ and other amyloid deposits at all stages. However, the nature of the Aβ-heparan sulfate proteoglycan binding has been difficult to elucidate because of the strong tendency of Aβ to self-aggregate. Affinity co-electrophoresis can measure the binding of proteoglycans or glycosaminoglycans to proteins without altering the physical state of the protein during the assay. We used affinity co-electrophoresis to study the interaction between Aβ and the glycosaminoglycan heparin and found that the aggregation state of Aβ governs its heparin-binding properties: heparin binds to fibrillar but not nonfibrillar Aβ. The amyloid binding dye, Congo red, inhibited the interaction in a specific and dose-dependent manner. The “Dutch” mutant AβE22Q peptide formed fibrils more readily than wild type Aβ and it also attained a heparin-binding state more readily, but, once formed, mutant and wild type fibrils bound heparin with similar affinities. The heparin-binding ability of aggregated AβE22Q was reversible with incubation in a solvent that promotes α-helical conformation, further suggesting that conformation of the peptide is important. Studies with another human amyloidogenic protein, amylin, suggested that its heparin-binding properties were also dependent on aggregation state. These results demonstrate the dependence of the Aβ-heparin interaction on the conformation and aggregation state of Aβ rather than primary sequence alone, and suggest methods of interfering with this association. A hallmark of both Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid β-peptide; HSPG, heparan sulfate proteoglycan; ACE, affinity co-electrophoresis; HCHWA-D, Hereditary Cerebral Hemorrhage with Amyloidosis, Dutch-type; HPLC, high performance liquid chromatography; CSF, cerebrospinal fluid; NaMOPSO, 3-[N-morpholino]-2-hydroxypropanesulfonic acid, sodium salt; LMW, low molecular weight; HFIP, (1,1,1,3,3,3)-hexafluoroisopropanol. and Down's syndrome is the presence of numerous extracellular deposits of the amyloid β-protein (Aβ), termed senile or neuritic plaques, in the brain parenchyma. In most cases, Aβ is also deposited in the walls of parenchymal and meningeal blood vessels. In these two types of deposits, Aβ exists largely in a fibrillar form consisting of 40 or 42 amino acid monomers aggregated into insoluble filamentous polymers. Aβ, which is derived by endoproteolysis from the β-amyloid precursor protein (1Kang J. Lemaire H.-G. Unterbeck A. Salbaum J.M. Masters C.L. Greschik K.-H. Multhaup G. Beyreuther K. Müller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3968) Google Scholar, 2Haass C. Schlossmacher M.G. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B.L. Lieberburg I. Koo E.H. Schenk D. Teplow D.B. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1767) Google Scholar), is by far the major constituent of plaques (3Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3699) Google Scholar, 4Selkoe D.J. Abraham C.R. Podlisny M.B. 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In were obtained with suggesting in or the that some Aβ fibrils are on the walls of and in the of Aβ to be at the are in of of and were by amino acid for were these of a on the of fibrils a of fibrillar or nonfibrillar Aβ were for at in In some cases, of Congo red or of were these were for The were to by a by one with The was and the bound on the was in a in were from the for from Aβ and heparin were to a for Aβ-heparin to measure the binding of Aβ to heparin were of wild type were in and were into the of a and was to the of Aβ is than that of heparin not a of heparin and Aβ to have a than that of this is the binding of Aβ to heparin have been as a of that were with Aβ In of Aβ on heparin was at peptide to These that Aβ and heparin do not the of the or that binding could not be detected because the of the Aβ-heparin is not from that of heparin the the of heparin and peptide in the A.D. P. A to Scholar). In this heparin was into the into a to the Because heparin is more than Aβ, the was between the heparin and the In this binding of heparin to Aβ have been as a of was by heparin However, as shown in in the of was at heparin to the could have to binding of heparin and Aβ in this is the of the was not from that of the in this Aβ. However, it is not for the of the to be from both that of Aβ and that of as Aβ and heparin have that are from the results in A and that Aβ and heparin do not other Because the association of heparan sulfate with Aβ deposits in AD brain suggested an important interaction between the two the that the of fibrillar Aβ a heparin-binding Aβ into of were in and into and for at of the the formation of in similar to the and of Aβ fibrils from Alzheimer's plaques J. Neuropathol. Neurol. PubMed Scopus Google Scholar, Wisniewski H.M. Masters C.L. K. Acta Neuropathol. PubMed Scopus Google Scholar). with Congo red, Aβ fibrils In of the nonfibrillar Aβ the presence of aggregated but not fibrillar Aβ. with Congo red, the nonfibrillar Aβ was not of the Aβ fibrils were into the of an and for heparin In to was with the low of fibrillar Aβ the of the heparin that the was to a specific binding interaction and not to physical of the heparin by a of that the binding to Aβ fibrils could be by aggregated similar to that shown in E. measure binding from of several The results from were to the A.D. P. A to Scholar, R.T. A.D. 1991; PubMed Scopus Google Scholar), which an of this is in of of Aβ an of the results obtained by ACE, a binding was also of fibrillar or nonfibrillar Aβ were with in which the were to a membrane by and to measure the fibrillar but not Aβ bound heparin with a low affinity Congo red is a used for the of of all types in The binding of Congo red to Aβ has been postulated to in on fibrillar Aβ Abraham D.J. 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In the an of binding is most readily the protein in the is high to the of heparin However, the binding were to a of Aβ to that the for Congo red of heparin binding with protein similar of Aβ were in the two a for Congo red than the may be to the of the Congo red of the well during the of in the the was from a or aggregated into fibrils as of were into the of and for binding The peptide not to heparin to peptide of fibrillar AβE22Q almost the of heparin of such as that in were and peptide of were as and results from several of fibrillar AβE22Q an of is in the low similar to the for heparin binding to wild type Aβ fibrils by the In Aβ was to a aggregation a in the heparin-binding of wild type and mutant In this peptide was for at at a of in and in the wild type Aβ in a binding state not However, the mutant peptide was to heparin and an affinity similar to that of AβE22Q We this as that AβE22Q a similar to that of wild type Aβ that been aggregated in with for the presence of this conformation from the that the interaction of heparin with the AβE22Q was by Congo red in a dose-dependent In addition, the ability of these to heparin was by incubation in for and is a solvent that is to or A. 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