Article

Kayed R, Head E, Sarsoza F, Saing T, Cotman CW, Necula M et al. Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers. Mol Neurodegener 2: 18

Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA. .
Molecular Neurodegeneration (Impact Factor: 6.56). 02/2007; 2(1):18. DOI: 10.1186/1750-1326-2-18
Source: PubMed

ABSTRACT

Amyloid-related degenerative diseases are associated with the accumulation of misfolded proteins as amyloid fibrils in tissue. In Alzheimer disease (AD), amyloid accumulates in several distinct types of insoluble plaque deposits, intracellular Abeta and as soluble oligomers and the relationships between these deposits and their pathological significance remains unclear. Conformation dependent antibodies have been reported that specifically recognize distinct assembly states of amyloids, including prefibrillar oligomers and fibrils.
We immunized rabbits with a morphologically homogeneous population of Abeta42 fibrils. The resulting immune serum (OC) specifically recognizes fibrils, but not random coil monomer or prefibrillar oligomers, indicating fibrils display a distinct conformation dependent epitope that is absent in prefibrillar oligomers. The fibril epitope is also displayed by fibrils of other types of amyloids, indicating that the epitope is a generic feature of the polypeptide backbone. The fibril specific antibody also recognizes 100,000 x G soluble fibrillar oligomers ranging in size from dimer to greater than 250 kDa on western blots. The fibrillar oligomers recognized by OC are immunologically distinct from prefibrillar oligomers recognized by A11, even though their sizes overlap broadly, indicating that size is not a reliable indicator of oligomer conformation. The immune response to prefibrillar oligomers and fibrils is not sequence specific and antisera of the same specificity are produced in response to immunization with islet amyloid polypeptide prefibrillar oligomer mimics and fibrils. The fibril specific antibodies stain all types of amyloid deposits in human AD brain. Diffuse amyloid deposits stain intensely with anti-fibril antibody although they are thioflavin S negative, suggesting that they are indeed fibrillar in conformation. OC also stains islet amyloid deposits in transgenic mouse models of type II diabetes, demonstrating its generic specificity for amyloid fibrils.
Since the fibril specific antibodies are conformation dependent, sequence-independent, and recognize epitopes that are distinct from those present in prefibrillar oligomers, they may have broad utility for detecting and characterizing the accumulation of amyloid fibrils and fibrillar type oligomers in degenerative diseases.

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    • "This result ruled out the possibility that EGCG interfered with the ThT assay without preventing amyloid formation, as in the case of certain quinone compounds[45]. We also confirmed, using OC antibody, which recognizes the epitope specific for amyloid fibrils regardless of their sequences, that apoA-I Iowa fibrils shared a common fibrilstructure with other amyloid species including a-synuclein, islet amyloid polypeptide, Ab and poly Q (Q36)[35]. Figure 2shows EGCG inhibited the formation of OC-reactive apoA-I Iowa fibrils, and these OC-reactive apoA-I Iowa fibrils disappeared after incubation with EGCG. Results of the ThT assay and AFM study that provided strong evidence that EGCG inhibited fibril formation of the apoA-I 1–83 fragments and disaggregated preformed apoA-I fibrils may exclude the possibility that EGCG interferes with recognition of the OC epitope. "
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    ABSTRACT: Introduction: Apolipoprotein A-I (apoA-I) amyloidosis is either a non-hereditary form with deposits of wild-type apoA-I proteins in atherosclerotic plaques or a hereditary form with progressive accumulation of mutant apoA-I proteins in different tissues. Several small polyphenolic molecules reportedly inhibited formation of fibrillar assemblies of some amyloidogenic proteins and their cytotoxicity, but small molecules that inhibit apoA-I fibril formation have never been reported.Methods: Our methods included a thioflavin-T-binding assay, atomic force microscopy and dot blot and cell-based assays.Results: We showed that (−)-epigallocatechin-3-gallate (EGCG), a tea-derived flavanol, inhibited in vitro fibril formation and disaggregated fibrils preformed by the N-terminal 1–83 fragments of wild-type (WT) apoA-I and the G26R point mutation of apoA-I (apoA-IIowa). We eliminated a common structure recognized by the anti-amyloid antibody OC by incubating apoA-IIowa with EGCG or treating apoA-IIowa fibrils with EGCG, which supported the above observation. In addition, EGCG rescued human embryonic kidney 293 cells from cytotoxicity and attenuated production of reactive oxygen species, which were induced by apoA-IIowa fibrils.Conclusions: Our results support the concept that EGCG inhibits amyloid fibril formation of various amyloidogenic proteins. Thus, EGCG may be a candidate for providing a structure to develop de novo inhibitors for amyloidosis treatment.
    No preview · Article · Dec 2015 · Amyloid
    • "For example , A11 polyclonal antibodies specifically recognize antiparallel -sheet A prefibrillar oligomers (PFOs), along with PFOs of other amyloid peptides sharing a common generic epitope arising from antiparallel -sheet aggregates[5,12]. Polyclonal rabbit serum OC recognizes parallel -sheet A fibrils and fibrillar oligomers, along with amyloid fibrils of other peptides sharing common epitopes arising from parallel , in-register -sheet structure[7,13]. Recently, we cloned 23 unique monoclonal antibodies from A 42 fibril vaccinated rabbits producing OC serum[14]. "
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    ABSTRACT: Recently we reported that several monoclonal antibodies that recognize linear segments of amyloid-β (Aβ) also recognize amyloid fibrils, but not monomers of unrelated sequences, indicating that recognition of a linear sequence segment is not a reliable indicator of sequence specificity. We asked whether any of the commonly used commercially available Aβ antibodies also recognize fibrils of unrelated sequence. Here we report that 4G8, which recognizes residues 18-23 of the Aβ sequence and is widely believed to be sequence-specific, also recognizes fibrils formed from α-synuclein and islet amyloid polypeptide (IAPP). The recognition of amyloid fibrils is aggregation-dependent because 4G8 does not recognize α-synuclein or IAPP monomer. 4G8 also stains fibrillar α-synuclein aggregates in human multiple system atrophy brain where it colocalizes with anti-α-synuclein monoclonal antibody LB509 immunoreactivity. We also found that LB509 recognizes Aβ fibrils, but not monomer, indicating that generic epitope-reactive antibodies are also produced in response to α-synuclein immunization. Taken together, our results indicate that generic fibril conformational epitope specificity may be a pervasive property among monoclonal antibodies raised against amyloid-forming antigens and that the specificity of their immunoreactivity should be rigorously established and otherwise interpreted with caution.
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    • "Reagents EGCG was obtained from Sigma-Aldrich and stock solutions of 10 mM were freshly prepared in aqueous buffer. The anti-amyloid antibody, OC (Kayed et al., 2007), was from Millipore. "
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