Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers

Kayed, Rakez; Head, Elizabeth; Sarsoza, Floyd; Saing, Tommy; Cotman, Carl W.; Necula, Mihaela; Margol, Lawrence; Wu, Jessica; Breydo, Leonid; Thompson, Jennifer L.; Rasool, Suhail; Gurlo, Tatyana; Butler, Peter C.; Glabe, Charles G.

doi:10.1186/1750-1326-2-18

Cited by 666 publications

(864 citation statements)

References 27 publications

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“…4 A and B) and oligomer-specific (A11; Fig. 4C) antibodies, revealing that they bind to unique conformational epitopes relative to previously identified conformation-specific antibodies (4,5,8). We conclude that Aβ gammabodies employ self-interactions between grafted amyloidogenic motifs and the same motifs within Aβ conformers to mediate conformation-and sequence-specific antibody recognition.…”

Section: Antibody Domains Displaying Amyloidogenic Aβ Motifs Recognizementioning

confidence: 55%

“…A breakthrough in this area has been the development of conformation-specific antibodies that selectively recognize uniquely folded conformers of amyloidogenic proteins (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Indeed, multiple conformation-specific antibodies have been reported that recognize structural features within amyloidogenic oligomers (5) and fibrils (4,6,8) in a sequence-independent manner. These and related antibodies have proven invaluable for identifying oligomeric and fibrillar conformers of several diseaselinked proteins both in vitro and in vivo (3-13).…”

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confidence: 99%

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Structure-based design of conformation- and sequence-specific antibodies against amyloid β

Perchiacca

Ladiwala

Bhattacharya

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

126

125

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Conformation-specific antibodies that recognize aggregated proteins associated with several conformational disorders (e.g., Parkinson and prion diseases) are invaluable for diagnostic and therapeutic applications. However, no systematic strategy exists for generating conformation-specific antibodies that target linear sequence epitopes within misfolded proteins. Here we report a strategy for designing conformation-and sequence-specific antibodies against misfolded proteins that is inspired by the molecular interactions governing protein aggregation. We find that grafting small amyloidogenic peptides (6-10 residues) from the Aβ42 peptide associated with Alzheimer's disease into the complementarity determining regions of a domain (V H ) antibody generates antibody variants that recognize Aβ soluble oligomers and amyloid fibrils with nanomolar affinity. We refer to these antibodies as gammabodies for grafted amyloid-motif antibodies. Gammabodies displaying the central amyloidogenic Aβ motif ( 18 VFFA 21 ) are reactive with Aβ fibrils, whereas those displaying the amyloidogenic C terminus ( 34 LMVGGVVIA 42 ) are reactive with Aβ fibrils and oligomers (and weakly reactive with Aβ monomers). Importantly, we find that the grafted motifs target the corresponding peptide segments within misfolded Aβ conformers. Aβ gammabodies fail to cross-react with other amyloidogenic proteins and scrambling their grafted sequences eliminates antibody reactivity. Finally, gammabodies that recognize Aβ soluble oligomers and fibrils also neutralize the toxicity of each Aβ conformer. We expect that our antibody design strategy is not limited to Aβ and can be used to readily generate gammabodies against other toxic misfolded proteins.misfolding | beta-amyloid | protein engineering A hallmark of protein misfolding disorders is that polypeptides of unrelated sequence fold into similar oligomeric and fibrillar assemblies that are cytotoxic (1). The structures of these enigmatic conformers have captured the imagination of many investigators who have sought to explain the molecular basis of proteotoxicity in conformational disorders such as Alzheimer's disease. Because misfolded proteins are typically refractory to structural methods such as X-ray crystallography and solution NMR, few high-resolution structures of full-length misfolded proteins have been reported (ref. 2 and references therein). The structures of oligomeric intermediates have proven especially difficult to characterize because these conformers are labile, transient, and, in many cases, heterogeneous.Given the complexity of high-resolution structural analysis of misfolded proteins, alternative biochemical approaches are critical for understanding structure-function relationships of aggregated proteins. A breakthrough in this area has been the development of conformation-specific antibodies that selectively recognize uniquely folded conformers of amyloidogenic proteins (3-13). Indeed, multiple conformation-specific antibodies have been reported that recognize structural features ...

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Section: Antibody Domains Displaying Amyloidogenic Aβ Motifs Recognizementioning

confidence: 55%

mentioning

confidence: 99%

Structure-based design of conformation- and sequence-specific antibodies against amyloid β

Perchiacca

Ladiwala

Bhattacharya

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

126

125

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“…6). 49 The OC binding further confirmed that the protein aggregates were at least partially composed of mature fibers containing cross β-sheet structure. The same experiment was measured by SAF microscopy directly in the flow cell with similar results (Supporting Fig.…”

Section: Structural Characterization Of the Protein Aggregatesmentioning

confidence: 73%

“…To further confirm the amyloid nature of the aggregates, the samples were stained with an Alexa-488 labeled OC antibody, 49 specific to the cross β-sheet amyloid structure regardless of the primary sequence of the protein of origin (Fig. 6).…”

Section: Structural Characterization Of the Protein Aggregatesmentioning

confidence: 99%

Mechanism of Membrane Interaction and Disruption by α-Synuclein

et al. 2011

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Parkinson's disease is a common progressive neurodegenerative condition, characterized by the deposition of amyloid fibrils as Lewy bodies in the substantia nigra of affected individuals. These insoluble aggregates predominantly consist of the protein -synuclein. There is increasing evidence suggesting that the aggregation of -synuclein is influenced by lipid membranes and, vice versa, the membrane integrity is severely affected by the presence of bound aggregates. Here, using the surface-sensitive imaging technique supercritical angle fluorescence microscopy and Förster resonance energy transfer, we report the direct observation of -synuclein aggregation on supported lipid bilayers. Both the wild-type and the two mutant forms of -synuclein studied, namely, the familiar variant A53T and the designed highly toxic variant E57K, were found to follow the same mechanism of polymerization and membrane damage. This mechanism involved the extraction of lipids from the bilayer and their clustering around growing -synuclein aggregates. Despite all three isoforms following the same pathway, the extent of aggregation and their effect on the bilayers was seen to be variant and concentration dependent. Both A53T and E57K formed cross--sheet aggregates and damaged the membrane at submicromolar concentrations. The wild-type also formed aggregates in this range; however, the extent of membrane disruption was greatly reduced. The process of membrane damage could resemble part of the yet poorly understood cellular toxicity phenomenon in vivo. AbstractParkinson`s disease is a common progressive neurodegenerative condition, characterized by the deposition of amyloid fibrils as Lewy bodies in the substantia nigra of affected individuals. These insoluble aggregates predominantly consist of the protein α-Synuclein. There is increasing evidence suggesting that the aggregation of α−Synuclein is influenced by lipid membranes and, vice versa, the membrane integrity is severely affected by the presence of bound aggregates. Here, using the surface sensitive imaging technique super critical angle fluorescence microscopy and Förster resonance energy transfer we report the direct observation of α−Synuclein aggregation on supported lipid bilayers. Both the wild-type and the two mutant forms of α−Synuclein studied, namely the familiar variant A53T and the designed highly toxic variant E57K, were found to follow the same mechanism of polymerization and membrane damage. This mechanism involved the extraction of lipids from the bilayer and their clustering around growing α−Synuclein aggregates. Despite all three isoforms following the same pathway, the extent of aggregation and their effect on the bilayers was seen to be variant and concentration dependent. Both A53T and E57K formed cross β-sheet aggregates and damaged the membrane at sub-micromolar concentrations. The wild-type also formed aggregates in this range; however, the extent of membrane disruption was greatly reduced. The process of membrane damage could resemble the yet poorly...

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“…57,58 In addition, we performed an antibody stain using a conformational specific anti-amyloid antibody (OC). 59 For this experiment, we employed unlabeled α-Syn protein and observed extensive antibody binding, indicative of the presence of amyloid-like structures ( Figure S5 in the Supporting Information). Since label-free α-Syn followed the same aggregation process as the acceptor or donor labeled protein, the influence of the fluorophores can be considered negligible, in line with what was reported in other studies.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

On-Surface Aggregation of α-Synuclein at Nanomolar Concentrations Results in Two Distinct Growth Mechanisms

Rabe

Soragni

Reynolds

et al. 2013

ACS Chem. Neurosci.

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The aggregation of α-synuclein (α-Syn) is believed to be one of the key steps driving the pathology of Parkinson's disease and related neurodegenerative disorders. One of the present hypotheses is that the onset of such pathologies is related to the rise of α-Syn levels above a critical concentration at which toxic oligomers or mature fibrils are formed. In the present study, we find that α-Syn aggregation in vitro is a spontaneous process arising at bulk concentrations as low as 1 nM and below in the presence of both hydrophilic glass surfaces and cell membrane mimicking supported lipid bilayers (SLBs). Using three-dimensional supercritical angle fluorescence (3D-SAF) microscopy, we observed the process of α-Syn aggregation in situ. As soon as α-Syn monomers were exposed to the surface, they started to adsorb and aggregate along the surface plane without a prior lag phase. However, at a later stage of the aggregation process, a second type of aggregate was observed. In contrast to the first type, these aggregates showed an extended structure being tethered with one end to the surface and being mobile at the other end, which protruded into the solution. While both types of α-Syn aggregates were found to contain amyloid structures, their growing mechanisms turned out to be significantly different. Given the clear evidence that surface-induced α-Syn aggregation in vitro can be triggered at bulk concentrations far below physiological concentrations, the concept of a critical concentration initiating aggregation in vivo needs to be reconsidered.

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Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers

Cited by 666 publications

References 27 publications

Structure-based design of conformation- and sequence-specific antibodies against amyloid β

Structure-based design of conformation- and sequence-specific antibodies against amyloid β

Mechanism of Membrane Interaction and Disruption by α-Synuclein

On-Surface Aggregation of α-Synuclein at Nanomolar Concentrations Results in Two Distinct Growth Mechanisms

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