Characterization of Systemic Amyloid Deposits by Mass Spectrometry

Murphy, Charles L.; Wang, Shuching; Williams, T; Weiss, Deborah; Solomon, Alan

doi:10.1016/s0076-6879(06)12004-2

Cited by 109 publications

(48 citation statements)

References 22 publications

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“…As evidenced, the epitope recognized by this antibody was present within the first 30 amino acids, and in studies of peptides encompassing this region (Figure 3a and b), it was further localized to the N-terminal 22 residues with an EC 50 of 0.24 ± 0.01 nM for Len , a value comparable to that of the intact V L (EC 50 , 0.18 ± 0.03 nM). Notably, there was no reactivity with Len (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). More precisely, the structure recognized by mAb 11-1F4 was contained within the N-terminal 18 residues (EC 50 Len (1-18), 0.30 ± 0.10 nM).…”

Section: Mab 11-1f Binds To a Conformational Epitope Located Within Fmentioning

confidence: 99%

Localization of a Conformational Epitope Common to Non-Native and Fibrillar Immunoglobulin Light Chains

et al. 2007

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Amyloid fibrils and partially unfolded intermediates may be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we had previously reported that the IgG1 mAb 11-1F4, generated by immunizing mice with a thermally denatured variable region fragment of the human Igκ4 Bence Jones protein Len, reacted specifically with light chain (LC) fibrils, irrespective of κ or λ isotype but, notably, did not with native molecules (Hrncic, R. et al. (2000) Am. J. Pathol. 157, 1239Pathol. 157, -1246. To elucidate the molecular basis of this specificity, we have used a europium-linked fluorescent immunoassay, where it was demonstrated through epitope mapping that mAb 11-1F4 recognizes a conformational determinant contained within the first (Nterminal) 18 amino acids of misfolded LCs. The nature of this epitope was evidenced in competition studies where the peptide Len (1-18), but not the intact protein or other LCs, inhibited the binding of the antibody to fibrils. This unique reactivity was dependent on the structural integrity of this portion of the molecule, particularly the presence of a highly conserved prolyl residue at position 8. On the basis of our experimental data, we posit that the mAb 11-1F4 binding site found on partially denatured and fibrillar LCs involves an inducible N-terminal main chain reversal that results in the formation of a proline anchored β-turn. Our delineation of this LC fibril-associated epitope provides the rationale for the design of novel amyloid-reactive antibodies with diagnostic and therapeutic potential for patients with LC-associated and other forms of amyloidosis. Amyloidogenesis involves a process by which normally soluble proteins or peptides form nonnative intermediates that eventually assemble into fibrils (1). All types of amyloid, regardless of amino acid sequence, share common tertiary structural features, that is, they are comprised of extended β-sheets oriented perpendicularly to the long fibril axis (2,3) and possess sites that bind the dyes thioflavin T (ThT1) and Congo red (4,5). Additionally, these components and their assembly precursors, irrespective of primary structure, contain generic conformational epitopes not present on native molecules (6-12), further indicating their physical homogeneity.The common epitopes present on amyloid fibrils and their intermediates offer targets for the design of novel diagnostic and therapeutic reagents. In this regard, we have generated a murine monoclonal antibody (mAb), designated 11-1F4 (13), which reacts specifically with light chain (LC) fibrils, but not soluble proteins, and has been shown in an experimental in vivo model to accelerate the removal of amyloidomas composed of human LC fibrils, an effect independent of the κ or λ isotype or the V L subgroup (9). This mAb was generated using as immunogen a heat-aggregated suspension of the human κ4 variable domain (V L ) Len (13,14) To localize and determine the nature of the neo-antigen recognized on conformationally altered LCs by...

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Section: Mab 11-1f Binds To a Conformational Epitope Located Within Fmentioning

confidence: 99%

Localization of a Conformational Epitope Common to Non-Native and Fibrillar Immunoglobulin Light Chains

et al. 2007

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“…[8][9][10][11][12] Although more sophisticated biochemical approaches have been used, they require quantities of tissue not readily available in a routine clinical setting and suffer from a lack of specificity because they contain nonamyloid tissues and serum, which are a rich source of amyloidogenic proteins. [13][14][15] In this study, we report the development of a specific and sensitive novel test for typing of amyloidosis in routine clinical specimens. To overcome the aforementioned difficulties, our approach combines specific sampling by laser microdissection (LMD) and analytical power of tandem mass spectrometry (MS)-based proteomic analysis.…”

Section: Introductionmentioning

confidence: 99%

Classification of amyloidosis by laser microdissection and mass spectrometry–based proteomic analysis in clinical biopsy specimens

Vrana

Gamez

Madden

et al. 2009

Blood

772

572

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The clinical management of amyloidosis is based on the treatment of the underlying etiology, and accurate identification of the protein causing the amyloidosis is of paramount importance. Current methods used for typing of amyloidosis such as immunohistochemistry have low specificity and sensitivity. In this study, we report the development of a highly specific and sensitive novel test for the typing of amyloidosis in routine clinical biopsy specimens. Our approach combines specific sampling by laser microdissection (LMD) and analytical power of tandem mass spectrometry (MS)-based proteomic analysis. We studied 50 cases of amyloidosis that were well-characterized by gold standard clinicopathologic criteria (training set) and an independent validation set comprising 41 cases of cardiac amyloidosis. By use of LMD/MS, we identified the amyloid type with 100% specificity and sensitivity in the training set and with 98% in validation set. Use of the LMD/MS method will enhance our ability to type amyloidosis accurately in clinical biopsy specimens. (Blood. 2009;114: 4957-4959)

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“…Neuritic plaque cores from AD brain were isolated as described (17). The chemical nature of patient-derived fibrils was determined by protein sequencing and tandem mass spectrometry (34). All of the amyloid and non-amyloid bovine elastin fibrils (Sigma-Aldrich) were sonicated (2 ϫ 30 s bursts) with a probe sonicator disruptor (Teledyne Tekmar, Mason, OH), aliquoted, and stored at Ϫ20°C.…”

Section: Methodsmentioning

confidence: 99%

Inherent Anti-amyloidogenic Activity of Human Immunoglobulin γ Heavy Chains

Adekar

Klyubin

Macy

et al. 2010

Journal of Biological Chemistry

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We have previously shown that a subpopulation of naturally occurring human IgGs were cross-reactive against conformational epitopes on pathologic aggregates of A␤, a peptide that forms amyloid fibrils in the brains of patients with Alzheimer disease, inhibited amyloid fibril growth, and dissociated amyloid in vivo. Here, we describe similar anti-amyloidogenic activity that is a general property of free human Ig ␥ heavy chains. A ␥ 1 heavy chain, F1, had nanomolar binding to an amyloid fibril-related conformational epitope on synthetic oligomers and fibrils as well as on amyloidladen tissue sections. F1 did not bind to native A␤ monomers, further indicating the conformational nature of its binding site. The inherent anti-amyloidogenic activity of Ig ␥ heavy chains was demonstrated by nanomolar amyloid fibril and oligomer binding by polyclonal and monoclonal human heavy chains that were isolated from inert or weakly reactive antibodies. Most importantly, the F1 heavy chain prevented in vitro fibril growth and reduced in vivo soluble A␤ oligomer-induced impairment of rodent hippocampal long term potentiation, a cellular mechanism of learning and memory. These findings demonstrate that free human Ig ␥ heavy chains comprise a novel class of molecules for developing potential therapeutics for Alzheimer disease and other amyloid disorders. Moreover, establishing the molecular basis for heavy chain-amyloidogenic conformer interactions should advance understanding on the types of interactions that these pathologic assemblies have with biological molecules. Alzheimer disease (AD)3 is approaching global epidemic proportions and is the most common of over 25 incurable protein misfolding diseases that are termed the amyloidoses (1, 2). A hallmark of the disease is deposition of amyloid fibril-containing neuritic plaques that are formed by abnormal processing of ␤-amyloid protein (A␤), a proteolyzed transmembrane 39 -43 fragment of amyloid precursor protein (3). Diverse experimental studies support a central pathogenic role for aggregated A␤, which in the brain initiates a cascade of events that ultimately lead to neuronal dysfunction and death (4 -6). The most neurotoxic A␤ species are low molecular weight oligomers (5), which include noncovalently linked species (7-9) and dityrosine cross-linked ␤-amyloid protein species (CAPS) (10).Active vaccination with A␤ or passive administration of anti-A␤ antibodies has shown promise in transgenic AD animal models and in some AD patients by inducing neuritic plaque clearance, neutralizing neurotoxic soluble A␤ oligomers, and/or improving cognitive functioning (6,8,(11)(12)(13). Immunotherapy has generally targeted linear sequence epitopes on A␤ (13). However, antibodies that do not distinguish between pathologic A␤ conformers and the monomeric peptide may have adverse effects because the native peptide has been implicated in normal lipid and cholesterol homeostasis (14). Of potentially greater use are antibodies that cross-react with conformational epitopes on pathogenic aggregated A...

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Characterization of Systemic Amyloid Deposits by Mass Spectrometry

Cited by 109 publications

References 22 publications

Localization of a Conformational Epitope Common to Non-Native and Fibrillar Immunoglobulin Light Chains

Localization of a Conformational Epitope Common to Non-Native and Fibrillar Immunoglobulin Light Chains

Classification of amyloidosis by laser microdissection and mass spectrometry–based proteomic analysis in clinical biopsy specimens

Inherent Anti-amyloidogenic Activity of Human Immunoglobulin γ Heavy Chains

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