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

Abstract

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...

Current and New Perspectives on the Molecular and Cellular Mechanisms of Amyloid Formation and Toxicity in Light Chain Amyloidosis

“…Immunohistochemical analysis revealed that mAB 11-1F4 recognized light chain fibrils regardless of their V L subgroup. The specificity of this antibody for AL fibrils (I, II, IV, 1, 3, 6, 8) was shown by Europium-Linked Immunosorbant Assay (EuLISA) where an EC 50 value (concentration of antibody at half maximum binding) for binding was ~130 ± 39 nM (O'Nuallain et al, 2007). The interaction of mAB 11-1F4 with native and fibrillar light chain LEN components was also checked by EuLISA and the antibody had similar avidity with both components.…”

“…The interaction of mAB 11-1F4 with native and fibrillar light chain LEN components was also checked by EuLISA and the antibody had similar avidity with both components. However, the fibrils had a ~2 fold reduction in signal (O'Nuallain et al, 2007). Peptide mapping was used to determine the cryptic epitope; it is located in the first 18 amino acids of the variable light chain domain and a prolyl residue at position 8 is necessary.…”

“…Peptide mapping was used to determine the cryptic epitope; it is located in the first 18 amino acids of the variable light chain domain and a prolyl residue at position 8 is necessary. A competition EuLISA was set up with mAB 11-1F4, and recombinant Wil fibrils were inhibited by a 50-fold molar excess of soluble LEN (1-22) peptide (O'Nuallain et al, 2007).…”

Inherent Anti-amyloidogenic Activity of Human Immunoglobulin γ Heavy Chains

“…Antibody Binding and Competition Assays-HC and antibody reactivity with amyloid fibrils, CAPS, and elastin fibrils was determined at 37°C using a europium (Eu 3ϩ )-based fluoroimmunoassay (EuLISA) (35) or ELISA. Antibodies or HCs were serially diluted in assay buffer (1% BSA in PBSA) and tested (100 l/well) in activated, high binding microtiter plate wells (COSTAR, Corning, NY) that were coated with 400 ng of target protein and blocked with 1% BSA (Sigma-Aldrich) in PBSA.…”

“…The statistical comparisons used paired and unpaired Student's t tests. (25) in our standard EuLISA (35). Following limiting dilution subcloning, two independent hybridomas, F1 and F2, were isolated that bound to LC fibrils with molar antibody concentrations that obtained half-maximum binding (EC 50 ) values of ϳ20 nM ( Fig.…”