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 Igkappa4 Bence Jones protein Len, reacted specifically with light chain (LC) fibrils, irrespective of kappa or lambda isotype but, notably, did not with native molecules (Hrncic, R. et al. (2000) Am. J. Pathol. 157, 1239-1246). 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 (N-terminal) 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 beta-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.

Figures

Figure 1

Binding of mAb 11-1F4 to LC amyloid fibrils. (Upper panel) Immunohistochemical analyses of Congo red- (left panels) and mAb 11-1F4- (right panels) stained tissue sections containing λ1, λ8, κ1, and κ4 amyloid deposits (Congo red fluorescence and immunoperoxidase reactivity, respectively; original magnification ×100 (λ1, λ8) and ×80 (κ1, κ4)). (Lower panel) EuLISA of mAb 11-1F4 binding to microtiter plate well-immobilized (left) κ1 (yellow □), κ2 (red rectangle), and κ4 (green △), and (right) λ1 (●), λ3 (blue ◇), λ6 (teal ◇), and λ8 (gray ○) AL fibril extracts.

Figure 2

Binding of mAb 11-1F4 to native and misfolded LC molecules. Antibody interaction, measured by EuLISA, to microtiter plate well-immobilized molecules: (a) native LC Len (○), native VL Len ●), and VL Len fibrils (▲); (b) VL Len in the absence (●) or presence of a 50-fold molar excess of soluble (○) or fibrillar (▲)VL Len; (c) immobilized mAb 11-1F4 binding to uncentrifuged (■) and centrifuged (□) biotinylated VL Len as well as solution-phase antibody binding to immobilized biotinylated (○) or unmodified VL Len (●); (d) immobilized VL Len in the presence of intact (△), F(ab′)2 (●), and F(ab) (○) 11-1F4.

Figure 3

LC epitope recognized by mAb 11-1F4. (a) N-terminal amino acid sequences of the first 30 residues of LC Len and the synthetic Len-related peptides as well as the EC50 and IC50 values of antibody and peptide reactivity, respectively, determined by EuLISA from the binding and competition curves shown in panels b through e. (b) Binding of mAb 11-1F4 to microtiter plate-immobilized VL Len and Len peptides. (c) Competition binding curves of the interaction of mAb 11-1F4 with immobilized VL Len in the presence of VL Len or peptides. (d) Comparison of antibody binding to immobilized Len (1–22) and the same peptide containing a serine for proline substitution at position 8 (P8S). (e) Competition binding curves of the interaction of mAb 11-1F4 with immobilized VL Len in the presence of Len (1–22) or P8S Len (1-22) peptides.

Figure 4

Inhibition of fibril binding by mAb 11-1F4. (a) Binding curves for the interaction of mAb 11-1F4 with microtiter plate-immobilized rVλ6 Wil fibrils in the presence of solution-phase competitors, soluble rVλ6 Wil (△), or Len (1–22) peptide (□), or in their absence (▲). (b) Inhibition of mAb 11-1F4 binding to human ALκ- and ALλ-related extracts by a 50× molar excess of soluble Len (1–22) peptide.

Figure 5

Proposed model for the conformational fibril-associated epitope recognized by mAb 11-1F4. (a) Amino acid sequence and X-ray crystallographic localization of the N-terminal A (4.1) and B (4.2) strands of native VL Len. (b) Hypothetical structural alterations in misfolded (i.e., partially denatured or fibrillar) VL Len involving a proline anchored loop flip of the N-terminal end of strand A (A′). The N-terminal 18 residues are bolded.