Phage display and peptide mapping of an immunoglobulin light chain fibril-related conformational epitope

Abstract

Amyloid fibrils and partially unfolded intermediates can be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we previously had reported that mAb 11-1F4, generated by immunizing mice with a thermally denatured variable domain (VL) fragment of the human kappa4 Bence Jones protein Len, bound to a non-native conformational epitope located within the N-terminal 18 residues of fibrillar, as well as partially denatured, Ig light chains (O'Nuallain, B., et al. (2006) Biochemistry 46, 1240-1247). To define further the antibody binding site, we used random peptide phage display and epitope mapping of VL Len using wild-type and alanine-mutated Len peptides where it was shown that the antibody epitope was reliant on up to 10 of the first 15 residues of protein Len. Comparison of Vkappa and Vlambda N-terminal germline consensus sequences with protein Len and 11-1F4-binding phages indicated that this antibody's cross-reactivity with light chains was related to an invariant proline at position(s) 7 and/or 8, bulky hydrophobic residues at positions 11 and 13, and additionally, to the ability to accommodate amino acid diversity at positions 1-4. Sequence alignments of the phage peptides revealed a central proline, often flanked by aromatic residues. Taken together, these results have provided evidence for the structural basis of the specificity of 11-1F4 for both kappa and lambda light chain fibrils. We posit that the associated binding site involves a rare type VI beta-turn or touch-turn that is anchored by a cis-proline residue. The identification of an 11-1F4-related mimotope should facilitate development of pan-light chain fibril-reactive antibodies that could be used in the diagnosis and treatment of patients with AL amyloidosis.

Figures

FIGURE 1

Multiple sequence alignments and consensus sequences for 11-1F4-binding phage peptides. Phage peptides were aligned (28) and divided into Groups I and II, based on the presence of one of the two proline (-X-Pro-X- and Pro-X-Pro) or cysteine (-le/Leu-Cys and Ile/Leu-X-Cys-) motifs, and each group adjusted to give the best alignment with the N-terminal sequence of VL Len. The hydrophobic and/or aromatic residues are bolded and prolines underlined. Phage peptide consensus sequences were determined using the program CONSENSUS (http://www.bork.embl-heidelberg.de/Alignment/). Abbreviations include: a, aromatic (F, H, Y, W); h, hydrophobic, (A, C, F, I, L, M, V); l, aliphatic (I, L, V); o, S or T; p, polar (D, E, G, H, K, N, P, Q, R, S, T, W, Y); s, small side chain (A, C, D, G, N, P, S, T, V) (31), and each uppercase letter represents the particular amino acid.

FIGURE 2

Sequence-position comparison of 11-1F4-binding phage peptides with the first 14 residues of VL Len. (a) Residue identity between phage peptides and VL Len; the number of identical (closed bars) and chemically similar (open bars) residues for each position are shown. The solid and open circles indicate the number of identical and chemically similar phage peptide residues expected to occur by chance, based on the observed frequency of amino acids in the random peptide phage display library (http://www.neb.com/nebecomm/ManualFiles/manualE8110.pdf). (b) Percentage of aromatic residues in the phage peptides at each position. The line dashed represents the percentage abundance of aromatic amino acids relative to all other residues in the random peptide phage display library. (c) Average residue hydropathicity values (33) for phage peptides (closed bars) relative to those of the Len(1–14) peptide (open bars). Each sequence position average hydropathicity value was determined from the sum of residue hydropathicity values divided by the number of total residues. The dashed line shows the average hydropathicity value for all 20 amino acids, corrected for the abundance of each in the random peptide phage display library. All sequence-position comparisons were determined using the multiple sequence alignments shown in Figure 1.

FIGURE 3

Binding of mAb 11-1F4 to plate-immobilized Len peptides. (a) Binding of 11-1F4 to Len peptides. (b) Competitive inhibition of 2 nM 11-1F4 binding to Len(1–22) in the presence or absence of Len peptides. Len(1–15), (△); Len(1–13), (▲); LenP8S(1–22), (♦); and Len(1–22), (○). The dashed line indicates 50% inhibition.

FIGURE 4

11-1F4-binding to plate-immobilized phage, VL Len, and synthetic phage peptides. NaSCN titration curves for antibody binding to (a) phage, protein Len, or (b) synthetic phage peptides. (c) 11-1F4 titration curves for binding to synthetic phage peptides. IC50 and EC50 values were determined from the midpoint of each titration curve. Len, (△); f11, (●); b2, (○); and a12, (♦) phage particles or peptide; and unamplified phage display library control (■).

FIGURE 5

Binding of mAb 11-1F4 to plate-immobilized Len(1–22) and phage peptides in the presence and absence of peptide inhibitors. (a) Binding of 11-1F4 to phage peptides or Len(1–22). (b) Competitive inhibition of 2 nM 11-1F4 binding to Len(1–22) in the presence of phage peptides or Len(1–22). The dashed line indicates 50% inhibition. a12, (▼); g4, (■); b2, (▽); a7, (□); and Len(1–22), (○).

FIGURE 6

Interaction of mAb 11-1F4 with plate-immobilized, Ala-substituted Len(1–18) peptides. (a) Binding of 11-1F4 to Len(1–18), (▲); IA2, (○); V3A, (●); T5A, (Δ); D9A, (□); and P8A, (■). (b) Antibody binding to Len(1–18), (▲); I11A, (▽); V13A, (♦); S14A, (◊); and I15A, (×).

FIGURE 7

Characterization of the specificity of anti-phage peptide immune serum. Reactivity of serum from an A12-immunized mouse (solid bars) and non-immunized mouse (open bars) against the immunogen, Len(1–22), as well as VL Len, and collagen fibrils.

FIGURE 8

Competitive inhibition of mAb 11-1F4 binding to Aβ40 fibrils. Binding of antibody to plate-immobilized Aβ40 fibrils in the absence of competitor (●) or in the presence of Jto fibrils (□) or Aβ40 fibrils (△). Competition experiments were performed by pre-incubating 11-1F4 with ~0.2 mg/ml fibrils.

FIGURE 9

Consensus sequence analyses and schematic representation of the conformational fibril-associated epitope recognized by mAb 11-1F4. (a) Comparison of Vκ4 with non-κ4 germ line consensus sequences (bolded residues are hydrophobic.). Abbreviations used in consensus sequences are: h, hydrophobic (A, C, F, I, L, M, V); o, S or T; p, polar (D, E, G, H, K, N, P, Q, R, S, T, W, Y); s, small residues (A, C, D, G, N, P, S, T, V), and u, minute residues (A, C, G, S). Consensus sequences were determined from VL germline sequences using CONSENSUS (http://us.expasy.org/tools/) analysis of CLUSTALW (28) multiple aligned sequences with 70% threshold. (b) Proposed model for the conformational epitope recognized by 11-1F4 involves a rearrangement of the first 9 VL residues centered on a cis-proline type VI β-turn or touch-turn induced by partial denaturation via surface adsorption or by fibrillogenesis. The N-terminal 15 residues are bolded.