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 (V L ) fragment of the human κ4 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. To define further the antibody binding site, we used random peptide phage display and epitope mapping of V L Len using wild-type and alaninemutated 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 V κ and V λ 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 κ and λ light chain fibrils. We posit that the associated binding site involves a rare type VI β-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.The amyloidoses are a group of over 20 protein misfolding disorders associated with often fatal sporadic and hereditary disorders, including Alzheimer's disease, type II diabetes, and primary (AL) amyloidosis (1-4). All types of amyloid, regardless of amino acid sequence, share tertiary structural and tinctorial features, i.e., they are formed from fibrils composed of extended β-sheets oriented perpendicularly to the long fibril axis (5,6) and bind the dyes thioflavin T and Congo red (7,8). Additionally, fibrils, as well as their assembly intermediates, contain generic conformational epitopes not present on the native proteins (9-16). These common characteristics reflect a similar fibrillogenic pathway(s) involving the assembly of soluble higher-order intermediates into fibrils that are structurally dissimilar to their precursors (2, 13,17).

Generation and Characterization of Anti-AA Amyloid-Specific Monoclonal Antibodies

“…Although proteolysis of the precursor protein is common in amyloid fibrils (Bohne et al, 2004; Monti et al, 2005; Röcken et al, 2006; Enqvist et al, 2009), this is the first example of fibril-specific mAbs that target a neo-epitope formed as a result of proteolytic cleavage. In contrast, other reported anti-fibril-mAbs bind conformational epitopes present on the non-native, fibrillar form of the amyloid precursor protein, but not the native, soluble, circulating molecule (O’Nuallain et al, 2005, 2007). …”

Radioimmunodetection of amyloid deposits in patients with AL amyloidosis

“…10,11 Thus, the inability of the radiolabeled antibody to bind certain AL deposits, both in vivo and in vitro, may reflect a Consecutive tissue sections from each patient (AL 2, lymph node; AL 11, bone marrow, and AL 12, liver) were subjected to histochemical (HC) staining with Congo red (CR, top) or immunohistochemical (IHC) studies using, as primary reagent, mAb 11-1F4 (middle), or, as a negative control, the antibody preincubated with a 22-mer peptide containing the conformational fibril-related epitope recognized by mAb 11-1F4 (bottom). Photomicrographs were acquired with a Leica DM 500 light microscope equipped with cross-polarizing filters.…”

“…10,11 Furthermore, when administered to mice bearing subcutaneous human AL amyloidomas, the antibody bound to the pathologic material and initiated an inflammatory response that led to elimination of the induced tumors. 12 Notably, we also demonstrated that m11-1F4, after radiolabeling with the positron-emitting isotope I-124, imaged the xenograft, as evidenced by micropositron emission tomography/computed tomography (PET/CT).…”

Anti-heparan Sulfate Peptides That Block Herpes Simplex Virus Infection in Vivo

“…To prove this point, we decided to perform alanine-scanning mutagenesis. It is commonly used to identify specific amino acid residues responsible for peptide function, stability, and conformation (36). Synthetic peptides were made whereby each residue of G2 was sequentially replaced with an Ala residue, and corresponding changes in G2 peptide were evaluated for the ability to affect viral entry (Fig.…”